escherichia coli genes regulated tramposable coliphage rotein...
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Escherichia coli Genes Regulated by the Tramposable Coliphage Ner Like Rotein-NLP
by Lisa Paterson
Dep artment of Microbiology and Immunology, McGill University
Montreal, Quebec, Canada
A thesis submitted to the Faculty of Graduate Studies and Research m partial fdfillment of the
requirements of the degree of Maaer of Science
O Lisa Alexandra Hunter Paterson, 1997
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Abstraet
The regdation of gene expression is essential to the Survival of cek The Ner (aegative -miy
~egulation) protek of temperate coliphages Mu and DIOS, the Nlp (Ner B e protein) proteins of the
Enterobacteriaceae, and the TMF (HIV- 1 TATA element modulatory gaor) protein of Homo
sqiem comprise a unique f b d y of DNA bmding proteins that contain a conserveci DNA bmdiog
domah The fimction of the ficherichia coli Nlp protein was mvestigated by identifjing genes
which Nlp may regulate. To iden* these gmes, lac Z gene fisions were generated m a strain of E
.coli - whose own nlp gene had been mternrpted by a ltcvlB tet msertion. Each clone withm this gene
tùsion collection had the lac Z gene mtegrated into a random location on the E. coli chromosome.
Clones m which the lac Z gene had insertal within a gene regulated by Nlp were predicted to have a
Werent lac Z phenotype on X-gal plates depending on the presence or absence of Mp expresseci
fiom a plasmid borne copy of the nlp gene. ûfthe 3360 clones screened, 5 were found to have a .
altered lac phenotype m the presence of Nlp. Three of these clones were found to have lac Z
expression mduced by Nlp, one was found to be repressed by Nlp, and one clone died upon Nlp
expression These putative Nlp-responsive genes were subcloned mto pBR322 to fàcilitate the*
identification. Restriction map aoaiysis, double stranded DNA sequencing and single stranded DNA
sequeochg were used to identay the Nlp responsive genes. Gaies that responded to NP,
overexpression were: 080 ('glycosol hydrol FS'), phB (which encodes a giyceroi-3-phosphate
acyltransferase), cscB (which encodes a sucrose pemiease), pgi (which encodes ghcoçe-&phosphate
isomerase), and mroF (which encodes the F subunit of NADH dehydrogenase). The role that Nlp
plays m the expression of these genes is hypothesid. Further mvestigation win provide msight mto
the function of Nip and fiutber c h a r a a h the NEWNP>/TMF farnih. of DNA bmding proteins.
Résumé
La régulation de la transcription est essentielle a la suivie des cellules. Les
protéines Ner (negative &y ~egdation) des phages tempérés Mu et D 108 d7Escherichia
colz avec les protéines Nlp mer--me protein) des Erzîerobacteriiaceue et TMF (HIV- 1
TATA element modulatory factor) de Homo sapiem définissent une famille unique de -
protémes qui se lient a l'ADN et dont les domaines de liaison sont conservés. L'élucidation
de la fonction de N p fut entreprise en identifiant les gènes qui sont régulés par Mp. Pour
se faire, des fusions du gène lac Z bent générées dans une souche d7E. coli dont le gène
nlp était interrompu (Seguin, 1995). Chaque clone de cette hirairie avait le gène lac Z
inséré à un difEerent site dans le chromosome d'E. coli. Les clones dans lesquels le gène
lac Z fit inséré directement en aval d'un gène régulé par Nlp auraient un phénotype
différent dépendant de la présence ou l'absence de la protéine Mp exprimée à partir d'une
copie du gène nlp sur un plasmide. Cinq des 3360 clones analysés démontraient une
altération du phénotype lac en présence de Nlp. Parmi ces cinq clones, trois montraient
une expression de lac Z induite par Nlp, un état réprimé par N p et un mourait lors de
l'expression de Mp. Ces gènes sensibles a Mp fùrent sous-clonés dans pBR322 pour
faciliter leur identification. La cartographie de restriction ainsi que les séquençages à
simple et à double brin furent entrepris pour identifier ces gènes sensibles à Mp. Les gènes
qui répondèrent à la surexpression de Nlp sont les suivants: 080 ('glycosol hydrol FS),
plsB (code pour une glycérol-3-phosphate acyltransférase), cscB (code pour une sucrose
perméase), pgi (code pour la glucose-bphosphate isomérase), et nuoF (code pour la sous-
unité F de la NADH déshydrogénase). L'hypothèse quant au(x) rôle(s) que joue Mp dans
l'expression de ces gènes est posée. Des recherches supplementaires apporteront des
précisions sur In fonction de Mp et aideront ahsi à mieux charactériser la famille
Ner/Nlp/TMF de protémes qui se k t à l'ADN.
iii
Table of Contents
Page #
* . ............................................................................................. Resume ................ .... ..ri
....................................................................................................... Acknowledgments M
... . . List of Abbrewations ............................................................................................... VUI
.......................................................................................... List of Figures and Tables - i ~
Chapter L Geaeral htroduction ............................................................................... 1
Chapter CL Materiais and Methods .................................................................... - 2 4
......................................................................... Bacterial Strains and Plasmids 24
. . ....................................................................................... Antii'biot~c Selection -24
............................................................... Restriction Endonuclease Digestions 24
. . ....................................................................................................... Ligauons -27
Preparation of Rubidium Chioride Cornpeteut C eils ......................................... 27
Transformation of Rubidium Chioride Competent CeUs .................................. 28
Cloning of NLP Respondmg Genes ................................................................ -29
Isoïation of Total Genomic DNA fiom Nlp Responrive Straim ........... 29
Subcloning the Nlp Respomïve Genes .................................................. 30
......................................................... Sequencing the Mp Responding Genes 30
........................................................... Dm6 le Stranded Sequencing - 3 0
.............................................................. Single S~anded Sequencing 3 1
Infection of the N p Responsive Clones (LF203020.LF203038. LF203040.
LF203047. and LF203057) with pBS5. pUC12Okan. pFIM2kan or pFT538 .... 32
Preprat im of the five M13K07 $sates .............................................. 32
Infection wirh Ml3K07 ..................................................................... -33
Chapter ïïL
.............................................................................................. Results -34
Clonhg a Region of the Mp Responsive Genes ............................................. 34
Restriction Endonuclease Digestions of p2O and p38 to Determine Orientation
and the Restriction Map of the Subcloned Portions of the Mp inducible
........................................................................................................ Genes -34
Double Stranded Sequencing Analysis of p20 and p 3 8 .................................. 42
The Identity o f N p Inducible Genes of Strahs LF203040 . LF203047, and
....................................................................................................... LF20305 7 45
M13K07 Infections with pFT528, pBS5, pFIM2Ka.u and pUC I20kan ........... 48
Chapter IV . Discussion ............................................................................................................... -57
................................................................................................................ References -78
Acknowiedgments
1 would iike to thank my supenisor, Dr.Michae1 DuBow for enthusiasm,
scientific expertise, and guidance throughout my Master's education. He has a keen
sense of how to capture an audience and make science both interesthg and
comprehensiile for scientists and non-scientists alüre. 1 would üke to thank hlln for
sharing his secrets m these areas.
1 would also like to extend my gratitude to Gina Macmtyre for her uubndled
enthusiasm, ashite scient& knowledge, support, and devotion to this project. 1 would
üke to also thank her for clonhg p40, p47,and p57 as weU as obtaining single manded
DNA for these clones. 1 would llso like to tbank Beatrice Seguin who did the initial
work for this project and whose hard work and perseverance got this project off of the
ground.
1 would also iike to extend my gratitude to the staff of this Depanment who
helped make my educational experience intereshg and enjoyable. 1 want to especiaily
thank the DuBow lab who have helped me over the past years and have made my
experience a memorable one. In particular, I would like to thank those that helped me
assemble this thesis: Madani Thiam for his translation of the abstract, Julie Guzzo for
constructhg the plasmid figures, Micheal Costanzo for giving me 'Power Point" tips
and helping me with the scanner. I'd also like to thank those that have helped me in
many h e s throughout my Master's education. These include, Caroline Diorio for her
scientific expertise, Felix Seider for his cornputer belp, Ian Siboo for his grasp of
'common sense', Doris Fortin for her taients m gel rescues, Jie Cai for ber sequencing
tips, David Alexander for helpmg me with sequence searches, Kirsty Salmon for her
support, organkational sküls, 're* fast and cleao' protocols, rides to the airport, and
enthusiasm, Julie Guuo for answering countless "how are we supposed
to.. ."questions (especially during my first T.A. experience), and Madani Thiam for bis
comy jokes, sound advice, patience (the list goes on.. .). 1 also want to thank Nicha for
her 'interesting' expressions, Stephanie Matheson for her unique and refieshing view on
life, Djenann St.Dic for her 'Yiood morning, Lisa!", and of course all DuBow lab
memben (present and past) with whom 1 have many fond memories most of which have
(unfortunately) been captured in photographs.
Finally, I would like to thank my hndy and fnends for theù consistent support.
More specificaily, 1 would like to thank my mother for her guidance and motivatiocal
sküls, my father for his advice, my sblings: Hüary, Feonagh, and Callum for their
humor and spirited personalities (which ahways cheer me up).I would also like to thank
my fnends Karen EUiott, Wendy Rose and Jan Luedecke who were always available to
kten to my hstrations, to offer advice and support and most importantly, to enjoy the
'finer' things in Me.
List of Abbreviations
amp ':
bp:
BSA:
CAMP:
cmr:
CRP:
DNA:
EDTA:
kanr :
kb:
kDa :
NMR
RNA:
SDS:
SSC:
tet ':
w/v:
ampicillin resistance
base pair(s)
bovine serum albumin
cyclic adenosine monophosphate
chloramphenicol resistance
CAMP receptor protem of E.coli; CAP
deoxyribonucleic acid
ethylene diamine tetraacetic acid
Kanamycin resist ance
kilobases
kilodaltons
nuclear magnetic resonance
nionucleic acid
sodium dodecyl sulfate
saturated sodium citrate
tetracyclin resistance
weight (in gr-) per volume ( 1 00 ml)
List of Figures and Tables
Page #
Chapter iï.
Table 1. Escherichia coli strains used m thi study.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -2 5
Table 2. Escherichia coli plasmids used in this study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -26
Chapter Tn.
Figure 1. Restriction endonuclease digestions of p20 and p38.. . . . . . . . . . . . . . . . . . . . . . . . . ... 36
Figure 2. Restriction map of Mud l lac. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -3 8
Figure 3. Redicted restriction endonuclease maps of p20 and p3 8. . . . . . . . . . . . . . . . . . . . . . -40
Figure 4. Restriction map for the Mp responsive gene and surroundmg sequence
subcloned into p20. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -. . . . - -43
Figure 5. Restriction map for the Mp responsive gene and surroundmg sequence
subcloned mto p38.. .. . . . . . . . . . . . . . .. . . . . .. . .. . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . ..46
Figure 6. Dideoxy-termination reactions on single stranded DNA fiom p 5 7sub. . . . . . . . .49
Figure 7. Restriction m p for the Nip responsive gene and surroundhg sequence
subcloned into p4O.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -5 1
Figure 8. Restriction map for the Nlp responsive gene and surroundmg sequence
subcloned mto p47. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -53
Figure 9. Restriction map for the Nlp responsive gene and surrounding sequence
subcloned mto p57.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -55
Table 3. The cornparison of lac phenotype of the N p responsive clones.. . . . . . . . . . . . . -59
Chapter L General Introduction
The regulation of gene expression is essential to the sumival and propagation of
organisms. Gene expression is regulated at several levels: transcription, translation, and
post-transcriptional or post-translational processing. Rokaryotic cells ofken use
transcription initiation as a means to control gene expression. Transcriptional initiation
is directed b y the RNA polymerase holoenzyme at sequences located immediat ely
upstream fiom expressed genes. These sequences are cailed promoters. Transcription
initiation is preceeded by several stepwise events. Fistiy, RNA polymerase recognizes
and binds to the promoters. Secondly, the DNA unwÎnds and changes conformation
from an initial closed complex to an open complex which facilitates DNA transcription.
RNA synthesis then begins and finally, the elongation complex is established
(Reviewed in Chamberlin, 1974).
In Esherichia coli, gene expression is initiated when RNA polymerase binds to
the promoter of the gene. RNA polymerase is a holoenzyme comprised of 5 subunit s:
a$pl a. The core enzyme (a$b') has the ability to synthesize RNA fiom the DNA
template (Burgess, 1969), but the sigma factor (o) is required to recognize and bmd to
the promoter (Travers and Burgess, 1969).The entire holoenzyme is therefore required
to initiate transcription, and the sigma factor is released fiom the core enzyme after the
RNA chiain has reached &9 nucleotides in fength. The majority of genes in E. coli are
recognized by the sigma factor 70 (0") wtiich specifïcaily bmds to consensus sequences
TATAAT and TTGACA centered 10 and 35 base pairs upstream fkom the start point of
transcription, respectively (Hawley and McClure, 1983). The sequence between these
consensus sequences is irrelevant, bowever the distance between them ( 16- 19 base
pairs) is critical in order to accommodate the stereochemistry of RNA polymerase.
Romoters that do not posses a -35 consensus sequence require the assistance of
accessory protemaceous factors for transcription activation (Reviewed m Raibaud and
Schwartz, 1984). Romoters that do not posses a - 10 consensus sequence require an
alternate sigma factor (ie. a3', or G" ) for transcription to proceed. These altemate
sigma factors are produced ody under certain environmentai conditions (ie. heat shock
or nitrogen starvation). Thus genes which require these specialized sigma fàctors are
invoived m specific environmental stress situations.
Recently, RNA polymerase was found to contai. another promoter bindhg
module, located m the C-tenninal domain of its a subunit, This domain was found to
bind to specific sequences upstream fiom the -3 5 consensus sequence and is a
determinant of promoter strength (Ross et al., 1993). Moreover, this domain also
contains the contact sites for Merent activator proteius, includmg CAMP receptor
prot ein (CRP) (discussed later).
RNA polymerase initiates transcription, but not every gene with a promoter and
both consensus sequences is constitutively expressed. M e r factors interact with RNA
polymerase or specinc DNA binding sites to control gene expression. T m actmg
regulatory proteins and cis-acting DNA sites are two components which fine tune the
control of gene expression. A tram-acting regulatory protem can act on any target
gene, where as a czs-acting site ody controls the expression of genes immediately
surrounding its location.
Gene expression is controlled either negatively or positively. Negative control
descriiles a gene that is active mtil a repressor protein bmds and prevents RNA
polymerase fiom bindmg or activating transcription. Negative control also involves
operators which are target DNA sequences for repressor protems and are usually
located upstream fiom the start site of transcription initiation. Repressor mediated
control is the most recognized fom of negative regdation, howwer there are many
other methods that cells utüize to inhibit transcription. These methods include: splitthg
the operon via gene remangement, mactivating a subunit of RNA polymerase (e.g. via
phosphorylation), mhibiting chah elongation by guanosine tetraphosphates, ter-ating
transcription during chah elongation, cleavage of the mRNA transcript, or inhibithg
translation initiation (Record Jr. et al., 1996). Conversely, m positive control a gene is
tumed off unless an aaivator protein is present. These activators increase the abüity of
RNA polymerase to recognize and bmd to a promoter since these genes typically have a
poor -35 consensus sequence.
The lactose fermentation pathway is the most completely documented exmple
of negative regdation in E.coli. It consists of one operon, lac ZYA, which is under the
negative control of a repressor, Lad. Lac1 bmds to an operator region which overlaps
the operon's promoter, specifically 0 1 . There are also two other psuedo-operators 02
and 0 3 (Gilbert et al., 1975) located at +401, and -82 from the transcriptional start
site, respectively. 02 and 0 3 have a lower a . 5 1 ~ for Laci but seem to help its
repressor actMty at 01. The mechanism of LacI repression is uncertain. Theories for
the mechanism of action for the lac repressor include blocking RNA polymerase fiom
binding to the IacZYA promoter, rnoâüjmg the initial transcription complex, and
promoter clearance. The efficiency of Lac1 repression is thought to be attrîbuted to
DNA loopmg between O1 and 03 (Sasse-Dwight and Gralia, 1988). This looping
induces a sharp bend at a TTTAT sequence located between the - 10 and -35 consensus
sequences which ükely directly affects transcription (Borowiec et ai., 1987). Expression
of the lactose fermentation genes is initiated in the presence of lactose. LacI has a
higher aflïnity for lactose and thus dissociates fiom the operator region in the presence
of this inducer. RNA polymerase can now activate transcription of the lacZYA operon.
The go1 operon, which encodes some of the enzymes involved in the
metabolism and m s p o a of galactose, consists of genes: galE. gdT and galX
Transcription of the operon occurs fiom two overlapping yet distinct promoters, Pl and
P2. The P2 start site is 5 base pairs upstream fiom the Pl start site. The gai operon is
controiled by CRP (discussed later) and the repressor proteh, G a R GalR bmds to two
operators, 0, (-60.5) and 0, (+53.5) which are separated by the aforementioned
promoters. GalR must bmd to both of these operators in order to repress transcription
of the gal operon. The synergistic repression of GaIR was originally poshilated to be
attriiuted to the formation of a 'looped complex' (Adhya and Majumdar, 1987). The
'looped complex' contorts the DNA backbone and creates a stiffcurvature. It is
hypothesized that under these conditions, RNA polymerase is unable to make the
necessary contacts to initiate transcription (Choy and Adhya, 1992). The mtergenic
region ( 1 13 base pairs) between the two operators c m be extended by 10-50 base pairs
without affecthg GalR repression. If GalR is unable to fom a DNA loop, it is bound to
0, and represses transcription fiom P 1, but activates transcription fiom P2. GalR
represses transcription fiom P 1 by fieezîng RNA polyrnerase (Majumdar and Adhya,
1989). GaiR repression of P l requires the C terminus of the a subunit of RNA
polymerase and thus is hypothesized to be the area of contact between GalR and RNA
polymerase. The mechanism by which GalR activates transcription at P2 is thought to
be indirect in that RNA polymerase is locally available to initiate transcription at P2
(Goodrich and McClure, 1992).
Genes mvoived in maltose fermentation are contained in 6ve operons: maiPQ,
ma& malZ. m a K lamBmaiM and malEFG. These operons code for receptors,
transporters, maltodextrio phophatases and amylomaltase. These five operons are under
the positive control of the inducer, MalT which is encoded fiom the gene ma17
immediately adjacent to the maiPQ operon. MalT bmds at the consensus sequence
ggGGAGîTGAgg which is characteristicaiiy centered around -37.5 or -38.5 base pairs
from the transcriptionai start site. This effectively covers the poor -35 sequence that is
typical of positively controlled operons. MaiT also binds to several distal sites in
addition to the proximal-3 5 bmding site. It is the interaction of MalT bound at distal
and proximal sites which detennines the activity of transcription (Danot and Raibaud,
1993). MalT is active ody m the presence of ATP and maltotriose (Richet and
Raibaud, 1989). In the presence of these two components, MalT binds to the major
groove and the two adjacent minor grooves. ATP mteracts with RNA polymerase to
form the initiation complex Though MalT acts as the sole activator of malPQ
transcription, it can act synergisiically with an accessory protem (CRP) as m
transcriptional activation of the divergent promoters of the malK and the malE operons.
The organization of MalT binding sites is different at these promoters (discussed
below).
The arabmose fermentation pathway is aiso under both positive and negative
controL The ara regulon consists of 3 operons: araBAD, a d and araF. G. These
operons encode for various processmg enzymes and transport machinery. The AraC
protem can act as both an activator and a repressor of this regulon. AraC has a cliffereut
conformation for each of this hctions. In the absence of arabinose, AraC is in its
repressor form and bmds as a dimer to the a& located -275 base pairs upstream fiom
the araBAD operon as weil as to oral1 site which is located 210 base pairs downstream
from araBAD start site. This effectively f o m a loop and represses transcription of this
operon (Martin et al., 1 986). However, when arabinose is available a conformational
change is induced m AraC which displaces it fiom a d . As concentrations of
arabinose and the activator form of AraC iticrease, the a r d site becomes occupied by
AraC. This site is located near the poor -35 site of the araBAD promoter and thus RNA
polymerase is stabilized by AraC and transcription fiom this promoter is activated.
Many sugar fermentation pathways (lactose, galactose, maitose and arabmose)
are primarily actnfated in response to the absence of glucose. Thus, when glucose is
available as an energy source, it is metaboüzed in preference to other available sugars.
This phenornenon bas been coined cataboüte repression and is attriiutable to the level
of cyclic AMP (CAMP) available m the celL When glucose is abundant m the cell, it
inhibits CAMP produaion as well as activates CAMP expulsion fiom the cell (Malunan
and Sutherland, 1965).The production of CAMP is catalyzed by the adenlyate cyclase
enzyme. The phosphoenolpynivate (PEP) dependent sugar phosphotransferase system
(PTS) is a signal transduction system that &bits the activity of adenylate cyclase
(Saier and Feucht, 1975). When giucose is present, it is phosphorylated by the IIA Glc
protem. Adenylate cyclase is allostericllly deactivated by the dephosphylation of IIA
Glc protem and thus CAMP is not produced-As the level of giucose decrease,
phosphorylated IIA Glc protem is available and activates adeniyate cyclase and CAMP
is produced. The presence of phosphorylated IIA Glc protein also activates membrane
permeases and therefore aiîows the transport of other sugars (Saier, 1989). In the
presence of a secondary carbon source, CAMP mteracts with its receptor protein, CRP
(a-ka CAP) and stimulates the transcription of CRP dependent genes.
CRP usually acts as a postive regulator which bmds to a specific symetrical site
on DNA in the presence of CAMP. When CRP bmds to its appropriate site it bends the
DNA - 100" .CRP may act synergisticdy with other activators (ie.AraC and MalT
discussed later) or it may act as the only activator of transcription (ie. iacZYA operon).
The crp gene is regulated by the CAMP-CRP complex (Cossari and Gicquel-Sanzey,
1985) as well as by antisense RNA (Okamoto et al., 1988). The transcription of crp is
also dependent on CAMP levels, thus the cAMP-CRP complex activates transcription
fkom crp when CAMP levels are hi@, but represses crp transcription when CAMP levels
are low (Hanamura and Aiba, 1992). This phenornenon enhances catabolite repression
and thus drastidly reduces the trenscription of CRP dependent genes in the presence
of glucose
The promoters of lac, malr and galP1 are activated primarily by CRP which
binds either 4 1.5, 6 1.5 or 7 1.5 base pairs upstream fkom the start of transcription and
directly mteracts with RNA polymerase. Thus CRP mduces a bend, contacts RNA
polymerase and this initiates transcription (Perez-Matin and Epmosa, 1993). in the lac
operon, CRP bends DNA and ailows the formation of a CRP-RNA polymerase
complex which mduces open complex formation and permits transcription to proceed
(Zinkel aud Crothers, 199 1). CRP seems to help in establishing the open cornplex. but is
not required for transcription activation after the open complex has been made (Tagami
and Aiba, 1995). The gai operon is cantroiled by CRP in a dinerent manner. The
products of the gai operon :ME, GalT, and GalK are transniied fiom two promoters
P l and P2 as mentioned previously. Transcription fiom P2 is negatively controlled by
CRP-CAMP, while P 1 is activated by it (opposite to the effect of GalR on the operon).
Intereaingly, the ratio of the gal operon products changes with the levels of CAMP
(Saier et al.. 1994). At high levels of CAMP, ail gal products are transcnbed fion Pland
translated at equivalent ratios. At low levels of CAMP however, P2 is used and the ratio
of gai operon products was shown as GalUGalPGaiK. This observed translational
polarity was not a characteristic of the transcriptional start site, but rather a
phenornenon that was repressed in the presence of CAMP-CRP. Studies indicated that
CAMP-CRP actually repressed Rho dependent htraoperooic transcriptional
termination, permitting the expression of the entire gd operon products in equivalent
proportions. At the malE and maiK divergent promoters, there is a 2 10 base pair
regdatory region which conssts of 2 MalT binding clusters separated by three
fùnctiond CRP bmding sites. The fist MalT bindmg ciuster con& of two bmding sites
(termed I and 2) which are upstream fiom the m a l . promoter. The second bmding
cluster consists of three MalT bmding sites (3,4, and 5) located upstream fiom the
maiK promoter. Bhding sites 3,4,and 5 overlap with three more MalT bmding sites
which are off- by three base pairs and are refered to as 3',4',and 5'. When CRP binds
its bmding sites between these MalT bmding clusters, it displaces MalT fkom sites 3,4
and 5 to sites 3',4',and 5' as weli as increases MalT bmding at sites 1 and 2.
Transcription fiom malK is iuitiated when CRP repositions MalT fkom the binding sites
3,4 and 5 to sites 3',4', and 5' . Thus, the interaction of at least five MalT monomeric
protehs, three CRP molecules and two RNA polymerases allow the expression of
genes fkom the malE and malK operons. C W s role m this nucleoprotein complex does
not require its specific interaction with MaiT or RNA polymerase since CRP c m be
replaced by another DNA binding protein, the Integration Host Factor (IHF) (Richet
and Sogaard-Anderson, 1994). MF is also documented to induce dramatic DNA
bending (discussed later). CRP also increases araBAD expression hy both breakhg the
repression loop and mcreasing the 28inity of AraC for the mal2 site. Therefore CRP
acts synergisticaly to interact with the activator conformation of AraC help to activate
arabinose fermentation. The mechanisms of action for cAMP-CRP are diverse and are
Eu fkom being completely resoived. The CAMP-CRP complex illustrates how an
organism c a . adapt to changing nutrient conditions and can produce the appropriate
enzymatic machinery for the appropriate metabolite.
Transcription many also be controlled by altermg ENA structure. In order for
transcription to occur, the double stranded DNA helix must separate. This is
accomplished by RNA polymerase. The degree to which DNA strands separate cm
therefore become a factor in gene expression. DNA supercoihg is controîled by
topoisornerase I and ïI (DNA gyrase). DurBig transcription, these topoisornerases
relieve torsional tension generated as RNA polymerase creates positive supercoils
ahead of its path and creates a trail of negative supercoils behind it (Lui and Wang,
1987). Though negative supercoiling seems to enhance strand separation and wodd
intuitively enhance promoter accessibility and subsequent gene expression, some
promoters are mhibted under these conditions ( h s s and Drlica, 1989).Regardless, the
degree of DNA supercoiling is a component of transcriptional regulation. Thus,
protems which interact with DNA and alter its structure are also important components
t O transcrip tional regulation, these proteins are collectively cded DNA binding
proteins.
DNA bmding proteins have mnny roles in the physiology of bacteria, including
gemme repücation, transcription, and repairing damaged DNA. DNA bmding proteins
such as IHF, HU and H-NS are thought to play important structural roles m the
nucler ' ' of prokaryotes.
The mtegration host factor (HF) facilitates DNA Dding. It has homology to
both the prokaryotic HU (30% sequence identity) protein and the eucaryotic TATA-
bhding factor (TFDII) (Nash and Granston, 199 1). IHF recogntes a specific DNA
sequence (Craig and Nash, 1984) and is impiicated in h DNA recombmation as well as
DNA replication at oriC (Polaczek, 1990). IHF u d y plays an activation role in gene
expression, though has also been reported to negatively reguiate gene expression (e.g.
ompC expression (Huang et al., 1990)). DNA bmds that are mduced by IHF facilitate
activator or repressor protems to mteract with RNA polymerase and help to assemble
higher order protein/DNA structures (Freidman, 1988).
HU is a histone iike protem which has üttie sequence specificity and is
poshilated to restrain DNA supercoils m nucleoids (Broyles and Pettijohn, 1986). It
consias of two 90 amino acid polypeptides HU- 1 and HU-2. HU prefers to bmd to bent
DNA and or cmcifonn structures (Pontiggia et al., 1993). It has also been reported to
bind one HU dimer per 9 base pairs iri vitro, though this binding mechanism couid only
be restricted to preferred binding sites in vivo (Broyles and Pettijohn, 1986). HU has
preferred bmdmg sites where it interacts with other regulatory proteins. HU is thought
to be mvohed in the initiation of replication at oriC, as weU as IM.UY other roles as an
a d a r y protein in recombination, inversion, DNA transposition and gene expression
(reviewed in Drlica and Rowiere-Yaniv, 1987).
H-NS is another histone-iike protem that is found in the prokaryotic nucleoid.
Like HU, H-NS bas littie sequence specificity and binds preferably to bent DNA. H-NS
seems to be connected to the regulation of many genes of apparent unrelated f'unction
(Yoshida et al., 1993). The mechanism by which H-NS operates is also unclear. There is
evidence that supports its h c t i o n as a direct repressor which mhibit s the formation of
open promoters (Ueguchi and MiPrno, 1993). There are also studies which bdicate
that it represses transcription by altering the level of DNA negative supercoiling
(Hulton et al., 1990).
Triple mutants of IHF, H-NS and HU are not viable, which suggests that no
other protein factor exists which complements the b c t i o n s of these protems
(Yasuzawa et al., 1992). However these three proteins do seem to complement each
other and may have overlappmg t'unctions, as single mutations in the genes encoding
these protems do not produce a lethal phenotype, albeit growth is slow and many
ceilular processes are hindered.
FIS is another abundant DNA bmding protein which bends DNA. Lüce lHF, it
bmds to specific sites, though the structure of DNA at these sites is as important as the
sequence (Finkel and Johnson, 1992). It was fist identified as the factor for bversion
stimulation for the Mu G-loop. FIS is aiso involved m the transcriptional activation of -
rRNA, and other genes involved in translation. Moreover, there is evidence that
FIS piays a role m DNA replication (Wold et al., 1996). F o o t p ~ t -dies have sbown
that FIS rernains bound to oriC except during replication initation irr vivo (Cassler et
al., 1995). The RS protem has aiso been s h o w to prevent initiation of oriC replication
bz vitro by forming an 'initiation preventive' complex (Wold et al., 1996).
Thus, DNA bhdmg proteins that are invohed m regulating gene expression and
ultimately control ce1 development, growth and Merentiation. The mechanisms by
which DNA bhding proteins recognize DNA is another interesthg area of research.
DNA bmding proteins are usually grouped into famüies based on the structural motifs
they use to bind target DNA. DNA bindmg protek f d e s mclude: helix-tum-helk
(HTH) protems, the homeodomains, mic h g e r protems, leucine zipper protems and
helix-loop-heüx protems (reviewed m Pabo and Sauer, 1992).
The a-heiix-twn-a-helix (HTH) structural motif is a feature of many
prokaryotic DNA binding proteins inciuding: Lac1 (Kaptem et d.. l985), Fis (Kostrewa
et al., 1990) CRP (McKay and Steitz, 198 l), AraC (Gallegos et al.. 1993) and k
phage repressor proteins Cro (Anderson et al., 198 1) and cl (Pabo and Lewis, 1982).
The motif con& of two helices jomed by a fhm. It is not a stable, independent
domain and aiways occurs as part of a larger DNA binding domain. DNA recognition
sites involve the HTH protem as weil as the larger DNA binding domain. Repressor-
operator structures have been resolved. For example, the k repressor binds a s a dimer
and each subunit (consisting of 5 a-helices) contacts halfof the bhding site. Heiices 2
and 3 fiom each subunit form the HTH motif and interact directly with the DNA. Some
residues fiom neighborsig h e b 1 make important contacts with the DNA backbone.
Though HTH protems encompass a variety of unique proteins, they do share some
common features. Firstiy, as with ?L repressor, they bind as dimers with each subunit
recognUmg halfof the DNA bindmg site. Secondly, helix 2 typicaiiy Lies within the
major groove while helix 3 lies above it . The N terminus of helix 2 is m close contact
with DNA bases while the N terminus of helk 3 contacts the DNA backbone. Finaily,
the binding site (operator region) is always B form DNA (Pabo and Sauer, 1992). The
protein-nucleic acid interactions involve van der Wads forces between short polar side
chahs or -NH group of the polypeptide backbone (helk 2) and the bases exposed in the
major groove. H e h huo is often referred to as the 'recognition' heüx Indeeâ, studies
have show that specinc amino acids within this helix are important for DNA bmdmg
(Ebright et al.. 1 984).
HomeodomaÎns are a structural motif of 60 amino acids found m many different
eucaryotic organisms (Scott et ai.. 1989). Though the homedomain contains a HTH
motif(Shepherd et al., 1984) its structure is stable and is able to bind DNA by itself
imlike the H.TH proteins (Qian et al., 1989). GeneraJly, the bomeodomam contains an
extended N-terminal a m and three a helices. Helix 1 and 2 are believed to mteract with
each other in an antiparailel arrangement, while the third h e h extends perpendicular to
1 and 2 and folds back agaha them (Kissinger et al., 1990). Helix 3 fits into the major
groove of DNA and forms the main contacts. Helices 1 and 2 span the major groove
but are fùrther away fiom the DNA and therefore only have two contact pomts with it.
The N terminal a m lies within the minor groove and makes additional contacts with the
DNA. The simildes between eucaryotic homeodomins and prokaryotic HTH
protehs have interesthg stmctural and evolutionary implications. Both proteins are
very similar, however the length of heiices m the eucaryotic homeodomain @es it a
more stable conformation and a slightly different DNA docking structure. Io HTH
protems, the N terminus of helix 3 has the closea contacts to DNA bases which makes
residues in the first tum of the helix critical for binding. In homeodomain proteins
however, the center of h e h 3 forms the critical contacts with DNA, which makes
residues of the second and third tum of the helix most important for DNA binding.
Moreover, m prokaryotic HTH proteins, a portion of h e k 2 fits into the major groove.
However, m homeodomains, this helix lies entirely above the major groove (Kissinger
et al., 1990). Homedomains were originally discovered m genes which regulate early
development m Drarophila (Scott et al., 1989). When homeodomains of protems are
exchanged, the DNA recognition specificity of the protem is Phered. However, no DNA
binding sequence has been identified for the homeodomain. Moreover, the
homeodomain may depend on flanlamg N and C terminal residues to modulate biuding
specifïcities (Kissinger et al., 1990). Homeodomains have also been reported to interact
with other proteins and form higher order recognition and regulaiton complexes
(Groutte and Johnson, 1988).
Zinc hge r protems contain a group of conserved amino acids which bmd a zinc
ion to form a DNA binding domain (Miner et al., 1985). The zinc finger protems
contain an antiparallel B pleated sheets and an a belix. The consensus sequence of a
zinc finger is C~~-X~-~-C~S-X~-P~~-X~-L~U-X~-H~SIX~-H~S. The zinc ion forms a
tetrahedral structure with the two Cys and two His residues which loops the intervening
amho acids, creating a 'figer' (Berg, 1986). Zmc finger proteins contain many of these
zinc h g e r motifs, protems which contain only one of the zinc fkger motifs may not be
a DNA binding protein. The interaction of DNA and many zinc finger proteins have
been resolved. For example, zifL68 contains three zinc h g e r motifs which fit in the
major groove and make contacts with three base pair subsites (Pavletich and Pabo,
199 1). Zinc fhger proteins are typically mvoived in cellular Merentiation, proto-
oncogenes, and growth signals.
The leucine zipper proteins typically con& of 60-80 amino acids which contain
two domains: a basic DNA binding dom* and a dimerization domain. The
dimerization domain has heptad repeats of leucine over 20-30 amino acids (Landschultz
et al.. 1988). The leucine zipper regions are believed to form two paraiiel a-heiices m a
coiled coil arrangement (O'Shea et al., 1989).The basic region contains many a r m e
and lysine residues. The structure of the peptide is predicted to resemble a 'Y" shape,
with the leucme zipper representing the stem and the basic DNA bindmg domains
coqrising the arms (O'Neil et al., 1990). This structure has been coined the bZIP
structural motX This structure explains why leucine zipper proteins target inverted
repeats. Smce leuche zipper protems have the ability to dimerize, they can combine
with themsehres or other Leucine zipper protems to generate different complexes with
difEerent regdatory effects on gene expression (Hai and Curran, 1 99 1 )
Helkloop-helix (HLH) proteins &are a sequence motif consisting of two
amphipathic a-helices (15- 16 amino acids m length) separated by a M e r region of 10-
24 rendues (Murre et al.. 1989). HLH have the ability to form dimers via hydrophobic
mteractions of the hydrophobic residues of the amphipathic helices (Voronova and
Baltimore, 1990). A highly basic region adjaceat to the HLH motifis required for DIVA
bindmg (Rendergast and Z a 1989). HLH proteins that posses this basic region are
referred to as bHLH proteins. The bHLH proteins are divided hto two classses: class A
comprises proteins that are ubiquitously expressed d e class B proteins are tissue
specific. Members of this protem f d y mclude E luE47, MyoD (mammalian
protehs) and da, AC-S (fly protehs). It is believed that the formation of homodimers
or heterodimers in bHLH provides another lwel of transcriptional regdation. DBerent
combmatiom of the bHLH proteins may have different functions within the cell, since
each combmation has a dinerent afnnity for DNA. Moreover, some members of the
HLH protein famiy fùnction as repressors when they dimerize with other HLH protems
(Barinaga, 199 1). This complex level of DNA transcriptional regulation iniplicates the
HLH protems m diffierentiation and development. For example, MyoD (a class B
protem) which signals muscle cell differeatiaton bbds DNA most effectively when it
dimerizes with E2A (a class A protemMWeintraub et al.. 199 L ) and is mactivated when
another HLH protein mterads with it (Benena et ai.. 1990). The level of
transcriptional regulation by this class of DNA bmding proteins is mdeed complex and
offers a new dimension to gene expression.
Families of DNA binding protems characteristicdly span many different
Kingdoms and are the underlying theme to organism growth, propagation and &al.
It is interesthg to look at these f d e s and study the evolutionary relationships that
they demonstrate. Nature has provided many solutions to the problem of protem-
nucleic acid interaction. The design and interaction of these protems has evolved hto a
flexible yet consistent system of gene regulation which in tum has allowed cells to
control the complex intricacies of life processes. The NER (negative early repressor)
protein of bacteriophages MU and D 108, the NLP (Ner-like-protem) of E. coli and
TMF (TATA- element-modulatory-factor)of human celis comprise a unique f d y of
DNA bhdmg protems that contain a conserved DNA bmding domain.
Mu and DL08 are temperate coliphages which exhiiit both lytic and lysogenic
life cycles. Duriog the lysogenic Me cycle, the phages infect and mtegrate their genome
mto a random location on the E. coli chromosome. The prophage is therefore replicated
dong with host DNA replication when the host cell divides. The lysogenic life cycle is
maintained by c repressor which is transcnied fiom P. and mhibits transcription fkom
P, and thus the eady genes for the lytic life cycle are not expressed. Durfng the lytic üfe
cycle, the coliphage amplifies and transposes its genome mtrachromosomaiiy. The
phage genomes are then encapsidated into assembled proheads and 50 - 100 cornpletc
phage particles are released upon host cell lysis (DuBow, 1987). The lytic cycle is
intitat ed b y the production of Ner (NegatRre-eariy- regulation ) which is transcnied fi-om
P, . The face to face promoters of P. and P, are both inhibted by the production of Ner.
Thus Ner represses transcription of c repressor as weU as autoregdates its owo
production. The repressim of transcription 6om P, by Ner is however overidden by the
presence of MF (mtegration host factor). The bhdmg of IHF to Pe activates
transcription of the early genes and the lytic cycle ensues.
The Mu Ner protein consists of 75 amino acids and is 50% homologous to the
DL08 Ner protem which consists of 73 amino acids The Mu ner-operator connsts of
two perfect 12 base pair mverted repeats separated by a 6 base pair spacer (Tolias and
DuBow, 1986). The Dl08 ner-operator however, contains two perfect 11 base pair
inverted repeats which are scparated by an 8 base pair spacer (Kukolj et al., 1989).
Raman spectroscopy of Dl08 Ner revealed that Ner changes its codonnation as weIl
as bends DNA upon bmding to its operator region (Benaides et al., 1994). Mu and
D 108 exhii 45% a-heücal secondary structure and the nonhelical structure is prïmady
B-çtranded (Benaides et al., 1994). A mode1 bas been proposed for the Dl08 Ner
protem mteraction demonstrating either a U-shaped or an S-shaped mode of
mteraction (Benevides et al., 1994). Mu Ner structure bas been examined via 3D and
4D heteronuclear magnetic resonance (HMR). This anaiysis revealed Mu Ner to
comprise of 5 a-heüces of which 2 showed a helix tum heüx motX This study also
showed that the helix turn heüx mot& were Wrely in contact with the Ner operator
region (Stzeleka et al., 1995). Though Ner appears to bend DNA in a manner similar
to helix turn heüw (HlM) mot& of DNA bmding protems, it possesses two unique
structural ciifferences: it behaves as a monomer in solution , and it covers a very large (5
tums of the DNA helix) operator regioo (Kukolji et al, 1989).
The unique Ner protein of Mu and D 108 coliphages has sequence homology to
a protein found in Esherichia coli cded Mp (Ner- Wre-protein) as weli as a protein
found m human cells caiied TMF (TATA element Modulatory Factor). TMF is a
transcriptionai factor which bmds to the TATA element of HIV- 1 LTR, in vitro. TMF
thus prevents the TATA Binding Rotem nom bhding to the TATA element and
transcription fiom the HIV- 1 LTR is repressed. The mif gene is located at position p 12-
p2 1 of human chromosome 3 which is a site associated with fiequent gene
rearrangements in lung, rend and other human cell carcmomas. TMF has also been
shown to negatively reguiate transcription of the adenoWus major late promoter
(Garcia et al., 1992). 'Ibe DNA binding domain of this 1093 amino acid protem is about
44% homologous to the prokaryotic Ner repressors.
The E.coli N p protein consisis of 93 amino acids and is 60% homologous to
the coüphage Ner protems, which is a higher level of homology than Mu and D 108
proteins have for each other (Choi et al., 1989). Mp however, does not bmd to the Mu
and D 108 Ner operators in vitro. or does t confer pseudoimmunity to phage
superinfection in vivo. Mp was discovered in 1989 wheu Choi et al. were attempting
to isolate a guanylate cyclase gene (cy@ in E. coi1 . The group used MK2000 l( cyo-
crp*') mutant main of E.cok The ctp*' mutation permits the fermentation of many
sugars without interacting with CAMP. Maltose fermentation, however still cannot
occur unless cGMP is present. Thus the group hoped to identify a cyg gene by
generating and transfor-g MK200 1 ceils with a myriad of plasmids each containhg a
different region of the E. coli chromosome. Ceiis which conferred a maltose positive
phenotype were beiieved to be harboring the cyg gene. Nlp however, was the gene that
complemented malt ose fermentation in MK200 1 ceîls.
Subsequently, the Ecoli Mp proteinwas shown not to be essential for viability
(Autexier and DuBow, 199 1 ) since the E. coli ri@ gene could be intempted with the
iuwAB reporter gene fiom Vibrio honieyi (Guzm and DuBow, 199 1). The transcription
of nlp was also monitored h o u @ light emission fiom the nlp: :lwulB transcriptionai
fùsion. Strabs LF7112 (W3 110 nlp::luwlBtet? and LF7114 (MIS200 1 nlp::lzaABtet')
were both show to actively transcnie Mp.
The transcriptional start site for nlp has been determined through Northem blot
as weil as primer extension analyses (Macintyre and DuBow, m preparation). These
analyses reveal a 340 nucleotide mRNA is transcnied 29 base pairs upstream of the
ATG start codon. These results indicate that Mp is an actively transcnbed
monocistronic gene.
The nlp gene is located at 69.3' on the E. coli chromosome. The gene
Umnediately upstrearn fiom nlp is ispB which encodes octaprenyl diphosphate synthase,
a component of isoprenoid biosynthesis. Though this gene is transcnibed m the same
direction as Np, there is a strong termination signal between the two genes and
does not appear to be mvolved in isoprenoid biosynthesis (Asai et al.. 1994). The gene
immediately downstream fiom Mp is mur2 which encodes a ZIDP-N-acetylglucosamine
enolpynivyl transferase. The mur2 gene is an essential gene m E-coli and is mvoived in
cell wail biosynthesis (Marquardt et al., 1992). These logistics support b a t Mp is a
monocistronic gme.
As mentioned before, Mp complements mahose fermentation in E-coli strain
MK200 1 and also bas been reported to stimdate the lac operon and thus has also been
coined sfs 7 for sugar fermentation stimulation gene (Kawamukai et al., 199 1 ).
Electrophoretic mobihy shift assays have s h o w that purined Mp binds DNA
containhg the mai and lac promoter regions (Macmtyre and DuBow, in preparation).
The Ner/Nip/TMF family of DNA binding motifs are consewed fiom coliphages
and prokaryotes through to human ceils. Thus fàr, the role of these DNA bmding
proteins remnsis elusive. However, the conservation of this DNA bbdmg domain
among otheMise unrelated organisns points to a global system of regdation yet to be
characterized.
This thesis focuses on the hc t i on of the Nlp protein. The Mud 1
bacteriophage (Casadaban and Cohen, 1979) was used to create random lacZ gene
fusions in a AIac strah of hcoii whose own Nlp gene was disrupted. Clones were
isolated that showed ahered P-galactosidase activity (lacZ gene product) m the
presence versus the absence of a plasmid borne 1249 gene. Five clones were found to
have a dinerent level of fbgalactosidase activity in the presence versus the absence of
Nlp (Table 3). Three of these clones exhibited an mcrease m lac2 transcritpion m the
presence of Mp. One clone showed a decrease m IacZ transcription, and one cloned
died in the presence of overexpressed Nlp.Total genomic DNA was isolated fiom these
clones and digested with Bgm enzyme then ligated with pBR322 or pBR328 linearized
at BamHI. The ligation products were then transformed into E.coli strain p90C (Alac)
and colonies that appeared blue on X-gal plates (thus containing the lac2 gene) were
picked and plasmid DNA was extracted. The isolated plasmid DNA was digested with
various restriction enzymes to detemine the restriction map of the subcloned, Mp
responsive genes.
To ve* that the Mp gene was the causitive agent for the obsewed changes in
lacZ expression, different portions of the Nlp gene were placed withui the clones. The
bacteriophage homologues of Mp, were also used to see if they could complement the
hc t ion of Np, to alter the expression of the Nlp responsive genes.
These experiments help to dari@ the fùnction of the Mp gene as well as m e r
characterize this unique fàmily of DNA bindmg proteins. The implications and relevance
of the Nlp responsive genes wiil be discussed and Nlp's contribution to gene expression
wül also be addressed. The hction of N p with respect to the NerMplllW: f d y of
DNA bmdhg proteins will also be examined. The implications of this f d y on the
evolution of gene expression wili also be discussed. Findy, fùture directions and
studies wiU be suggeaed.
Chapter LL Materials and Methods
Bacterial Strains and Plasmids
AU bacterial strains and plasmids used in this work are listed in Tables 1 and 2,
resp ectively.
Antibiotic Selection
The concentrations of antibiotics used for bacterial growih were 40 pghl of
ampicillin (amp; Novophann), 50 pg/d of kanamycin (km; Boehringer Mannheim), 10
&mi of tetracyclin (tet; Boehringer Manneheim). Chloramphenicol (cam; Sigma), was
used at a concentration of 75 pghl for plasmid amplification during plasmid isolation
and 50 pghl in liquid culture and plates. Isopropyl 8-d-thiogalactopyrauoside (JPTG;
Diagnostic Chernicals Limited) was used at a concentration of 1 m M for the induction
of cloned gene expression.
Restriction Endonuclease Digestions
DNA samples were digested using 3 units of enzyme pet pg of DNA. DNA was
hydrolyzed in bovine serum albumin (BSA) [25 pghl BSA (Miles Scientific) in 10 mM
Tris-HCl pH 7-51, and digestion bder (6mM Tris-HCI pH 7.5,6mM MgCl2, 75 mM
NaCl, and 6 m M pmercaptoethanol). The digestion reaction was c k d out for 2-4
hours m a 37 OC water bath. Digested DNA çamples were then either loaded mto
agarose gels or
Table 1. Escherichia coli strains used in this study
LF203020, LF20303 8, LF203040, LF203047 and LF203057
Characteristics Origin
AproAB-lac, trp8am, Bukhari and Metlay, P L 1973
recAl supE44 e d A 1 Vieira et al., 1985 h d R17 gyr A96 relA1 thi A(luc-proAB)
ara, A (gpt-lac)5, th , MiUer, 1992 F, lac'. pro-. thf (CSH142)
thi Campbeii et aL, 1978
W3 110 nlp::luxAE Tetr Autexier, 199 I
40 nlp: : IurAB Tetr Seguin, 1995
LF20300 containhg Seguin, 1995 pOX3 8cam
F, Mu c ts d 1 (arnpr, Casadaban, 1978 lac) M u cts, A@roAB, IacIPOZYA) XIII, SPA
Derivatives of strain Seguin, 1995
LF2030 1 containhg the Mud 1 ~ansducing phage fiom strain Mal 1 03. These strains are NLP responsive.
Table 2. Escherichia cdi phsmids used in tbis study
Plasmid
pdp 1.1
pBR322 pBR328 pBS5 pFuSl&c p u c 118
p u c 119
p u c 120
p20, and p38
p40,p47 and p57
Characteristic
ampr, Ecoli nlp (1.1 kb Afm-Hindm) in puci 18 ampr, tetr cloniag vector pBR322 plus camr Kanr pdp 1.1 lacZYA., tetr ampr lac P/O, clonmg and sequencmg vector same as pUC 1 18 except MCS is m opposite orientation same as pUC 1 18 plus an NcoI site in the MCS Kanr
pUC 120, Kanr cassette mserted into ScaI site lkb NcoI-Hindm fiagment in p UC 120k pBR.322 derived, amp: ner, cts, lac 475bp DraI-EcoRI fiagment fiom pKG528 into PUC 1 19 l3,gfI.I fiagrnent f?om LF203020 or LF203038, respectively- spanning the Mud junction, cloned mto the BamHI site of pBR322 Bgm fiagment tiom LF203040,47,or 5 7, respectively-sponnsig the Mud junction, and cloned mto BamHI site of pBR328 I kb Hindm., 1 kb &RI and 2.4 kb HpaI fragments î?om p40,p47 and p57 respectively, subcloned into put I i 8
Origin
Autexier, 199 1
SutcWe, 1979 Covarrubias et al., 198 1 Se@, 1995 A.Guzu, Vieira and Mes- 1987
Vieua and Messbg, 1987
Vieua and Messing, 1 987
Vieira and Messing, 1982
this work
this work
Kukolj and DuBow, 1992
J. Lemieux
this work
G. Mcintyre
G. Mcintyre
purilied by extractmg once with an equai volume of phenol and twice with an equal
volume of ether. The DNA was then precipitated by adding O. 1 volumes of 2 S M
ammonium acetate and 2.5 volumes of absolute ethanol, placed in dry ice for 30
minutes and centrifiiged at 4 OC for 30 minutes at 12 000 x g. Ethanol was removed
fiom the pefleted DNA, the DNA was washed with 70% ethanol, desiccated in a
vacuum and resuspended in 1 x TE (IO mM tris-HCI pH 7.5 and L m M EDTA).
Restriction enzymes used were purchased from Giico BRL, Phartnacia, or New
England Biolabs.
Liga tions
A total of 1 pg of DNA fragments were mked with 1 unit of T4 DNA ligase
(Gibco BRL) , 2 pL of Lmker Ligation BufEer (60 m . Tris-HCI pH 7.5, 10 m M
MgCl2, 15 mM DTT, 1 mM spennidine, 0.75 mM ATP and 50 pg/ml autoclaved
gelath). The DNA was mcubated overnight at 11 OC, and then heat inactivated at 65 OC
for 10 minutes. The ligation mumire was then diiuted two fold in deionized water and
used to transform competent E-coli.
Reparation of Rubidium Chloride Competent CeUs
A fie& overnight bacteriai culture was diluted ( 1:20) into 100 ml of Luria-
Bertani (LB) broth ( 1% tryptone, 1% NaCl, 0.5% Bacto yeast extract, and 2 m M
NaOH). Cells were grown in a 37 OC s h a b g mcubator to an absorbance (A of
0.50. Cells were then chüled on ice for 5 minutes and centntùged at 4000 x g for 15
minutes at 4 OC. The cell pellet was resuspended in 40 ml of TFB 1 [ 30mM
CH3COOK, 100mmol RBCB 1ûmM CaClr2H20, 5OmM MnClr4H20, 15% glucose ;
pH 5.81 lefi on ice for 5 minutes and centdùged at 4000 x g for 15 minutes at 4 OC.
The ceil pellet was resuspended m 4 mL of TFB II [lOmii MOPS, 75mM CaC12-
2H20, lOmM RbCh, 15% glycerol; pH 6-31, left on ice for 15 minutes. This suspension
was dMded into 200 pL aliquots, quick-fiozen m an ethanol dry ice bath and stored at - 70 OC.
Transformation of Rubidium Chloride Competent CeU
An aliquot of rubidium chloride competent cells was thawed at room
temperature and then put on ice for 10 minutes. No more than 100 pg of DNA was
added, mixed and left on ice for 30 minutes. The ceils were heat shocked at 42 O C for
90 seconds and put on ice for another 1-2 minutes. 4 volumes of LB (800 pL for 200
p L of ce&) was added and the suspension was incubated at 37 OC for 1 hour. Cells
were plated onto the appropriate selective medium and mcubated overnight at 32 OC.
Cloning of the MP Respoading Genes
Isolation of Total Genomic DNA from NLP Responsive StraUis
Chromosornal DNA fiom mains LF203020,LF203038, LF203040, LF203047,
and LF203057 was isolated. Ovemight cell cultures were centrifbged in 50 ml conical
tubes for 15 minutes at 5000 x g and 4 OC. Cell pellets were resuspended m 1.4 r d of
IO X TE. Volumes of 200 pi, of 10% (wlv) SDS and 200 pi, of RNase A (1mglmL in
10 mM Tris-HCL pH 7.6) were added and mixed by inversion. The suspension was
incubated at 37 OC for 2 ho1us. LOO pl of pronase (20 m g h l in 10 m M Tris-HCL pH
7.6) was then added to the mixtures and incubated for another 2 hours. An equal
volume of buffer saturated phenol was added, mixed by inversion and then centrifuged
at 5000 x g for 15 minutes (4 OC ) to allow phases to separate. The upper aqueous
phase was removed with a wide-bore pipette tip and transferred to a 14 ml Falcon tube.
Two more phenol extractions were then performed as descnLbed above. Then the DNA
was extracted three times with an equal volume of ether. Ethanol precipitation of
extracted DNA was done by adding two volumes of absolute ethanol. The fibrous DNA
precipitate was spooled out with a 1-5 pl micropipette sealed at one end. Total genomic
DNA was dried in a vacuum desiccator and resuspended in 50 pi of L x TE.
Subcloning the Nlp responsive genes
Chromosomal DNA fiom each NLP responsive straPi was digested with Bgm
and ligated into pBR322 vector hearized with BamHI. Each ligation was transfomied
into competent p90C ceils and plated onto LB based agar plates which contained 40
mg/L X-gal and 40 p%/ml of ampicüün. Petri dishes were mcubated ovemight at 32 OC.
Blue colonies containing the recombiuaat pBR322 plasmid with !a&? and the junction of
NLP responsive genes mserted were isolated as d e m i e d by Morelie ( 1989).The
plasmids (named 38, 20,40,47, and 57) were digested with various enzymes to
determine orientation of the chromosoma1 msert within the tetracyclin gene of pBR322
as well as to determine a restriction endonuclease map of the unhowu genes.
Sequeochg the NLP Responsive Genes
Double Sbanded DNA Sequencing
Plasmid DNA was prepared by QIAGEN mini prep spin c o l ~ s and 5pl each
of p20,p38,p40yp47and p57 were denatured by alkaline denaturation (Sambrook et al.,
1989) and sequenced by the dideoxy chah tennination method (Sanger et al., 1977)
using the Pharmacia Sequenase kit, with primer pBR1 or pBR2 (Eom either the left or
ngbt side of the h H I insertiou site in the tetracycline gene of pBR322) and a-"S
dATP (500 Cihnmol; NEN DuPont Canada Inc. ). Reactions were subjected to
electrophoresis through a 8% polyacrlyamide-7M urea denaturing gels, dried under a
vacuum and exposed to Kodak XAR-5 film on DuPont Cronex intendjing screens at - 370 C for 2-3 days.
Single Stranded Sequencing
Isolation of single sshanded DNA
Mp inducible genes were subcloned kom p20, p40, p47, and p57 mto vectors
pUC 1 19 or pUC 118 as descnied m table 2. 0.1 ml of each ovemight culture
containhg JM 109 ceUs transformed with one of these constmcts was diluted in 9.9 ml
of 2xYT broth [ 1.6 % (w/v) Bacto-tryptone, 1% (w/v) Bacto-yeast extract, O. 5%
(w/v) NaCI, pH 7.01 and grown at 37 OC in a shaking air incubator. When cells reached
a density of 0 . 5 ~ 108 cells/ml (ASs=O. 1) they were infected with the M l3KO7 belper
phage (3 x 10" PFU/ml) and mcubated for one hour m a 37 OC in a shakmg air
incubator. Eight ml of the culture was removed and 8ml of fie& 2 x YT was added in
its place. Kanamycm was also added to a final concentration of 70 pg/mL. The cells
were then incubated ovemight at 37 OC . The foilowîng monimg, 2 x .3 ml of each
culture was transferred to a 1.5 ml conical cenhifuge tube and centrifùged at 4 OC, 15
000 x g for Sminutes in a Fisher microtùge. The supematants were transferred to new
tubes and 200 pi of 20 % (w/v) polyethylene glycol (PEG 6000) in 2.5 M NaCl was
added. The tubes were &&en at room temperature for 15 minutes and centrifùged in a
microfùge for 5 minutes. The Wai pellets were resuspended m 50 p l 1 x TE1 tube and
then the contents of the 2 tubes were pooled. 50 pi of phenol was then added and
vortexed and mcubated at room temperature for 15 minutes. The tube was then
cenaaùged in a microfùge 15 000 x g for 15 minutes The aqueous layer was
subsequentiy removed and transferred to a new tube, and extracted twice with ether.
Then 1/10 volume of 3M sodium acetate and 2 volumes of ethanol was added. The
single stranded DNA was precipitated m a dry ice ethanol bath for 15 minutes. The
tubes were then centrifuged for 15 minutes at 15 000 x g. 4 OC to peilet the DNA. The
pellet was then washed with 70% ethanol, dried in a vacuum desiccator and
resuspended m 25 pi of 1 x TE. One pi was then run on an agvose gel to ver@ the
concentration.
Sequencing single stranded DNA
The DNA sequences of the Mp responsive genes were determined by the
dideoxy chah temination method descnied by Sanger ( L 977). Reactions were
performed with Sequenase enzyme version 2.0 (Amersham Life Science), according to
the maniifactwer's itistnrctions.
Infection of the Mp Responsive Clones (LF'203020, LF203038, LF203040, LF203047, LF'203057) with pBSS, pUClZOkan, pFïM2kan or pFI'538
Preparation of five M13K07 lysates
Ml09 cells containhg plasmid pBSS, pUClZOkan, pFIM2kan, pFT538, or no
plasmid were grown ovemight in 10 ml of LB plus kanamycin. Cultures were diluted 5
ml of culture in 250 ml of superbroth [3.5% (w/v) bactotryptone (Difco), 2% (wh)
Bacto yeast extract (Difco), 0.5% (wh) NaCl, adjust pH to 7.5 with NaOW and grown
to an Asso = O. 1 . Cells were then infected wîth 0.5 ml of Ml3KOî helper phage ( 1.65 x
10'~ PFU/ml) and grown for one hour at 37 OC in a s h u g air incubator. Kanamycm
was then added (2.5 ml) and the cuitures ere grown ovemight m the 37 OC shaking air
mcubator. The next day, the celis were transferred to 300 ml centrifiige tubes and
centrifùged 2X for 1 5 &tes at 5 000 x g and 4 OC. The supematants were collected
and iiltered (Millipore, 0.45 p).
Infection of M13K07
LF203020, LF203038, LF203040, LF203047, and LF203057 ceUs were
mfected with single stranded M l3KO7 phage containhg pBS5, pUC LPOkan,
pFWkan, pFT538, (or no plasmid) were used to test whether the mduction or
repression of lacZ gene expression was attributable to Mp or Ner and not anti-sense
murZ which is contained in pBS5. Clones were plated onto LB agar plates containhg
tetracylcine, chloramphenicol, ampiciliin, kanamycin and 40 pg!d X-gal (Vector
Biosystems) covered with 2.5 ml of 0.75% soft agar [1% (w/v) tryptone, 0.5% (w/v)
Bacto yeast extract, 1% (w/v) agar and and 2mM NaOHl coataining 200 pl of single
stranded M MC07 plus pBSS, pUC 120kan,pFIM2kan, pFT538 or no plasmid at all.
Plates were incubated ovemight at 32 OC. Colonies were then observed for changes in
lac phenotype.
Chapter LLL Resillts
Cloning a Region of the N p Responsive Genes
Total chromosomai DNA fiom strains LF203020, 38, were isoiated and
hydrolyzed with Bgm restriction endonuclease, The restriction fragments were then
ligated with a linearized pBR322 vector (hydrolyzed with BumHI). The ligation was
then used to transform comptent p90C cells (AlacZYA). The transfomiants were plated
onto plates containing amp, mcubated ovemight at 32 OC and then mastered onto plates
containing amp, IPTG, and X-gai. M e r an ovemight mcubation at 32 OC, many white
and some blue colonies appeared on the plates. The blue colonies were selected for
M e r investigation and the subcloned Mp responsive gene constnicts were named p20
and p3 8 based on their parent strain (either LF203020, or LF2O3O3 8).
Restriction Endonuclease Digestions of p20 and p38 to Determine Orientation
and Restriction Map of the Subcloned Portions of the N p Inducible Genes
Plasmid DNA @20 and p38) was isolated and electrophoresed on a 0.75%
agarose gel. The construct size was estimated to be approximately 17.7 kb for p20 and
20 kb for p38. Since the Mudl insert is 9.7kb and the pBR322 vector is 4.36 1 kb, the
subcloned Nlp responsive genes were estimated to be approximately 3.7 kb and 6 kb
respectively. The p20 and p38 constnicts were then digested with various restriction
endonucleases and subjected to electrophoresis through a 0.75% rgarose gel ovemight
(figure 1). Smce the restriction maps of both Mudl (figure 2) and pBR322 are known,
a restriction map for p20 and p38 could be constructed based on restriction fiagrnent
size estimation. Moreover, double digestions of these constructs were done to
determine the orientation of the subcloned Mudl/Nlp responsive genes m the pBR322
vector (data not sûown). Both inserts (Mud 1 plus Mp responsive gene) were
predicted to be in the same orientation with the Mud 1 portion of the msert closest to
the EcoRV in pBR.322 ( located at 185 bp) and the Mp responsive gene closer to the
BglI site in pBR322 (located at 929 bp). As mentioned previously, both mserts were
placed mto pBR322 at a BamM site located at 375 bp. The predicted restriction maps
for p20 and p3 8 were determined (figure 3).
Figure 1. Restriction endonuekase digestions of p20 and p38
Restriction endonuclease digestions were done on p20, and p38 and
electrophoresed on a 0.75% agarose geL In (A), 1pg of p20 was digested with 3 Units
of Bgm (he2), BamHI (lane 3), H i d i I I (lane 4), EcoRI (lane 5) , EcorV (lane 6) ,
Bgfi (lane 7), i@nI (ianeg), Pst1 (lane 9), &II (lane IO), and with no restriction
endonuclease (lane 1). In (B), 1 pg of p38 was digested with 3 Units of BamHI (lane
2), Bgm (lane 3), Pst1 (lane 4), H i a (lane 5), EcoRI (lane 6), EcorV (lane 7), BgA
(lane 8), KpnI (lane 9), Atun (lane 10) and with no restriction endonuclease (lane 1 ).
h= 0.3 3 3 pg of h DNA digested with 3 lmas of H i d used as a size marker. Marker
&es are as foilows (fiom the top ofthe gel) 23.1 kb, 9.4 kb, 6.6 kb, 4.4 kb, 2.3 kb, 2.0
kb, and 0.5 kb.
Figure 2. Restriction map of Mud llac
Restriction sites were detemmied fkom submitting lacZYA, and trpAB sequences
through "webcutter". The size of each portion was estimated fiom a schematic of Mud 1
(O'Connor and Malamy, 1988). Oniy the portion of Mud 1 lac which was subcloned into
pBR322 is represented (e.g. nom the Bgm site). The Sze of the Bgm portion of the lac
msert is 8.17kb (O'Connor and Malamy, 1988). The numbers mdicate distances in kb
between restriction sites. G=Bgn, U=PMin, R=EcoRI, RV=EcoRV, D=Hindm, and
H=HpaI, L*=location of former Bgm restriction endonuclease site
Figure 3. Redicted restriction endonuclease maps of p20 and p38
Restriction sites were determined Grom restriction endonuclease digestions (figure
3). The totai number and size of fragments generated for each restriction endonuclease
were calculated and restriction sites m pBR322 (4.3 kb) or Mudl lac (approximately
8.7 1 kb) were accounted for. B=BamHI, FPsti, K=KpnI, H=Hindm, E=EcoRI,
EV=EcoRV pBR1 and pBR2 =pimers used for double stranded sequencmg. (A) p38 ( N p
responsive insert is 7.1 kbXB) p20 (Nlp responsive insert is 3.8 kb). Not aii sites in
pBR322 or Mud liac are represented. See New England Biolabs catalogue for a complete
map of pBR322 and figure 2 for a complete map of the subcloned portion of Mud liac.
Double Stranded Sequencing Analysb of p38 and pZO
Both p20 and p38 constnicts were dmatured and subjected to dideoxy-chah
termination reactions as descried m Materials and Methods. Two difEerent sets of
reactions were performed for p38, each wiih a different primer (shown m figure 3),
since at the time of sequencing, the orientation of this insert was not certain. The
sequence for p20 was read and entered mto the N[H genebank 'BLAST search" site on
the World Wide Web. A match for this sequence was found and corresponded to a
region between 87.8 and 89.2 minutes on the Ecoii chromosome. The sequence of this
region was then placed into a 'Webcutter' program to generate a restriction map of this
area. This data was used in conjunction with the predicted restriction map of the
construct to predict the approximate location of the Mudi insert and determine the
identity of the Nlp mducible gene. The Mp mducible gene m clone LF203020 is
predicted to be either a . open readmg fiame named O80 or O8 1 .The restriction map
for this area is shown m figure 4. As predicted, a HindlII site was present in the Mp
responsive area of p2O. The Bg(I and PvuiI sites were not predicted, however, the
fiagments resulting fiom these digestions would be too small and difiicult to detect on
an agarose geL Moreover, both PvuII and BglI are enzymes which cut several times in
pBR322 and Mud llac which makes these digestions (figure 1) difncult to correctiy
assess. The k7roI and SalI sites were not originaliy tested, however, subsequent
digestion of p20 with these enzymes yielded the appropriate fiagments (data not
shown).
Figure 4. Restriction map for the N p responsive gene and surroundhg sequence
subcloned into p20.
Restriction sites were determined by submitting the sequence of the Mp
responsive region of p20 into 'webcutter'. The numbers hdicate the distance in kb
between restriction endonucleae sites. B=BamHI, R=EcoRI, RV=EcoRV, U=PvuII,
K=KpnI, PPstI, F undetermined distance (depends on where Mud 1 hserted h t o the
genome).
A shilar procedure was carcied out for each sequence generated for p38 (one
nom each primer). The first primer @BRI) generated a sequence which corresponded
to gene cynS, ~s gene is located just upstream of lacZYA and is part of the Mudl
section of the msertion. The second primer @BU) generated a sequence for p38 that
corresponded a region 89.2 to 92.8 minutes on the E. coli chromosome. Again, the
sequence was piaced into 'webcutter' and the generated restriction map (figure 5) was
compared to the restriction map predicted for p38 (figure 3). The predicted map
corresponded weli to the generated map for the Mp responsive area of p38. Again, the
only discrepancies could be attributable to fkagments that were too small to detect on
an agarose gel. The Mudl phage was predicted to have insexted upstream fkom the
plsB promoter, thusplsB is believed to be a Mp responsive gene.
The Identity of NLP Responsive Genes of Strains LF203040,47, and 57
The remaining clones (LF2030409LF203047, and LF203057) were subcloned
(Macintyre, unpublished). Here, the Mudl plus Nlp responsive gene were digested with
Bgm and ligated with pBR328 linearized at its BarnHi site (375 bp).The ligation
mixture was transformed into p90C and plated onto amp, IPTG, and X-Ga1 containing
plates. Blue colonies were seleaed for fùrther study and named p40, p47, p 5 7
Digestions with various enzymes were also done (Macintyre et al., m preparation) and
restriction maps were predicted for these constnicts. The predicted sizes of the
subcloned Mp responsive gene portion of the insert were 33 kb, 1 1, and 9 kb
respectively (&a Macmtryre, 1996).
Figure S. Restriction map for the Mp responsive gene and surroundhg sequenee
subcloned into p38.
Restriction sites were dete&ed by subtcütting the sequence of the Nlp
responsive region of p38 mto 'webcutter'. The numbers mdicate the distance m kb
between restriction endonuclease sites. B=BumHI, R=EcoRI, RV=EcoRV, U=PvuCI,
K=ICpnI, PPs t i , x= undetermined distance (depends on where Mud 1 inserted into the
genome)
Double shanded sequencing of these constmcts proved to be unsuccessfùl,
perhaps because of the large size of the constructs. These genes were therefore
subcloned mto pUCll8 (see table 2) and single stranded DNA was generated and then
sequenced (figure 6). The sequences for each gene were submitted to the NDI-
genebank BLAST search database and were identified as &.dC or cscKA3 (depending
on wether or not the s t rah is sucrose positive-see discussion), pgi, and moF,
respectively . The restriction maps corresponded weil with the predicted restriction sites
of the Mp responsive areas of these clones. The restriction maps for the Mp responsive
regions of p40, p47 and p 5 7 are show 8i figures 7, 8 and 9 respectively.
Figure 6. Dideoxy-termination reactions on single stranded DNA kom p57sub
Smgle stranded sequencing reactions were done on single m d e d DNA generated
from p57sub. The sequence is 'read' fiom the bottom to the top of the gel Reactions m
lanes 57(l) were electrophoresed for 3 hours, while reactions in lanes 57 (2) were
electrophoresed for 7 hours. C = cytosine, T = thymine, A= adenosine, G = guanine
Figure 7. Restriction mrp for the Mp responsive gene and surrounding sequence
subcloned into p40.
Restriction sites were determined by subminmg the sequence of the Nip
responsive region of p40 into 'webcutter'. The sequence of this area has not entirely been
determined and therefore restriction sites were determined fiom 'EcoMap6 '( Rudd,
1992).The numbers mdicate the distance in kb between restriction endonuclease sites. in
order to simpi@ the map EcoRV, EcoRI, Bgn, PvuII restriction endonuclease sites were
removed, the number of sites for these enzymes are 1 1,6,12, and 6 respectively.
D=Hindm, FPstI, B=BamHI, K=KpnI, L*=missed B g A restriction endonuclease site
and F undetermined distance (depends on where Mud 1 mserted into the genome )
Figure 8. Restriction map for the Mp responsive gene and surroundhg sequence
subcloned hto p47.
Restriction sites were determined by submittmg the sequence of the Nip
responsive region of p47 mto 'webcutter'. The numbers mdicate the distance in kb
between restriction endonuclease sites. For ciarity, 8 BgiI and 8 EcoRV sites are not
shown. D=HidiU, U=A>uII, P-Pstl R=EcoRI, B=BamHI, K=Kpnï, and x=
undetermined distance (depends on where Mud 1 mserted mto the genome )
Figure 9. Restriction map for the Mp responsive gene and surroundiog sequence
subcloned into p57.
Restriction sites were detenrtined by submittmg the sequence of the Mp
responsive region of p57 mto 'webcutter'. The sequence of this area has not been entirely
determined and therefore restriction sites were determined fiom 'EcoMW" (Rudd, 1992).
The numbers mdicate the distance in kb between restriction endonuclease sites. For clarity,
3 BgfI, and A.uIIy Pst1 restriction endonuclease sites were removed. The number of Pvun
and Pst1 sites m this area depends upon the distance 'y'. DcHindm, R=EcoRi, B=BamHI,
K=KpnI, RV=EcoRV, G=Bgli, H=HpaI, L*= unhydrolyzed Bgm site. x= undetermined
distance (depends on where Mud ! hserted mto the genome ), and y-determhed
distance to next BgOI (this euzyme is not documented m Rudd (1992))
M13kW Infections with pFTS38, pBSS, pFIMZkan, and pUCl2Okan
The original Mudl lac fision library was tested with a construct which
contained the entire Mp gene as well as some downstream sequence overlappîug with
mur2 (by about 1 kb) which is transcribed in the opposite direction to nip. Thus, the
change m lac phenotype could potentially be due this downstream sequence and not
related to Mp expression at all. The downstream sequence of the nlp gene in this
construct was therefore isolated and înserted mto pUC l2Okan. The effect of Nlp's
homologue, DL08 Ner, was aiso tested to see if it couid complement the fùnction of
Mp in these Mp responsive clones Five M 13 Lysates were made containing either
plasmid pFT538 (hahoring the D 108 ner gene), pBS5 (harboring the nip gene and
approximately 1 kb of downstream sequence), pFlM2Kan (containing the later half of
the Nlp gene and downstream sequence present in pBS5), pUC 120kan (control) and
finally an M 13 lysate which did not contain any plasmid. StrPms LF203020,38,40,47
and 57 were infected with each of these lysates and screened for changes in lac
phenotype. The p UC l2Okan containhg lysate, and the lysate that did not contain any
plasmid, were used as controls. The controls verified that changes m lac phenotype
were not attributable to pUC, M13, infection or plasmid establishment or expression.
Ner was not found to have any effect on lac phenotype of any of these clones, though a
more sensitive P-galactosidase assay should be performed in order to detect small
differences in IacZ transcription. The downstream region was also found to have no
effect on their lac phenotype, and Nlp was observed to have the same changes
originally reported ( Beatrice Seguin, 1995).lbe Iac phenotype changes nom this
experiment are reported in table 3.
Table 3. The cornparison of lac phenotype of the N p responsive clones
Strahs LF20302O,LF203038,LF203049,LF203047, and LF203057 were
repiica plated onto LB based plates containhg Cam, amp, te?, X-Ga1 and overlain
with M% agar (0.75%). The soft agar contained 200 pi of an M 13KO7 lysate which
harbored either Nlp @BSS), Ner @FT53 8), or anti-sense murZ (pFIM2K).
Controls {vector oniy (pUCl20k) and M13K07 lysate only) were also doue. The
I d acthdy was determhed by the color of the clone on the plates f i e r overnight
incubation at 32 OC.
M13K07 Lysate Harboring t- Ly sate Harbo ring
Lysate Harboring pFT528 M13K07 Lysate Harboring pFIM2K
white pale blue white white paleblue white
white
blue blue
white pale blue white
white paleblue white
LF203047 LF203 05 7 blue white blue I
Chapter IV. Discussion
Nlp was iirst identified by Choi et al. ( 1989) as stimulahg expression of the
maltose and lactose operons m a E. coli va- crp mutant, MK200 1. The fùnction of Nlp
m wild type Exoli bas yet to be d e t e h e d . Nlp is conserved among closely related
bacterial species, but is not required for ceU viabüity (Autexier and DuBow, 199 1 ). Nlp
also does not seem to be required for the metabolism of many sugars under minimal
nutrient conditions m wild type E.coli (Autexier, 199 1 ) . However, Mp is actively
transcnied in wild type Exoli and therefore may have a function The fhction of Mp in
mahose metabolism in strain MK200 1 may be a consequence of DNA bmdmg due to
overexpression. However, when IHF (a protem which has been characterized as a DNA
binding and bending protein) was overexpressed m this strain, maltose metabolism was not
observed, thus Mp does seem to be speciaiized for this fùnction. Other proteins have been
reported to complement maltose metaboüsm m MK200 1 and classified as the sj i genes
(Kawamukai et al., 199 1). Nip is not required for mitose metabolism in wild type Exoli,
nor in cya, c p , and cya crp mutant strains (Autexier, 199 1). Thus Mp and other sfs genes
seem to complement maltose metaboüsm when CRP is mutated to the CRP *' phenotype.
The CRP *' mutation permits CRP to act in a manner similar to the CRP-CAMP cornplex.
However, both lac and malQ expression by CRP*' are not equivaleot to CRP-CAMP
expression of these genes (Aiba et al., 1985) unless Mp is overexppressed (Choi et
a[., L989). The hc t ion of Nlp m wild type E.coli may be as a global regulator. Nlp could
bind to DNA and mhibit or enhance gene expression. Putative a-helix-tum-a-helix motifs
have been identified in Mp (Choi et al., 1989). Moreover, Ner (which is homologous to
N p ) is known to bmd and bend DNA (Benevides et al., 1994). Nlp, howwer, bas yet to be
conclusively determined as a DNA bmding protesi.
The hc t ion of the Mp protein m Escherichia coli was mvestigated by creating
random lac2 gene fùsions in a strain of E.coli whose own nlp gene had been mtempted
by the Hisertion of a IuxAB tetr cassette. A liirary of 3360 Exoli ceiis, each with the l a d
gene mserted mto a different random location in the E-coli chromosome was created.
Each clone of this library was also Alac and therefore any Pgalactosidase actMty would
be due to the expression of the gene into which the Mud lanrp lac gene had inserted. The
expression of these genes could thus be monitored m the presence vernis the absence of a
plasmid bound nlp gene (pBS5). Changes in P-galactosidase a c t ~ t y of 5 of the 3360
clones in the presence versus the absence of Nlp identified 5 potential genes that could be
regulated by Mp. Changes in IacZ expression was measured by B- galactosidase assays
(MacIntyre et al., unpublished) and lac phenatype on X-gal containing plates (table 3).
The five clones were called LF203020,3 8,4O,47 and 57.
The identity of the Mp responsive genes was determined by subcloning the lac
portion of Mud lamp lac by digestmg total genomic DNA with Bgm restriction
endonuclease. Thus Bgm fragments fiom the clones genome, of which one included the
lac geae, Mp responsive gene and gene sequence until the next Bgïii restriction
endonuclease site were inserted into pBR.322 at BamHI. The vectors which contained the
lac gene plus Nip responsive gene were identified by transforming p90C (A lac) E.coli
cells and plating these ceils onto amp and X-gal contaiuing plates. Blue colonies must be
expressing the IacZ gene and therefore harbor the pBR322 consmict containing the lac
genes and the Nlp responsive gene. The f i e Nlp responsive genes were isolated in this
mamer. Subsequent identification was based on restriction endonuclease map anaiysis
and double or single stranded sequencmg.
Double stranded sequencing of p20 identifïed a genomic region between 87.8 and
87.9 minutes on the E.coli chromosome. Tbis region has been characterized by Plmkett et
al. (1993) and a restriction map of this uea is s h o w in figure 4. A Bgl II site is located
immediately upstream fiom the sequences read. Smce the Mp responsive insert was
predicted to be between 3.7 and 3.9 kb, the Mudliac - Mp responsive gene junction is
believed to be just downstream nom 080 and 08 1. The prediaed restriction map
corresponded to this region (compare figures 3 and 4) and the midentified PMdI and Bgn
sites can be justified since both of these enzymes cut numerous times in either the vector or
in Mud 1 and therefore the fragments generated would be too small to Msualize on a
polyacqlamide gel. Restriction digests with Xhol and XhoI plus S d were also done and
the resuhs were consistent with this identified area (data not shown). Mud lamp lac was
predicted to have mserted approxirnately 100-400 base pairs downstream fkom the XhoI
site. Thus the Mp responsive gene in clone LF203020 is either 080 or 08 1 both are
transcnbed in a direction that would permit IacZ expression from Mud 1 amp lac. Both of
these open reading fiames have not been characterized, however, 080 shows homology to
'glycosol hydrol F S . This protein is referenced in the Swiss protein data base 'PROSITE'
but has no more mformation published on it. Both 08 1 and 080 are predicted to encode
smali gene products of approximately 8 1 amho acids. Interestingly there is a '%end site'
predicted to be immediately upstream fiom 08 1. Two bends of 77.5 1 and 87.15' have
been predicted at this area (Phinkett et al., 1993). Perhaps Nlp has an sffiMV for bent
DNA (I ie prokaryotic DNA binding protein HU) and thus it enhances transcription of 08 1
or 080 or both by bmding at this site. hdeed &gaiactosidase activity of clone LF203020
indicates an increase in expression of these gene m the presence of plasmid bound Mp. The
hct ion of Nlp m the expression of 080, or 08 1 could be better hypothesized ifmore
information was availrble concerning these genes. For example, studies whicb identified
the cellular location of the gene products of 080 and 08 1 (ie. membrane versus
cytoplasmic prot eins), or which determined under which environmental circumstance these
genes are transcnbed (m both mutant and wild type E-coli strains) or hally whether or not
these gene products were essential for E.coli viability. Further investigation into these
genes, as well as the fùnction of Mp m their expression, will heip characterize the Nip
protem.
Double stranded sequencmg of p38 revealed an area corresponding to
approxhately 92 minutes on the E. coli chromosome. This area has also been
characterized (Blattner, F. R et al., 1993) (Heidi et al., 1993). A Bgm site is located just
downstream fiom the sequeoce read. The predicted size of the Nlp responsive gene insert
was 5.5 - 6.4 kb, and the gene located this distance fiom the Bgm insert is plsB which
encodes glycerol-3-phosphate acyltmsferase (WiJkison et al., 1 986). The predicted map
of p38 corresponds with the generated restriction map of this area (compare figures 3 and
5). Moreover, plsB is transcniied in a direction that would d o w lacZ expression fiom
Mud lamp lac. Interestingiy, there are two bend sites immediately upstream fiom plsB:
one of 74" and one of 84". Thus, it is plausible that Nlp could be bmdmg to bent DNA
and activating transcription of this gene. The j3-gaiactosidase actMty of LFîO3038 in the
presence versus the absence of N p indicltes that Mp activates transcription ofplsB.
Glycerol-3-phosphate acyltran&ease is an mtegral membrane protem that catalyzes the
transesterification of a fatty acyl group fiom acyl coenzyme A to the sn- 1 position of sn-
glycerol3-phosphate (Wilkison et ai., 1986). This is referred to as the committed step m
phosphohpid biosynthesis. E. coli cells which overproduce this enzyme form intraceMar
tubular structures composed of ordered an-ays of this protein (Wilkison et al.. 1986). The
stringent response (due to amino acid starvation) m E. coli inhiibts phospholipid
biosynthesis (Ray and Cronan, 1975). Moreover, the overexpression of p l d reüeved this
inhibition, thus giycerol3-phosphate is believed to be the target of ppGpp (guauosine-5'-
diphosphate-3'-diphosphate) which is a key component to the stringeut response (Heath et
al.. 1994). PeptidogScan biosynthesis is also inhibited m the stringeut response, and the
plsB gene product has also been reported to relieve this phenornenon in E. coli (Heath et
al.. 1994).. Penicillin tolerance is also obsewed for E. coli ceils during the stringent
response which is also relieved by the overexpression ofpkB (Rodionov and Ishiguro,
1995). Both peptidogiycan biosynthesis and penicillin tolerance are thus consequences of
the inhibition of phospholipid biosynthesis in the stringent response. The mur2 gene is
located immediateiy downstream from nlp and is transcribed in the opposite direction to
nlp. Its gene product catalyses the nrst committed step m peptidoglycan biosynthesis and
is an essentiai gene in E.coli (Brown et al., 1995). Since the original experiment of
identifjing clones that changed I d expression in the presence versus the absence of Mp
were done with pBS5 which includes the nlp gene plus a downstrearn region (containing
about 40 base pairs of anti sense nnuZ gene), it is possible that the effect of mcreased
expression ofplsB is not due to Nlp but rather anti-sense mur2 which may be iuhiiiting
the translation of this essential gene. Transcription fiomplsB may be activated m order to
coqensate for this loss. Perhaps Nlp and MurZ are not as mdependent as origin*
hypothesized and may be part of a regdon mvohed in cell waii biosynthesis or are perhaps
components of the stringent response. Studies Bito whether or not the overexpression of
Mp relieves ppGpp inhiiition of peptidoglycan biosynthesis or prevents ppGpp dependent
peniciUm tolerance could be examined to determine Nlp ' s role m plsB expression.
Double stranded sequencing of p4O was unsuccessful therefore a 1 kb HindITI
fiagrnent was subcloned mto pUC 1 18 and single stranded DNA was generated and
sequenced. The sequence showed homology two regions, one located at 25.3 minutes and
the other to 5 1.6 minutes. By examining restriction map analysis it was determined that the
region at 5 1.6 minutes was the subcloned area of the Nlp responsive gene. The estimated
Sze of the Nlp responsive gene m p40 was 33 kb. The restriction fiagments generated
fiom the HN>dm digestion also clarified the location of the Mudlamp lac msertions
because there were Bgm sites located on either side witbin 1 kb of the identified seguence.
The restriction map (Rudd, 1992) of this area was compared to a photograph of restriction
fiagments generated through restriction endonuclease digestions of p4O. These restriction
fragments were consistent with the restriction map of this area (figure 7). It is likeiy that
the extra BgiiI site m this region is due to mcomplete digestion of LFZO3O4O total
genomic DNA with this enzyme and the subsequent subcloniug of this fiagrnent into
pBR328 (MacIntyre and DuBow, unpublished). Digestion of p4O with BgiiI does suggea
that there is a BgiiI site m this plasmid. The restriction map for p40 is shown in figure 7.
The Mp inducile gene was deduced to be either cscB or cscK (Bock- et al.. 1992) or
drdA dependmg on whether or not the LF series are capable of sucrose or Dserine
metabolism (Bloom et al., 1975). The cscB/cscK genes are both t ransded m the same
direction and would allow the expression of the l a d gene of Mudlamp lac. These genes
are part of the csc ( & . r o m o s o ~ coded =rose genes) regulon which is controlled by a
sucrose specific repressor, CscR The regulon contasis structural genes cscA and cscK
which encode sucrose hydrolase and Dhctokkase, the repressor (cscR) and a transport
protein cscB which encodes sucrose pemease. This regulon is ody present in about 50%
of E.coli wild type isolates and is located at 5 1 minutes on the E. coli chromosome. The
presence of the csc genes causes the mhibition of neighboring gene, drdA (the structural
gene for Dserine deaminase). The insertion of the csc genes into the intergenic region
between drdA and &dC in E.coli strairïs that do not possess these genes also causes the
inhibition of dsdA. It bas been hypothesized that these genes fùnction as 'chromosomal
alternatives' (Boclrmann et ai., 1992). It is possible that Nlp has a role in activating both
&dC and cscB or cscK, or perhaps only has a role in the expression of one of these
systems. It must be determhed whether or not the E. coii strains used in this study are able
to grow on sucrose and thus whether or not they posses the csc operon. I f the strains do
not posses the csc operon, then Nlp's role in the expression of dsdC shouîd be
investigated. The Ad operon encodes the regdatory protein (dsdC) and the structure
(&A) for Dserine deaminase. Dserine is converted to pynivate and arnmonia by D S -
aminase (hdA). The DsdC regulatory protein acts as a positive regulator and activates the
transcription of dSd A. The drd operon is active when the inducer (Dserine) is present.
The CAMP-CRP complex has also been reprted to mduce this operon (Bloom et al..
1975). Thus Nlp's role m this operon could tentatively be to complement the activty of
CAMP-CRP. If these strains are sucrose positive, then Mp's role in the csc operon could
be investigated. The csc operon is poorty expressed, which is most iikely due to the
inefficient uptake of sucrose by CscB. Thus, the effect of Mp on the efficiency of sucrose
uptake could be mvestigated. Most sucrose transport system that are plasmid based or
found in the chromosomes of other related bacteria (e.g. K-pnemoniae) phosphorylate and
upt ake sucrose via a phosphoenolpyruvate (PEP)-dependent, sucrose-6-
p hosp hotransferase ( sucrose PTS) system (Postma and Lengler, 1 985). The sucrose is
transported into the cytoplasm and modified to sucrose-dphosphate, and subsequently
hydrolysed by an invertase into D-glucose dphosphate and Dhctose . The Dhc to se
substrate is then phosphorylated by a h c t o h a s e (Postma and Lengler, 1985). The csc
sucrose transport pathway is a non-PTS metabolic pathway, though it does posses an
invertase, a hctokinase and a repressor. CscB actively transports sucrose and is specific
for this sugar (Sahin-Toth et al., 1995). Interestingly, the CscB permease shows 3 1.2%
identity at the amho acid level to LacY (the transport protem of the lac operon). The
potential role of Nip in this system couid be similar to the role of CR.-CAMP in the lac
operon. Overexpression of Nlp h this clone resulted in celi death, thus Mudl amp lac has
inserted into a gene which is required for E. coli viability if Mp is overexpressed. If' Mp
inactivates transcription of this gene then the Survival of the cells is dependent on the
expression of this gene. If Nlp activates this gene then the Mudl omp lac msertion has
disrupted the generation of a f'unctiona.1 gene product. For example, the mtemption of a
membrane bound protem could result m a cytoplasmic buüd up which could be toxic to the
ce& Altematively, disruption of a component of the sucrose metabolic pathway
(ie.hctokinase) could resutt in the buifd up of a precursor metabolite which could be toxic
to the cell. This effect could be compounded by an mcrease in the expression of the
downstream permease. Ifthe strains are not sucrose pontive, then Nlp is involved m the
transcription of &dC . However, it would still be interestmg to mvestigate Mp's role in the
csc regulon, since these operons are predicted to be alternates for each other. Moreover,
since Mp has been previously implicated in the activation of other sugar operons, it would
be a plausible hct ion to M e r examine.
Double stranded sequencing was not successful for p47. A Ikb, EcoRl, piece of
the Mp responsive gene was therefore subcloned hto pUC 1 18 and single strauded DNA
was isolated and then sequenced. The area sequenced was located to arouud 9 1.5 miautes
on the E - d i chromosome. The restriction fkagments generated the direction of the
Mud lump lac insert was detexmined and a unique BglII was found approximately 1 1 kb
upstream which corresponded to the predicted size of the Nlp responsive gene msert . The
Mp responsive gene is ükely pgi which encodes glucose-6-phosphate isomerase. Again,
there is a predicted bend of 79" located immediately upstream frompgi. A restriction map
for this region is shown in figure 8. The kgalactosidase a c t ~ t y of the clone LF2O3 047 in
the presence versus the absence of Nlp mdicat es that Nlp inhibits the expression of this
gene. Glucose-6-phosphate isomerase is also referred to as p hosphoglucose isomerase. It
is iuvolved m glycolysis or glucose metaboüsm (Fromm et al., 1989) by catalping the
mterconversion of glucose-6-P and -ose-6-P. In anaerobiosis, transcription fÎompgi is
stightly repressed (Schreyer and Bock, 1980). Interestingly, anaerobiosis is associated
with higher CAMP b e l s and thus sugar operons dependent on CRP-CAMP activation are
expressed m these conditions. It is intereshg that Np, which has been associated with the
metabolism of CRP-cAMP dependent sugar fermentation pathways, also inhiibàs the
expression of a gene which is mvolved glycolysis. The function of Mp m this system lends
support to the theory that Mp acts m concert or as a back up system for CRP-CAMP gene
expression for some genes.
Attempts to sequence p57 fiom a double stranded template were also unsuccessful
and therefore the Mp mducible region had to be subcloned into pUC 1 18. A 2.4 kb HpaI
fiagment was isolatecl, subcloned into pUC 1 18, and single stranded DNA was generated
and sequenced. Like p40, the sequence located to a region at 5 1.6 minutes and a region at
25.1 inmutes on the E-coli chromosome. The region located at 5 1.6 ainutes was more
consistent with the results obtained fiom restriction map analysis. Also similar to p40, is
the presence of a BgiII site which can only be explained by mcomplete digestion of the
total genomic DNA of strain LF203057. Digestions of p57 with Bgm enzyme also
revealed that this plasmid Wrely contains a BgiII site. The subcloned HpaI fiagrnent of the
Mp responsive gene also contained the Mud llac juuction and thus the location of the Mp
responsive clone could be conclusively determined. The Mp responsive gene in LF203057
is nuoF. A restriction map of enzyme sites in this region is shown in figure 9. The Bgm
site is predicted to lie approximateiy 9 kb downstream fiom nuoF on the E-coli
chromosome. Unfortunately, this region has not been sequenced and therefore the exact
location of this site cannot be c o h e d . The restriction map (Rudd, 1992) of this area
correlates to data fiom restriction digestions of p57 (MacIntryre and DuBow,
unpublished). Nlp stimulates the expression of this genes as shown by the increase m P-
gakactosidase actMty of LF2O3O7 in the presence versus the absence of plasmid borne Nlp
(table 3). NuoF is a subunit of the membrane bound NADH dehydrogenase in E. coIi.
NADH dehydrogenases donate electrons to ubiquinone which are transfmed to a
terminal oxidase complex (Anaraku and Gennis, 1987) and thus believed to generate a
proton motive force to permit a number of energy dependent h c t i o n s such as active
transport m E.coli. There are two separate genetic loci which encode NADH
dehydrogenase, one maps to 5 1.5 and 5 1.8 minutes (mo) and the other maps to 25.1
minutes (ncDi ) on the E.coli chromosome (Calhoun and Gennis, 1993). NADH
dehydrogenase II actMty is associated with the mih locus, whüe NADH dehydrogenase I
activity is associated with the nuo locus (Weiduer et al., 1993). . This explains why
sequences generated fkom p40 and p57 showed homology to two different regions on the
Ecoli chromosome. Single mutants of each of these loci have growth rates comparable to
wdd type strains. Growth is impaired in strains containhg mutations at both of these loci.
The di6erence between NADH dehydrogenase 1 and II is that energy via electron flow
through NADH-dehydrogenase 1 will conserve more energy due to the correlation of a
coupling site in this enzyme (Matsushita et al, 1987). Thus, it has been hypothesized that
energy recovered kom NADH oxidation may by controiled regulating the level of tnro
versus expression. Nlp may fùnction as a regulator of rmo and possbly d.
Expression £iom ndh has been reported to decrease during late exponential growth. Late
exponential growth is Plso when CRP-CAMP dependent operons are expressed, which
could lend support to the function of Nlp as a synergistic regulator to CRP-CAMP gene
regdation. Thus the eEect of Nlp on both NADH-dehydrogenase 1 and II couid be
investigated and the hc t i on of Mp in this system could be determined.
Smce nlp is a gene in many Enferobacteracea (Autexier and DuBow, 199 1 ) it
should have an important physiological fùnction. This study has discovered 5 genes which
are eEected by the overexpression of Nlp. Three of these genes were found to have a bend
site of 74"-84". The remaining genes ( m F and drdClcscB or cscK) may also possess
bend sites which have not yet been documented. These bend sites msy be required for Nlp
binding. Band retardation assays on these areas could be done to determine if Nlp bmds to
these areas. This would conclusively estabiish whether or oot Nlp is a DNA bmding
protein. Moreover, genes with bend sites of 74"-84' could be tested to see ifMp bmds
specifically to this site. ifthis is true, then Mp acts as a global regulator like HU in binding
specifically to bent DNA. [fMp ooly bmds to the five genes identified by this study, then
perhaps Nlp plays a more specific regdatory role. The majority of the Nlp responsive
genes (cscB/K or &dA, pgi, and nuoF) discovered in this study are also CAMP-CRP
dependent operons. This may implicate Nip in a complementary fiuiction to CAMP-CRP.
This theory is also supported by previous observations that Mp complements maltose and
lactose fermentation m MK200 1 (cya crp* 1) (Choi et al., 1989). This possibility could be
fùrther investigated by isolating CAMP-CRP inducible genes and examining whether or
not Mp bhds to these area andfor effect transcription of them
Mp may have a specific function for 08 1/080, plsB, cscB/K or pgi, and
moF transcription. Perhaps these genes have an underiying common theme and form part
of an indentified regdon, or perhaps Nlp acts as a global regulator which affects the
transcription of many dflerent unrelated genes. Fuither investigation into Nlp's role in the
regulation of these genes, as wel as other genes (CAMP-CRP induced or with similar
bend sites), will ansver many questions conceming gene regulation and may idente a
novel I backup system of gene regulation in E-coli. Smce this study could ody iden*
Nlp responsive gene that are not essentid to Ecoli viability? an aitemative method should
be used to identify any Mp responsive genes that are essential.
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