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Investigación1 Y QIK

The yqiHIK gene cluster from Bacillus subtilis is predicted to encode an extracellular lipoprotein(YqiH), a secreted !acetylmuramoyl!"!alanine amidase (YqiI), and a cytoplasmicglycerophosphodiester phosphodiesterase (YqiK)# $e%erse transcriptase &'$ ($T!&'$) analysissho ed that the yqiHIK genes are transcribed as an operon# 'onsistent ith the in silicoprediction, e found that the purified YqiI protein exhibited hydrolytic acti%ity to ardpeptidoglycan sacculi# Transcription studies ith yqiH!tre reporter fusion strains re%ealed thatthe expression of yqiHIK is sub*ected to finely tuned osmotic control, but enhanced expressionoccurs only in se%erely osmotically stressed cells# &rimer extension analysis pinpointed theosmotically responsi%e yqiHIK promoter, and site!directed mutagenesis as employed to assessfunctionally important sequences required for promoter acti%ity and osmotic control# &romoter

%ariants ith constituti%e acti%ity ere isolated# deletion analysis of the yqiHIK regulatoryregion sho ed that a + !bp T!rich - segment positioned ./0 bp upstream of the ! +sequence is critical for the acti%ity and osmotic regulation of the yqiHIK promoter# Hence, theexpression of yqiHIK is sub*ected to genetic control at a distance# 1pon the onset of gro th ofcells of the B# subtilis ild!type strain in high!salinity medium (.#2 3 a'l), e obser%ed grossmorphological deformations of cells that ere then re%ersed to a rod!shaped morphology again

hen the cells had ad*usted to the high!salinity en%ironment# The products of the yqiHIK genecluster ere not critical for reestablishing rod!shaped morphology, but the deletion of thisoperon yielded a B# subtilis mutant impaired in gro th in a defined minimal medium and athigh salinity#

4tructure and genetic organi5ation ofthe yqiHIK gene cluster# ( ) 6eneticmap of the yqiHIK genes in thegenome of B. subtilis # The length andthe position of the different &'$!amplified fragments are indicated#

bent arro indicates the promoterregion and the directionof yqiHIK transcription7 the positionof a putati%e factor!independenttranscriptional terminator sequenceis mar8ed by a lollipop# (B) $T!&'$!

based analysis of theputati%e yqiHIK operon# Theamplification reactions using theindicated - primers (&. to &9)

ere conducted on differenttemplates: c- , genomic - as apositi%e control, and $ as a

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negati%e control# Total $ as isolated from cells of B. subtilis strain .;/ gro n in 433# 3,molecular mar8er#

'ell all hydrolase acti%ity of the purified YqiI<4trep!tag!II protein# ("eft) 'oomassie blue!stained 4-4!& 6= gel of the affinity purified

YqiI<4trep!tag!II protein# ($ight) >ymogramanalysis ( .; ) using purified B. subtilis .;/sacculi# 4taining of the polyacrylamide gel ithmethylene blue and destaining ith distilled

ater detected areas of peptidoglycan lysis#=ight micrograms of the purified YqiI<4trep!tag!II protein as applied for both 4-4!& 6=and the 5ymogram#

In silico analysis of the YqiI protein# ( ) lignment of the amino acid ( ) sequences(catalytic domain only) of the YqiI, "yt', ' l', ' l-, and Yr%? proteins from B. subtilis andthe ' l@. protein from P. polymyxa %ar#colistinus # Blac8 boxes highlight amino acids thatare in%ol%ed in the coordination of a 5inc ion in the acti%e site of amidases ( .+ ,+9)# (B)-omain organi5ation of B. subtilis proteins homologous to YqiI# The lengths of the proteinsare indicated# Boxes ith hatch mar8s represent 4ec!type signal sequences7 they are presentin all amidases sho n except for ' l'# The amidaseA ( miA ) domain (& 0.+020) ismar8ed# Blac8 boxes and gray triangles represent different types of cell all!binding

domains: cell all!binding domain 2 ('CBA2) (& 09.22) of "yt', the spore domain(4&D$) of ' l' (& 0+0 ;), the 4H b domain of Yr%? (43002/E), and the amine domain( 3I ) (& ..E9.) of ' l@# (') In silico model of YqiI based on the crystal structure of thecatalytic domain of the ' l@. protein (&-B accession number .?CF) from P.

polymyxa %ar#colistinus # The amino acids H!.+, =!2G, H!/2, and =!.9/ are predicted tocoordinate a 5inc ion in the acti%e center of the protein and contribute to en5yme acti%ity#

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6ro th properties of yqiHIK deletionmutant strain K B/ and strain K B E ( lytC cwlC cwlD yrvJ yqiI )# ( and B) 'ulturesof B. subtilis ild!type strain .;/ (blac8circles), its ( yqiHIK ::neo ) deri%ati%e strain

K B/ ( hite circles), and the lytC cwlCcwlD yrvJ yqiI quintuple mutant strainK B E (gray circles) ere gro n in 433alone ( ) or in 433 containing .#2 3 a'l(B)# (') The morphologies of B.subtilis strains .;/, K B/, and K B E ereanaly5ed by phase!contrast microscopy atdifferent time points (indicated by arro s)during the gro th of the cultures# 4cale bar,+ m#

luorescence and scanning electronmicroscopy of salt!stressed B. subtilis cells#( ) 'ells of ild!type strain .;/ and itsmutant deri%ati%es K B/ ( yqiHIK ) andK B E ( lytC cwlC cwlD yrvJ yqiI ) ere

gro n in 433 ith .#2 3 a'l for .2 h (timepoint 9) ( ig# 9B ) and obser%ed by bothphase!contrast and fluorescence (afterstaining ith the "i%eJ-ead Bac"ight

bacterial %iability 8it) microscopy# 4cale bar,+ m# (B and ') 'ultures of strain .;/ eregro n to the early exponential phase(D- +E/ of 0#E to 0#G) in 433 alone (B) or in433 containing .#2 3 a'l (') and ere

obser%ed by scanning electron microscopy# The magnification as set at /,000, and thescale bar represents 9 m#

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Influence of the T!rich regionlocated upstream ofthe yqiHIK promoter region onosmotic induction of yqiH-

treA reporter gene expression# ( )- sequence of the 00!bpfragment fused to thepromoterless treA reporter gene instrain K B.+# The T!rich region ishighlighted by a blac8 box# The - fragment sho n is defined as 0#

rro s indicate three differenttruncations ( . to ) of the 00!bpregion originally used to construct

the yqiH-treA gene fusion# The positions of the L + and L.0 yqiHIK promoter regions, thetranscriptional initiation site, the ribosome!binding site of the yqiH gene, and itstranslational start codon of the yqiH reading frame are indicated# (B) &romoter acti%ity andosmoregulation of reporter strains carrying the %arious deletion constructs in cellsculti%ated either in 433 or in 433 containing .#2 3 a'l#

Influence of the T!rich region locatedupstream of the yqiHIK promoter region

on osmotic induction of yqiH-treA reporter gene expression# ( ) -sequence of the 00!bp fragment fusedto the promoterless treA reporter gene instrain K B.+# The T!rich region ishighlighted by a blac8 box# The -fragment sho n is defined as 0# rro sindicate three different truncations ( .to ) of the 00!bp region originally

used to construct the yqiH-treA genefusion# The positions of the L + and L.0 yqiHIK promoter regions, the transcriptionalinitiation site, the ribosome!binding site of the yqiH gene, and its translational start codonof the yqiH reading frame are indicated# (B) &romoter acti%ity and osmoregulation ofreporter strains carrying the %arious deletion constructs in cells culti%ated either in 433 orin 433 containing .#2 3 a'l#

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2 YQIK HYPOTHETICAL PROTEIN [ E SCHERICHIA COLI STR . K-12

SUBSTR . W3110 ]

2.1 S UMMARY

Gene symbolyqiK

Gene descri !ion hypothetical protein

Loc"s !#$ Y75_p2977Gene !y e

Protein codingRe%&e' s!#!"s PROVISIONA

Or$#nism !"cherichia coli "tr# K$%2 "&'"tr# ()%%* +"train, K$%2- "&'"train, ()%%*- old$na.e, !"cherichia coli"tr# K%2 "&'"tr# ()%%*/

Line#$e 0acteria1 Proteo'acteria1 a..aproteo'acteria1

!ntero'acteriale"1 !ntero'acteriaceae1!"cherichia

2.2 G ENOMIC C ONTEXT

&e'"ence( N3_**7779#% +)%9%52*##)%9)%4%/

2.3 G ENOMIC REGIONS , TRANSCRIPTS , AND PRODUCTS

Genomic &e'"ence( N3_**7779

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2)* + I+LIOGRAPHY

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