pbpb, a gene coding for a putative penicillin-binding ... · southern analysis of chromosomal dna...

JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.0010 DOI: 10.1128/JB.183.2.628–636.2001

Jan. 2001, p. 628–636 Vol. 183, No. 2

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

pbpB, a Gene Coding for a Putative Penicillin-Binding Protein,Is Required for Aerobic Nitrogen Fixation in theCyanobacterium Anabaena sp. Strain PCC7120

SARA LAZARO, FRANCISCA FERNANDEZ-PINAS, EDUARDO FERNANDEZ-VALIENTE,AMAYA BLANCO-RIVERO, AND FRANCISCO LEGANES*

Departamento de Biologıa, Facultad de Ciencias, Universidad Autonomade Madrid, 28049 Madrid, Spain

Received 26 July 2000/Accepted 26 October 2000

Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, whichis unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate).Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of theoriginal mutant. The transposon had inserted within an open reading frame whose translation product showssignificant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins(PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similaritysearch allowed us to identify at least 12 putative PBPs in the recently sequenced Anabaena sp. strain PCC7120genome, which we have named and organized according to predicted molecular size and the Escherichia colinomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal as pbpB andits product as PBP2. The wild-type form of pbpB on a shuttle vector successfully complemented the mutationin SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen,nitrate and N2, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substitutedby other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, therole of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.

Filamentous cyanobacteria such as Anabaena sp. strainPCC7120 perform oxygenic (i.e., higher plant-type) photosyn-thesis. When grown in the presence of fixed nitrogen, all thecells have similar morphology and are known as vegetativecells. When the filaments are deprived of nitrogen, a small per-centage of the vegetative cells of Anabaena sp. strain PCC7120differentiates into nitrogen-fixing heterocysts (36) that are semi-regularly spaced along the filament generating a pattern (5,37, 38, 41, 43). Protection of nitrogenase from inactivation byoxygen appears to depend, to a great extent, on a barrier to thediffusion of oxygen and other gasses through the envelope ofthe heterocyst. This envelope consists of a layer of polysaccha-ride surrounding a layer of glycolipid, which in turn surroundsa cell wall that is presumed to correspond to that of normalgram-negative vegetative cells (41).

In order to identify mutants in which nitrogen fixation isaffected, we mutagenized Anabaena sp. strain PCC7120 withTn5-1063 (40). The transposon was introduced by conjugation(10, 39). Exconjugants were selected in the presence of anti-biotics and nitrate and then transferred to nitrate-free me-dium. Mutants unable to grow with dinitrogen as the solenitrogen source were selected for further study (14). In thepresent study, we report the physiological and molecular char-acterization of one of these strains, which we have designatedSNa1. The transposon in SNa1 inserted within an open readingframe (ORF) whose predicted protein sequence shows signif-

icant homology to a family of proteins known as high-molec-ular-weight penicillin-binding proteins (HMW PBPs), whichare involved in the synthesis of the peptidoglycan layer of thecell wall. A BLAST search of the recently sequenced Anabaenasp. strain PCC7120 genome has allowed us to identify 12 pu-tative PBPs that we have named and organized according topredicted molecular size and the Escherichia coli nomenclaturefor PBPs (19, 33). Based on this nomenclature, we have namedthe gene interrupted in SNa1 pbpB and its product PBP2. Thewild-type gene has been cloned, and the mutation has beensuccessfully complemented. The expression of pbpB has beenstudied in vivo using a pbpB-luxAB fusion.

The evidence presented indicates that the Anabaena sp.strain PCC7120 putative HMW PBP described here, encodedby pbpB, is required for growth under aerobic nitrogen-fixingconditions.

MATERIALS AND METHODS

Strains and growth conditions. Anabaena sp. strain PCC7120 and its deriva-tives (Table 1) were grown at 28°C in the light, ca. 90 microeinsteins m22 z s21,on a rotary shaker in 50 ml of medium AA/8 (23) supplemented with nitrate(5 mM) in 125-ml Erlenmeyer flasks. Constructions (Table 2) were introducedinto cyanobacterial strains by conjugation (10, 39), and single and double recom-binant strains were selected as described by Cai and Wolk (6).

The different strains were grown in the presence of appropriate antibiotics.Mutant SNa1 was grown in liquid with 40 mg of neomycin sulfate (Nm) per ml.Single recombinant SR2028-8 and double recombinant DR2028-6 were grownwith 2 mg of spectinomycin dihydrochloride (Sp) per ml plus, respectively, 1 mgof erythromycin (Em) per ml and no additional antibiotic. Mutant SNa1 bearingplasmid pBG2030 was grown with 1 mg of Em per ml and 40 mg of Nm per ml.Aldehyde, the substrate for bacterial luciferase, was generated internally by theproducts of Xenorhabdus luminescens luxC, luxD, and luxE borne on plasmidpRL1472a (15). Em, at 1 mg z ml21, was incorporated in the media to select forthat plasmid.

* Corresponding author. Mailing address: Departamento de Biolo-gıa, Facultad de Ciencias, Universidad Autonoma de Madrid, 28049Madrid, Spain. Phone: 34-91-3978176. Fax: 34-91-3978344. E-mail:[email protected].

628

on Decem

ber 2, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

http://jb.asm.org/

Assays of nitrogenase. Liquid cultures were deprived of combined nitrogenunder air (aerobic conditions) or under an N2/CO2 (99:1) gas phase (microaero-bic conditions; cultures were sealed with rubber stoppers and continuously bub-bled with the N2/CO2 gas mixture) for 24 to 48 h, at which time heterocysts, ifthey were to form, were clearly discernible. Nitrogenase activity was measured inwhole cells by the acetylene reduction technique (35). For aerobic assays, 10 mlof cell suspensions deprived of nitrogen under air was placed in stoppered 40-mlvials; to begin the assay, 4 ml of air was withdrawn and replaced with 4 ml ofacetylene. The vials were incubated for 30 min at 28°C on a rotary shaker undera constant irradiance of 100 microeinsteins m22 s21. For microaerobic assays,the gaseous phase of the cultures deprived of combined nitrogen under N2/CO2

was replaced by 10% (vol/vol) acetylene in N2 and was incubated as indicatedabove. At the end of the 30-min incubation period, 0.5-ml samples were with-drawn and their ethylene content was determined by injection into a ShimadzuGC8A gas chromatograph.

Recovery of transposon-containing plasmids and construction of derivativesof these plasmids. Plasmid pBG2002 (Tn5-1063 and contiguous Anabaena DNA)(Table 2) was obtained by digestion of chromosomal DNA from mutant SNa1with EcoRV and then by recircularization of the fragments with T4 DNA ligaseand transfer to E. coli HB101 by electroporation. Colonies that grew on L agarplates with 50 mg of kanamycin sulfate (Km) per ml were analyzed further (40).Approximately 3.2 kb of Anabaena sp. strain PCC7120 genomic DNA was re-covered in pBG2002.

In order to generate the SNa1 mutation in wild-type Anabaena sp. strainPCC7120 (Table 2), most of the transposon was removed from pBG2002 bycutting with PstI and BamHI. The remaining 4.1-kb piece of DNA was ligatedwith pRL759D (4) that had been cut with PstI and BamHI, generating pBG2027.Plasmid pRL1075, which bears the conditionally lethal gene sacB that allows forselection of double recombinant strains (6), was cut with FspI, and a fragment of5.6 kb was inserted into pBG2027, which had been cut with EcoRV, to generatepBG2028 (Table 2).

Cloning of pbpB and assays of complementation of the mutant strain. A PCRclone of wild-type Anabaena sp. strain PCC7120 DNA bracketing the transposon

in mutant SNa1 was generated with the primers 59-ATCATCGCCACGGCAAAATT-39 and 59-GTGTAGCACCAGCACAACTA-39. The resulting 2.4-kbPCR fragment was first cloned in the vector PCR2.1TOPO (Invitrogen, Carls-bad, Calif.) producing plasmid pBG2029 (Table 2). From pBG2029, that samefragment was cut and inserted between the BamHI and XhoI sites of pRL1342(Cmr Emr; RSF1010-based plasmid obtained from C. P. Wolk [unpublisheddata]) generating plasmid pBG2030 (Table 2), which can replicate in Anabaenasp. strain PCC7120. In parallel, the 2.4-kb Anabaena sp. strain PCC7120 DNAwas also inserted between the SmaI sites of pRL1404 (16), producing plasmidpBG2031 (Table 2), which can also replicate in Anabaena sp. strain PCC7120.

Plasmids pBG2030 and pBG2031 were transferred with pRL1342 andpRL1404, respectively, as controls from E. coli to cells of mutant strain SNa1 asdescribed previously by Wolk et al. (39) by using helper plasmid pRL623 (whenpBG2031 or pRL1404 was transferred) (Table 2) (13) or pDS4101 and pRL1124(when pBG2030 or pRL1342 was transferred) (Table 2) (7, 17). Selection wasmade on petri dishes of agar-solidified AA medium (1, 23) containing 5 mg of Spper ml and 200 mg of Nm per ml (pBG2031 or pRL1404) or 10 mg of Em per mland 200 mg of Nm per ml (pBG2030 or pRL1342).

The green colonies that appeared on the filters were further restreaked toplates of the same medium to assess their ability to grow in the absence ofcombined nitrogen.

Southern analysis. Southern analysis of chromosomal DNA made use of theGenius system (Boehringer Mannheim GmbH, Mannheim, Germany). DNAprobes were labeled with digoxigenin-11-dUTP from random primers.

Sequence analysis. Automated sequencing (ABI Prism 377 DNA Sequencer;Perkin-Elmer, Norwalk, Conn.) was performed on fragments that were sub-cloned from pBG2002 and pBG2029. The initial sequencing from the ends of thetransposon in pBG2002 was performed from specific primers for the left andright ends of the transposon (4, 16). Sequence analysis was performed with theUW GCG version 7 package of the University of Wisconsin Genetics ComputerGroup (9). Amino acid sequence analysis was performed with the DAS trans-membrane prediction package (Proteomics tools) at the ExPASy MolecularBiology Server (http://www.expasy.ch). Database comparisons and alignments of

TABLE 1. Anabaena strains used in this study

Strain Derivation and/or salient characteristics Source or reference

PCC7120 Wild type C. P. WolkSNa1 Nmr Fox2 Fix1 This studySNa1(pBG2030) Nmr Emr This studyDR2028-6 Spr Smr Ems of double homologous recombination of plasmid pBG2028 with wild-type PCC7120 This studyDR2028-6(pRL1472a) Spr Smr Ems, double recombinant strain expressing luxCD-E (in a plasmid) This studySR2028-8 Spr Smr Emr product of single homologous recombination of plasmid pBG2028 with wild-type PCC7120 This study

TABLE 2. Plasmid constructs used in this study

Plasmid Derivation and/or salient characteristics Source orreference

Bluescript Cloning vector Stratagene (SanDiego, Calif.)

pBG2002 Circularized EcoRV fragment from Anabaena sp. strain SNa1 that bears Tn5-1063 This studypBG2027 Anabaena DNA-containing PstI-BamHI portion of pBG2002 fused to the PstI-BamHI portion of pRL759D that con-

tains oriV, bom, luxAB, and Smr Spr determinantThis study

pBG2028 Product of ligation of pBG2027 linearized at EcoRV, with the FspI portion of pRL1075 that contains sacB and Cmr

Emr determinantsThis study

pBG2029 2.4-kb PCR fragment bearing wild-type pbpB as its only ORF cloned in the PCR2.1TOPO vector This studypBG2030 Wild-type pbpB on a BamHI-XhoI fragment from pBG2029, inserted into pRL1342 cut with BamHI and XhoI This studypBG2031 Wild-type pbpB on a BamHI-XhoI (blunted) fragment from pBG2029, inserted between the SmaI sites bracketing

Cmr cassette C.C1 in pRL1404This study

PCR2.1TOPO Cloning vector InvitrogenpDS4101 Apr, helper plasmid based on ColK, carries mob (ColK) 17pRL623 Helper plasmid bearing methylase genes M.AvaI, M.Eco47II (whose product methylates AvaII sites), and M.EcoT22I

(EcoT22I is an isoschizomer of AvaIII)13

pRL759D One of a family of BLOS plasmids with bom, V. fischeri luxAB, oriV and Smr Spr determinant for in vitro replace-ment of all but the termini of a transposon

4

pRL1075 Contains a sacB-oriT (RK2) Cmr Emr cassette that is separated from oriV by inverted polylinkers 4pRL1124 Kmr derivative of pACY177 that bears the same methylase genes as pRL623 7pRL1342 Cmr Emr RSF1010-based plasmid C. P. WolkpRL1404 Vector based on Nostoc plasmid pDU1 that replicates in Anabaena sp. strain PCC7120, with Cmr cassette C.C1 iso-

lated from pRL171 (11) inserted in its BamHI site, providing the only SmaI, KpnI, and SstI sites in the plasmid16

VOL. 183, 2001 A PUTATIVE PBP AND N2 FIXATION IN CYANOBACTERIA 629

on Decem


.org/D

ownloaded from

http://jb.asm.org/

the DNA and predicted protein sequences were performed by using the defaultsettings of the algorithm developed by Altschul et al. (2) at the National Centerfor Biotechnology Information with the BLAST network service programs.

In vivo monitoring of the expression of pbpB. Fifty milliliters of rapidly growingcultures of strains DR2028-6 (bearing pRL1472a) and SR2028-8 was washed atleast three times with AA/8 and then resuspended in 50 ml of AA/8 withoutantibiotics. For measurements with nitrate as the nitrogen source, AA/8 plus 5mM nitrate was used to wash and resuspend culture samples. Filaments wereexamined periodically with a microscope, and the luciferase activity of aliquotswas measured at specified times as a measure of the transcription of pbpB. Theluminescence of SR2028-8 was measured with supplementation of exogenousaldehyde (12); the luminescence of DR2028-6(pRL1472a) was measured withand without supplementation with exogenous aldehyde (12, 15). In the lattercase, the addition of aldehyde did not change luminescence, indicating thatendogenous aldehyde was not limiting. Luminescence was measured using adigital luminometer (Bio Orbit 1250 luminometer; Turku, Finland). The lumi-nometer was calibrated by setting the background counts to zero and the built-instandard photon source (a sealed ampoule of the isotope 14C with activity of 0.26mCi) to 10 mV. Calculations made according to the method described by Hast-ings and Weber (21) indicated that 1 U (1 mV) corresponded to a light emissionof 6.7 3 105 quanta/s from the vial.

Nucleotide sequence accession number. The nucleotide sequence of pbpB is inthe GenBank database under accession number AF076847.

RESULTS

Phenotype of mutant SNa1. Transposon-bearing (i.e., anti-biotic-resistant) exconjugant colonies grown with nitrate as thenitrogen source were transferred to petri dishes lacking com-bined nitrogen. Colonies that after several days yellowed and

therefore were nitrogen starved were selected for furtherstudy. Microscopic observation of one such colony on an N2

plate showed a clearly distorted morphology of both vegetativecells and heterocysts (Fig. 1B to D). Filaments were yellow andusually short and twisted, with vegetative cells unequal in sizeand even shape. The heterocysts or heterocyst-like cells thatcould be identified in such filaments appeared rather distortedwith thin envelopes, and in most cases, no cyanophycin gran-ules could be distinguished at their poles (Fig. 1B to D). How-ever, when the same strain was grown with combined nitrogen(nitrate), no significant differences in cell shape and morphol-ogy with respect to the wild type were observed (not shown).

The doubling time (hours), estimated from the increase indry weight of the wild-type and mutant SNa1 strains whengrown in liquid AA/8 medium supplemented with 5 mM ni-trate, was nearly equal (32.2 h for the mutant strain versus32.3 h for the wild type). However, when deprived of a sourceof combined nitrogen, the mutant strain bleached and died; infact, the nitrogenase activity, as measured by the acetylenereduction method (35) after 24 h of nitrogen deprivation underaerobic conditions of the mutant strain, was around 0.5% ofthat of the wild-type strain (1.9 nmol of C2H4 per mg [dryweight] per h versus 356 nmol of C2H4 per mg [dry weight] perh). However, under microaerobic conditions, the mutant strainreduced acetylene to levels similar to those found in the wild-

FIG. 1. Light micrographs of wild-type Anabaena sp. strain PCC7120 (A) and mutant strain SNa1 (B to D) grown in AA medium lacking nitrate(micrographs for A and B were taken after 48 h of nitrogen deprivation; micrographs for panels C and D were taken after 96 h). The filamentsof strain SNa1 (B to D) are short and twisted; vegetative cells vary in size. Cells that may be presumptive heterocysts are designated (h). Bar, 5mm.

630 LAZARO ET AL. J. BACTERIOL.

on Decem


.org/D

ownloaded from

http://jb.asm.org/

type strain under the same conditions (52 nmol of C2H4 per mg[dry weight] per h versus 64 nmol of C2H4 per mg [dry weight]per h).

The microscopic observations of the mutant strain, as well asthe bleaching of the cultures on N2 and the nearly background-level nitrogenase activity values, indicated that the mutantstrain SNa1 is unable to fix atmospheric N2 under aerobicconditions. But as it reduces acetylene under microaerobicconditions, the mutant strain can be classified as phenotypi-cally Fox2 Fix1 (14).

Southern analysis showed that only one copy of the trans-poson had inserted into the Anabaena sp. strain PCC7120genome within an EcoRV fragment of approximately 11 kb(not shown).

Reconstruction of the mutation in mutant SNa1. To deter-mine whether the phenotype of SNa1 was the result of inser-tion of the transposon, rather than the result of a secondarymutation, the transposon insertion was reconstructed (see ref-erence 4). Transposon Tn5-1063 (7.8 kb), together with ap-proximately 3.2 kb of contiguous Anabaena sp. strain PCC7120DNA, was recovered from mutant SNa1 upon excision withEcoRV and circularization and transfer to E. coli by electro-poration, producing plasmid pBG2002 (from which plasmidpBG2028 was constructed) (Table 2; Materials and Methods).Southern analysis of DNAs from three strains, derived frompresumptive recombination of pBG2028 with the wild-typestrain Anabaena sp. PCC7120, showed that the original muta-tion had been reconstructed (not shown). The phenotype ofthe three double recombinant strains also matched that ofmutant strain SNa1 (not shown). One such strain was desig-nated DR2028-6 and was used for subsequent in vivo geneexpression studies (see below).

Analysis of the gene interrupted by the transposon in strainSNa1. The transposon in strain SNa1 was found to interrupt anORF of 2,289 bp (not shown). The transposon had inserted1,263 bp 39 from the first ATG codon of the ORF, generatinga 9-bp repeat (59-GTACGCGTC-39). A putative ribosomebinding site (59-AGGG-39) is located 9 bp upstream of the firstATG codon. A putative prokaryotic Rho-independent termi-nator sequence (59-GAAATTTCTAAGCCTACCCCCTATTCTGAAAAATTTC-39) is located immediately following thetwo stop codons.

The Tn5-interrupted ORF encodes a predicted protein of763 amino acids with an expected molecular mass of 85.1 kDaand an expected pI of 9.17. It shows significant homology withHMW PBPs, which are involved in the synthesis of the pepti-doglycan layer of the cell wall of gram-positive and gram-negative bacteria (18).

Figure 2 shows a comparison of the predicted sequences ofthe putative PBP from Anabaena sp. strain PCC7120 and twoother sequences that produced significant alignments: PBP1Afrom E. coli, the gram-negative bacterium of reference in thestudy of PBPs, and a presumptive PBP (PBP1B) from theunicellular cyanobacterium Synechocystis sp. strain PCC6803,whose complete genome has been recently sequenced (24, 25).The PBP from Anabaena shares 18.9% identity (31.5% simi-larity) with PBP1A, the product of ponA from E. coli, and22.5% identity (37.2% similarity) with the presumptive PBP1B,putatively encoded by mrcA, from Synechocystis. Based on thewhole protein sequence, the homology, although significant, is

around 30%; however, when we consider the two functionaldomains of these proteins, a higher homology arises. (i) The Nterminus is a putative transglycosylase domain that catalyzesglycan chain elongation. In this region, the identity is 27.6%(50.3% similarity) with PBP1A of E. coli and 22.2% (41.1%similarity) with the presumptive PBP1B from Synechocystis. (ii)The C-terminal domain is the PB domain that acts as atranspeptidase and catalyzes peptidoglycan cross-linking. Inthis region, the identity is 18.6% (33.5% similarity) withPBP1A of E. coli and 27.9% (41.4% similarity) with the pre-sumptive PBP1B from Synechocystis. HMW PBPs are sub-grouped in classes A and B following the method described byGoffin and Ghuysen (19), where the amino terminus of class Acontains six conserved motifs, while the amino terminus ofclass B contains only four conserved motifs. As indicated be-low, the PBP of Anabaena sp. strain PCC7120 falls within classA of HMW PBPs.

The N-terminal domain of the PBP of Anabaena sp. strainPCC7120 contains two main and characteristic features: thehydrophobic sequence assumed to function as a membraneanchor (the 20-amino-acid region underlined in Fig. 2) (18)and the amino end core, comprising the transglycosylasenon-PB (n-PB) module, which can be defined as the sequenceextending from the amino end of motif 1 to the carboxy end ofmotif 6. In Fig. 2, within unshaded boxes, the regions compris-ing residues 90 to 99, 121 to 128, 141 to 145, 158 to 167, 224 to229, and 283 to 295 correspond to motifs 1, 2, 3, 4, 5, and 6,respectively, as described for class A HMW PBPs by Goffinand Ghuysen (19). Amino acid residues E and D in motif 1 andE in motif 3 are conserved in all class A PBPs and are consid-ered essential components of the transglycosylase activity (19).However, the core of the n-PB module of the PBP of Anabaenasp. strain PCC7120 diverges from that of the class A consensusin that the first amino acid residues of motifs 3 and 6 aredifferent (Fig. 2).

Within the C-terminal domain of the PBP of Anabaena sp.strain PCC7120, three motifs can be found that are highlyconserved in all members of the penicilloyl serine transferasesuperfamily (Fig. 2, shaded boxes). These motifs include theSXXK tetrad containing the active-site serine at residues 358to 361, the SXN triad at residues 417 to 419, and the K[T/S]Gtriad at residues 548 to 550. In the folded protein, these con-served motifs generate an active site that interacts with b-lac-tam antibiotics. The serine catalyzes the rupture of a peptidebond, forming a serine ester-linked acyl derivative. The core ofthe PB module can be defined as the sequence starting 60amino acid residues upstream from motif SXXK and termi-nating 60 to 70 amino acid residues downstream from motifK[T/S]G or at the carboxy end of PBPs which have no carboxy-terminal extensions. Such extensions are present when thesequence between motif K[T/S]G and the carboxy end of theprotein is more than about 60 to 70 amino acid residues long.This extension is usually rich in N and Q and the chargedamino acid residues D, E, K, and R. This extension in the PBPof Anabaena sp. strain PCC7120, together with the one forPBP1C of E. coli (19), is the largest of all known HMW PBPs.

During the course of our study, a preliminary sequence ofthe Anabaena sp. strain PCC7120 genome became available atthe Cyanobase website (http://www.kazusa.or.jp/cyano) in theform of unedited sequence files (contigs).


on Decem


.org/D

ownloaded from

http://jb.asm.org/

We sought putative PBPs within the Anabaena sp. sequencefiles by using BLAST searches with the amino acid sequencesof PBP1a, -1b, -1c, -2, -3, -4, -5, -6, and -7, DacD, AmpC, andAmpH of E. coli; PBP1, -2a, -2b, -2c, -2, -4 and -5 of Bacillussubtilis; presumptive PonA (sll0002), MrcA (sll1434), MrcB(slr1710), FtsI (sll1833), DacB (slr0804 and slr0646), PBP4(sll1167), and slr1924 of Synechocystis sp. strain PCC6803; andthe putative PBP of our study.

We found 12 putative Anabaena sp. PBPs, which we haveordered in Table 3 according to their predicted molecularsizes. Genes and proteins have been designated by basicallyfollowing the E. coli nomenclature for PBPs. The object pro-tein of our study is the second largest in size and accordinglyhas been designated PBP2, and the gene interrupted in SNa1has been designated pbpB. Anabaena sp. PCC7120 has at leasteight HMW PBPs (six belonging to class A and two to class B)and four low-molecular-weight (LMW) PBPs. The ORF in the

sequence file C304 is located at one of the extremes of thesequence file, and the ORF is interrupted in the N terminus.The incomplete ORF fragment shows homology with PBP4 ofE. coli and with the presumptive products of dacB genes(slr0646 and slr0804) of Synechocystis.

Table 3 also shows the position of the hydrophobic se-quences in the N terminus that are assumed to function as amembrane anchor. Only PBP9 lacks this region, and in the caseof the PBP within fragment C304, whose N terminus we havebeen unable to localize in any other sequence file, we cannotverify whether it has the transmembrane region.

Figure 3 shows the alignments of the 12 putative AnabaenaPBPs in three classes (HMW PBPs [classes A and B] andLMW PBPs), as previously described by Goffin and Ghuysen(19). Regarding the class A HMW PBPs, the nucleotide se-quence of pbpA (PBP1) possibly starts with a UUG codon thatis preceded by an evident ribosome binding site (AGGGG

FIG. 2. Similarity of PBP2 of Anabaena sp. strain PCC7120 (Ana) to PBP1A of E. coli (Eco) and to the presumptive PBP1B of Synechocystissp. strain PCC6903 (Syn). The symbols , and . indicate parts of the E. coli protein that have been omitted because they show no similarity toPBP2. Dashes indicate gaps introduced to maximize similarities. The putative transmembrane domain of PBP2 is underlined at the N terminus ofthe translated protein. The presumptive active-site serine is indicated by an asterisk. The six conserved motifs (from 1 to 6) characteristic of theN-terminal regions of multimodular class A HMW PBPs are indicated with unshaded boxes, and the three conserved motifs (from 7 to 9) at theC-terminal region are indicated with shaded boxes. The identities defining the amino acid sequence signatures of the motifs are indicated inboldface below each box (19). Consensus residues that differ from the Anabaena amino acid are italicized.


on Decem


.org/D

ownloaded from

http://jb.asm.org/

GA). PBP1 presents a carboxy-terminal extension rich in theamino acids N, Q, E, K, and R, but it is shorter than the onefound in PBP2. The n-PB module of PBP3 diverges in the firstamino acid residues of motifs 5 and 6 (Fig. 3, underlineditalics) from the class A motif 5 and motif 6 signatures. pbpD(PBP4) possibly starts with a GUG codon. The six identifiedclass A HMW PBPs of Anabaena are highly homologous toclass A PBP1a, -1b, and -1c of E. coli; to PBP1, -2c, and -4 ofB. subtilis; and to those presumptively encoded by genes ponA(sll0002), mrcA (sll1434), and mrcB (slr1710) of Synechocystis.

There are two clear class B HMW PBPs in Anabaena (Fig.3), PBP5 and PBP6. PBP6 is the only one of the eight HMWPBPs that does not present any dicarboxylic acid (D or E) im-mediately downstream from the hydrophobic sequence at theN terminus; PBP6 also diverges in the 2nd amino acid residueof motif 1 and the 11th residue of motif 4 (Fig. 3, underlineditalics) from the class B motif 1 and motif 4 signatures. The twoclass B HMW PBPs are highly homologous to PBP2 and -3 ofE. coli; PBP2a, -2b, and -3 of B. subtilis; and that presumptivelyencoded by ftsI (sll1833) of Synechocystis.

The LMW PBPs PBP9 and PBP11 (Fig. 3) are very similarto the b-lactamases AmpC and AmpH of E. coli and to the pre-sumptive products of gene pbp (sll1167) and slr1924 of Syn-echocystis. As already shown in Table 3, PBP9 does not have atransmembrane region in the N terminus. In PBP11, the thirdmotif differs from the consensus sequence K[T/G]G. PBP10 ishighly homologous to PBP4 of E. coli and to the presumptiveproducts of gene dacB (slr0804 and slr0646) of Synechocystis.

Cloning of the wild-type version of pbpB and complementa-tion of the mutation. A 2.4-kb PCR fragment of Anabaena sp.strain PCC7120 DNA was shown to contain the pbpB gene asits only ORF by sequencing (not shown). The sequence of thecloned gene was identical to that obtained from the transpo-son-mutagenized form of the fragment recovered on pBG2002(Table 2). The 2.4-kb fragment bearing the PCR-amplified wild-type pbpB was cloned in shuttle vector pRL1404 as pBG2031a.In that vector, pbpB is close to cyanobacterial replicon pDU1and is oriented to permit transcription of pbpB from pDU1.Plasmid pBG2031a was transferred by conjugation to mutantSNa1 but proved highly toxic to mutant SNa1 even when ni-trate was used as the nitrogen source; this toxic effect could be

due to a very strong promotion coming from the internal pro-moter of pDU1. In parallel, we also cloned that same fragmentin the RSF1010-based plasmid pRL1342 (C. P. Wolk, unpub-lished data), generating plasmid pBG2030, which can replicatein Anabaena sp. strain PCC7120. Plasmid pBG2030, after con-jugal transfer to mutant SNa1, successfully complemented themutation: Emr colonies form mature and functional hetero-cysts that appear in long filaments with normally shaped veg-etative cells and grow aerobically with N2 as the sole nitrogensource (not shown).

In vivo expression of pbpB. Transposon Tn5-1063 generatestranscriptional fusions between Vibrio fischeri luxA and luxBgenes, which encode luciferase, and genes into which the trans-poson becomes inserted (40), thus permitting monitoring ofgene expression in vivo, provided that the transposon is cor-rectly oriented. However, the transposon in mutant SNa1placed luxAB antiparallel to the gene pbpB (not shown). Plas-mid pBG2028 (Table 2; Materials and Methods) was con-structed in order to reconstruct the mutation placing luxABparallel to the direction of transcription of pbpB. Single recom-binant (Emr Spr Smr [sucrose sensitive] Fox1 Fix1) and doublerecombinant (Ems Spr Smr [sucrose resistant] Fox2 Fix1)strains were obtained. Single recombinant SR2028-8 and dou-ble recombinant DR2028-6 were selected for the in vivo ex-pression studies. Plasmid pRL1472a, which bears the aldehydebiosynthetic genes luxCD-E (15), was introduced by conjuga-tion into the double recombinant strain DR2028-6. The in vivoexpression of pbpB from cell suspensions of strains DR2028-6and SR2028-8 was monitored as a function of time using ni-trate or N2 as nitrogen sources (Fig. 4).

Although pbpB of Anabaena sp. strain PCC7120 is essentialfor nitrogen fixation under aerobic conditions, the pattern ofexpression of the gene in the single (Fig. 4B) and double (Fig.4A) recombinant strains is very similar, regardless of the choiceof nitrate or N2, during the first 10 h of culture. An initial four-fold induction is followed by an equally extensive decrease inexpression. The subsequent impression that, in the double re-combinant strain, the gene is transcribed more in the presencethan in the absence of nitrate could be due to the inability ofthe strain to fix N2 aerobically. Such an effect is not observedin the single recombinant strain, which is capable of aerobic N2

TABLE 3. Putative PBPs found in the genome of Anabaena sp. strain PCC7120

Anabaena sp. strainPCC7120 genome

sequence filesa

ORF (nucleotideposition in thesequence file)

Amino acidresidue no.

Molecularmass (kDa)

Suggested genedenomination

Suggested proteindenomination PBP class Position of the trans-

membrane region

C374 203934366 776 86.357 pbpA PBP1 HMW class A 69–85C372 29389327101 763 85.171 pbpB PBP2 HMW class A 15–34C376b 33383335653 756 82.742 pbpC PBP3 HMW class A 163–176C331 13567311639 643 71.054 pbpD PBP4 HMW class A 41–65C329 774535916 610 67.486 pbpE PBP5 HMW class B 24–45C259 19381317555 609 67.256 pbpF PBP6 HMW class B 48–64C331 1090339089 605 66.891 pbpG PBP7 HMW class A 7–19C353 26459324651 603 65.963 pbpH PBP8 HMW class A 7–17C365 15916314306 537 60.683 pbpI PBP9 LMW nfb

C378 60290358830 487 52.917 pbpJ PBP10 LMW 8–18C242 577334532 414 45.570 pbpK PBP11 LMW 9–20C304 31832331074 ? ?c ? ? LMW ?

a Kazusa DNA Research Institute (http://www.kazusa.or.jp/cyano).b nf, not found.c ?, unknown at present.


on Decem


.org/D

ownloaded from

http://jb.asm.org/

fixation. Therefore, PBP2 is probably present under both growthconditions but is essential only when N2 is used as the solenitrogen source and cells start to differentiate into heterocysts.

DISCUSSION

We describe for the first time the cloning and molecularcharacterization of a gene, pbpB, presumptively encoding acyanobacterial PBP. Remarkably, this protein appears to berequired specifically for aerobic nitrogen fixation. A mutationin pbpB results in no evident phenotypic difference with thewild-type strain when cells are grown aerobically with or mi-croaerobically without nitrate. However, when deprived of com-bined nitrogen under aerobic conditions, filaments become shortand yellow and are comprised of vegetative cells of unequal size,and the heterocysts that differentiate are nonfunctional.

The protein predicted by the ORF interrupted by the trans-poson in SNa1 resembles class A HMW PBPs (19). The onlyinformation available about the occurrence and number of

PBPs in cyanobacteria comes from the complete chromosomalsequence of the unicellular cyanobacterium Synechocystis sp.strain PCC6803 (24, 25) and contigs representing the genomicsequences of Anabaena sp. strain PCC7120 (http://www.kazusa.or.jp/cyano) and Nostoc punctiforme (http://spider.jgi-psf.org).Eight Synechocystis genes that presumptively encode PBPswere identified on the basis of sequence similarity. Four ofthese (ponA, mrcA, mrcB, and fts1) encode presumptive HMWPBPs, and the others (dacB [slr0804 and slr0646], pbp [sll1167],and slr1924) encode presumptive LMW PBPs. We have iden-tified 16 putative PBPs from N. punctiforme contigs; 7 corre-spond to class A HMW PBPs, 4 to class B HMW PBPs, and 5to LMW PBPs. Of 12 putative PBPs identified from Anabaenacontigs, 6 correspond to class A HMW PBPs. To our knowl-edge, Anabaena sp. strain PCC7120 and N. punctiforme inparticular show the highest number of class A HMW PBPsever reported from a single organism. No mutagenesis studyhas heretofore been undertaken, and no physiological role hasbeen ascribed to any putative cyanobacterial PBP.

FIG. 3. Amino acid sequence analysis of putative PBPs of Anabaena sp. strain PCC7120 according to the method described by Goffin andGhuysen (19). Conserved motifs (19) and amino- and carboxy-terminal extensions of putative PBPs of Anabaena sp. strain PCC7120 are shown.Asterisks at the bottom of each PBP group define the amino acid signature of the motifs. Amino acid residues that do not obey the amino acidsignature are in underlined italics. PBP2, the protein of our study, is in boldface. The intermotif distances are given as the number of amino acidresidues.


on Decem


.org/D

ownloaded from

http://jb.asm.org/

Mutant strain SNa1 was successfully reconstructed, and themutation was complemented with a 2.4-kb fragment that bearspbpB as its only ORF. The next ORF 39 from pbpB is oppo-sitely directed in the chromosome and encodes a putative pro-tein that is highly similar to a hypothetical protein of Synecho-cystis (s76621). We conclude that the mutant phenotype ofSNa1 is due to the interruption of pbpB by Tn5-1063 and notto a polar effect of that mutation.

PBPs of a given species often overlap functionally. As a re-sult, a mutation in a single PBP-encoding gene in E. coli (3, 8,22, 33, 34), B. subtilis (29–32), and Pseudomonas aeruginosa(28) often does not produce a phenotype different from that ofthe wild-type strain. In contrast, mutation of Anabaena sp.gene pbpB results in a distinctive phenotype.

The heterocyst is a specialized cell whose interior becomesmicroaerobic, permitting nitrogen fixation to take place in anaerobic environment. The development of a microaerobic in-terior entails major modifications of the original vegetative cell(41). In addition, the analysis of Fox2 mutants of Anabaena sp.strain PCC7120 defective in the synthesis of lipopolysaccharidehas provided evidence of a role for the cell wall of the devel-oping heterocyst as a determinant for heterocyst differentiation(42). In Streptomyces griseus, several distinct PBPs may be re-quired for septation during vegetative growth and sporulation(20). Perhaps during heterocyst differentiation, changes in thestructure of the cell wall similarly require the activity of dif-

ferent PBPs. Because pbpB is expressed in both the presenceand absence of nitrate, PBP2 is probably present in vegetativecells. However, during growth on nitrate, PBP2 is either dis-pensable or replaced by other PBPs. PBP2 is apparently es-sential for aerobic growth on N2. We suggest that this proteinmay be needed to create a specific structure of the peptidogly-can layer in heterocysts, perhaps by altering the number ofcross-links between glycan chains and/or the degree of elonga-tion of chains, to facilitate deposition of the additional cell walllayers needed to protect nitrogenase from entry of O2.

Because there is evidence of a relationship between thedifferentiation of heterocysts and of spore-like cells called aki-netes (26, 27, 37), we are currently trying to disrupt the corre-sponding gene of cyanobacterial strains that are capable ofboth differentiation processes. It will be of interest to deter-mine whether ORFs that we have identified as possibly encod-ing PBPs are essential for the viability or processes of cellulardifferentiation.

ACKNOWLEDGMENT

This work was supported by Direccion General de Ensenanza Su-perior PB96-0487.

REFERENCES

1. Allen, M. B., and D. I. Arnon. 1955. Studies on nitrogen-fixing blue-greenalgae. I. Growth and nitrogen fixation by Anabaena cylindrica Lemm. PlantPhysiol. (Rockville). 30:366–372.

2. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res. 25:3389–3402.

3. Baquero, M. R., M. Bouzon, J. C. Quintela, J. A. Ayala, and F. Moreno. 1996.dacD, an Escherichia coli gene encoding a novel penicillin-binding protein(PBP6b) with DD-carboxypeptidase activity. J. Bacteriol. 178:7106–7111.

4. Black, T. A., Y. Cai, and C. P. Wolk. 1993. Spatial expression and autoreg-ulation of hetR, a gene involved in the control of heterocyst development inAnabaena. Mol. Microbiol. 9:77–84.

5. Buikema, W. J., and R. Haselkorn. 1993. Molecular genetics of cyanobacte-rial development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44:33–52.

6. Cai, Y., and C. P. Wolk. 1990. Use of a conditionally lethal gene in Anabaenasp. strain PCC 7120 to select for double recombinants and to entrap insertionsequences. J. Bacteriol. 172:3138–3145.

7. Cohen, M. F., J. C. Meeks, Y. Cai, and C. P. Wolk. 1998. Transposonmutagenesis of heterocyst-forming filamentous cyanobacteria. Methods En-zymol. 297:3–17.

8. Denome, S. A., P. K. Elf, T. A. Henderson, D. E. Nelson, and K. D. Young.1999. Escherichia coli mutants lacking all possible combinations of eightpenicillin binding proteins: viability, characteristics, and implications forpeptidoglycan synthesis. J. Bacteriol. 181:3981–3993.

9. Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set ofsequence analysis programs for the VAX. Nucleic Acids Res. 12:387–395.

10. Elhai, J., and C. P. Wolk. 1988. Conjugal transfer of DNA to cyanobacteria.Methods Enzymol. 167:747–754.

11. Elhai, J., and C. P. Wolk. 1988. A versatile class of positive-selection vectorsbased on the nonviability of palindrome-containing plasmids that allowscloning into long polylinkers. Gene 68:119–138.

12. Elhai, J., and C. P. Wolk. 1990. Developmental regulation and spatial pat-tern of expression of the structural genes for nitrogenase in the cyanobac-terium Anabaena. EMBO J. 9:3379–3388.

13. Elhai, J. A., A. Vepritsky, A. M. Muro-Pastor, E. Flores, and C. P. Wolk.1997. Reduction of conjugal transfer efficiency by three restriction activitiesof Anabaena sp. strain PCC7120. J. Bacteriol. 179:1998–2005.

14. Ernst, A., T. A. Black, Y. Cai, J.-M. Panoff, D. N. Tiwari, and C. P. Wolk.1992. Synthesis of nitrogenase in mutants of the cyanobacterium Anabaenasp. strain PCC 7120 affected in heterocyst development or metabolism.J. Bacteriol. 174:6025–6032.

15. Fernandez-Pinas, F., and C. P. Wolk. 1994. Expression of luxCD-E inAnabaena sp. can replace the use of exogenous aldehyde for in vivo local-ization of transcription by luxAB. Gene 150:169–174.

16. Fernandez-Pinas, F., F. Leganes, and C. P. Wolk. 1994. A third genetic locusrequired for the formation of heterocysts in Anabaena sp. strain PCC7120.J. Bacteriol. 176:5277–5283.

17. Finnegan, F., and D. Sherratt. 1982. Plasmid ColE1 conjugal mobility: thenature of bom, a region required in cis for transfer. Mol. Gen. Genet. 185:344–351.

FIG. 4. Luciferase activity of suspensions of cells from the doublerecombinant strain DR2028-6 (A) and single recombinant strainSR2028-8 (B). Cells were washed three times in AA/8 medium plus 5mM nitrate and resuspended in the same medium (F) or were washedthree times and resuspended in AA/8 medium lacking nitrate (E).Samples were taken and their luminescence was measured at the timesindicated. chl, chlorophyll.


on Decem


.org/D

ownloaded from

http://jb.asm.org/

18. Ghuysen, J. M. 1991. Serine b-lactamases and penicillin-binding proteins.Annu. Rev. Microbiol. 45:37–67.

19. Goffin, C., and J. M. Ghuysen. 1998. Multimodular penicillin-binding pro-teins: an enigmatic family of orthologs and paralogs. Microbiol. Mol. Biol.Rev. 62:1079–1093.

20. Hao, J., and K. E. Kendrick. 1998. Visualization of penicillin-binding pro-teins during sporulation of Streptomyces griseus. J. Bacteriol. 180:2125–2132.

21. Hastings, J. W., and G. Weber. 1963. Total quantum flux of isotropic sources.J. Opt. Soc. Am. 53:1410–1415.

22. Henderson, T. A., K. D. Young, S. A. Denome, and P. K. Elf. 1997. AmpC andAmpH, proteins related to the class C b-lactamases, bind penicillin andcontribute to the normal morphology of Escherichia coli. J. Bacteriol. 179:6112–6121.

23. Hu, N.-T., T. Thiel, T. H. Giddings, and C. P. Wolk. 1982. New Anabaena andNostoc cyanophages from sewage settling ponds. Virology 114:236–246.

24. Kaneko, T., S. Sato, H. Kotani, A. Tanaka, E. Asamizu, Y. Nakamura, N.Miyajima, M. Hirosawa, M. Sugiura, S. Sasamoto, T. Kimura, T. Hosouchi,A. Matsuno, A. Muraki, N. Nakazaki, K. Naruo, S. Okumura, S. Shimpo, C.Takeuchi, T. Wada, A. Watanabe, M. Yamada, M. Yasuda, and S. Tabata.1996. Sequence analysis of the genome of the unicellular cyanobacteriumSynechocystis sp. strain PCC6803. II. Sequence determination of the entiregenome and assignment of potential protein-coding regions. DNA Res. 3:109–136.

25. Kaneko, T., S. Sato, H. Kotani, A. Tanaka, E. Asamizu, Y. Nakamura, N.Miyajima, M. Hirosawa, M. Sugiura, S. Sasamoto, T. Kimura, T. Hosouchi,A. Matsuno, A. Muraki, N. Nakazaki, K. Naruo, S. Okumura, S. Shimpo, C.Takeuchi, T. Wada, A. Watanabe, M. Yamada, M. Yasuda, and S. Tabata.1996. Sequence analysis of the genome of the unicellular cyanobacteriumSynechocystis sp. strain PCC6803. II. Sequence determination of the entiregenome and assignment of potential protein-coding regions (supplement).DNA Res. 3:185–209.

26. Leganes, F. 1994. Genetic evidence that hepA gene is involved in the normaldeposition of the envelope of both heterocysts and akinetes in Anabaenavariabilis ATCC 29413. FEMS Microbiol. Lett. 123:63–68.

27. Leganes, F., F. Fernandez-Pinas, and C. P. Wolk. 1994. Two mutations thatblock heterocyst differentiation have different effects on akinete differenti-ation in Nostoc ellipsosporum. Mol. Microbiol. 12:679–684.

28. Liao, X., and R. E. W. Hancock. 1997. Identification of a penicillin-bindingprotein 3 homolog, PBP3x, in Pseudomonas aeruginosa: gene cloning andgrowth phase-dependent expression. J. Bacteriol. 179:1490–1496.

29. Popham, D. L., and P. Setlow. 1993. Cloning, nucleotide sequence, andregulation of the Bacillus subtilis pbpF gene, which codes for a putative class Ahigh-molecular-weight penicillin-binding protein. J. Bacteriol. 175:4870–4876.

30. Popham, D. L., and P. Setlow. 1994. Cloning, nucleotide sequence, mutagen-esis, and mapping of the Bacillus subtilis pbpD gene, which codes for peni-cillin-binding protein 4. J. Bacteriol. 176:7197–7205.

31. Popham, D. L., and P. Setlow. 1995. Cloning, nucleotide sequence, andmutagenesis of the Bacillus subtilis ponA operon, which codes for penicillin-binding protein (PBP) 1 and a PBP-related factor. J. Bacteriol. 177:326–335.

32. Popham, D. L., and P. Setlow. 1996. Phenotypes of Bacillus subtilis mutantslacking multiple class A high-molecular-weight penicillin-binding proteins. J.Bacteriol. 178:2079–2085.

33. Spratt, B. G. 1977. Properties of the penicillin-binding proteins of Esche-richia coli K12. Eur. J. Biochem. 72:341–352.

34. Spratt, B. G., and K. D. Cromie. 1988. Penicillin-binding proteins of gram-negative bacteria. Rev. Infect. Dis. 10:699–711.

35. Stewart, W. D. P., G. P. Fitzgerald, and R. H. Burris. 1967. Acetylenereduction by nitrogen-fixing blue-green algae. Arch. Mikrobiol. 62:336–348.

36. Wolk, C. P. 1982. Heterocysts, p. 359–386. In N. G. Carr and B. A. Whitton(ed.), The biology of cyanobacteria. Blackwell Scientific Publications, Ox-ford, United Kingdom.

37. Wolk, C. P. 1996. Heterocyst formation. Annu. Rev. Gen. 30:59–78.38. Wolk, C. P., and M. P. Quine. 1975. Formation of one-dimensional patterns

by stochastic processes and by filamentous blue-green algae. Dev. Biol. 46:370–382.

39. Wolk, C. P., A. Vonshak, P. Kehoe, and J. Elhai. 1984. Construction ofshuttle vectors capable of conjugative transfer from Escherichia coli to ni-trogen-fixing filamentous cyanobacteria. Proc. Natl. Acad. Sci. USA 81:1561–1565.

40. Wolk, C. P., Y. Cai, and J.-M. Panoff. 1991. Use of a transposon withluciferase as a reporter to identify environmentally responsive genes in acyanobacterium. Proc. Natl. Acad. Sci. USA 88:5355–5359.

41. Wolk, C. P., A. Ernst, and J. Elhai. 1994. Heterocyst metabolism and devel-opment, p. 769–823. In D. Bryant (ed.), Molecular biology of the cyanobac-teria. Kluwer Academic Publications, Dordrecht, The Netherlands.

42. Xu, X., I. Khudyakov, and C. P. Wolk. 1997. Lipopolysaccharide dependenceof cyanophage sensitivity and aerobic nitrogen fixation in Anabaena sp. strainPCC7120. J. Bacteriol. 179:2884–2891.

43. Yoon, H. S., and J. W. Golden. 1998. Heterocyst pattern formation controlledby a diffusible peptide. Science 282:935–938.


on Decem


.org/D

ownloaded from

http://jb.asm.org/

pbpb, a gene coding for a putative penicillin-binding ... · southern analysis of chromosomal dna...

Documents