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J Bacteriol. Feb 2011; 193(4): 896–908.
Published online Dec 17, 2010. doi:  10.1128/JB.00594-10
PMCID: PMC3028666

The Putative Hydrolase YycJ (WalJ) Affects the Coordination of Cell Division with DNA Replication in Bacillus subtilis and May Play a Conserved Role in Cell Wall Metabolism [down-pointing small open triangle]

Abstract

Bacteria must accurately replicate and segregate their genetic information to ensure the production of viable daughter cells. The high fidelity of chromosome partitioning is achieved through mechanisms that coordinate cell division with DNA replication. We report that YycJ (WalJ), a predicted member of the metallo-β-lactamase superfamily found in most low-G+C Gram-positive bacteria, contributes to the fidelity of cell division in Bacillus subtilis. B. subtilis ΔwalJwalJBsu) mutants divide over unsegregated chromosomes more frequently than wild-type cells, and this phenotype is exacerbated when DNA replication is inhibited. Two lines of evidence suggest that WalJBsu and its ortholog in the Gram-positive pathogen Streptococcus pneumoniae, WalJSpn (VicX), play a role in cell wall metabolism: (i) strains of B. subtilis and S. pneumoniae lacking walJ exhibit increased sensitivity to a narrow spectrum of cephalosporin antibiotics, and (ii) reducing the expression of a two-component system that regulates genes involved in cell wall metabolism, WalRK (YycFG), renders walJ essential for growth in B. subtilis, as observed previously with S. pneumoniae. Together, these results suggest that the enzymatic activity of WalJ directly or indirectly affects cell wall metabolism and is required for accurate coordination of cell division with DNA replication.

All cells are faced with the fundamental challenge of duplicating their genetic information and accurately partitioning a complete copy into each new daughter cell. To ensure that the chromosome is stably transmitted with every cell division, all stages of the cell cycle—including DNA replication, cell growth, and cell division—must be carefully coordinated. Bacteria maintain the fidelity of cell division through the combined activity of multiple regulatory pathways. For example, a number of mechanisms function to ensure proper localization of the cytokinetic Z-ring and control the assembly and activity of the division machinery at the septum (1, 3, 24, 29, 46). During the late stages of cell division in Bacillus subtilis, a DNA translocase (SftA) helps ensure that chromosomes are fully segregated into daughter cells before septation is completed (8, 36). Failure to properly coordinate the stages of the cell cycle can lead to aberrant division events where the septum forms over the chromosome and results in at least one daughter cell lacking a complete copy of the genome. B. subtilis can recover from these “nucleoid bisection” events through the activity of SpoIIIE, a membrane-bound DNA translocase (64). Although many bacterial cell cycle regulators have been described, our understanding of how the cell cycle is coordinated remains incomplete.

Central to the process of cell growth and division is the formation and assembly of new cell wall material along the length of the cell and at the division septum. The Gram-positive cell wall is a dynamic structure made of a thick (~50-nm) layer of cross-linked peptidoglycan (murein) cables that wrap around the cell membrane (33). B. subtilis cells expand via the integration of cell wall material along the length of the cell. New peptidoglycan is integrated in a helical pattern, which is dependent on the association of the peptidoglycan synthetic machinery with three actin-like proteins: MreB, Mbl, and MreBH (19, 37, 38). During a defined time period prior to division, extensive peptidoglycan synthesis is also observed at the division site as the division septum is being formed between the new daughter cells. The enzyme complexes responsible for division site synthesis and cell elongation are distinct and contain different enzymes and regulatory proteins (17, 73, 79).

Cell wall elongation and cell division are regulated by mechanisms controlling the expression and localization of the peptidoglycan biosynthetic machinery. In B. subtilis, one signaling pathway regulating cell wall metabolism is the essential WalR/WalK (YycF/YycG) two-component signaling system (TCS) (9, 10, 23, 34). WalK is a histidine sensor kinase that, when stimulated, phosphorylates the WalR response regulator; phosphorylated WalR in turn can function as a transcriptional regulator. The WalRK TCS modulates the expression of at least 10 genes with putative roles in cell wall metabolism, including hydrolases and peptidoglycan deacetylases (10, 22, 23). WalK is active only when localized to the division septum, where it associates with the FtsZ ring (27). The nature of the signal sensed by WalK is unknown, but it has been hypothesized to respond to levels of lipid II (a key intermediate in peptidoglycan synthesis) or other signals within the cell membrane or at the septum (10, 22, 27, 68). In Streptococcus mutans, the orthologous WalK kinase has been reported to respond to oxidative stress (20, 22).

walRK orthologs are found in most low-G+C Gram-positive bacteria (22, 25). WalRK is the only essential TCS in B. subtilis under standard laboratory growth conditions (25, 42). It is generally thought that cells require the expression of several WalRK-dependent genes for viability (10, 22). Similarly, the essentiality of the walRK TCS in Streptococcus pneumoniae, walRKSpn (vicRK), is due to its role in regulating expression of the pcsB cell division gene (4, 54). In B. subtilis, walRK is coexpressed with four other genes: yycH and yycI, which regulate the activity of WalK; yycJ (walJBsu), a putative hydrolase belonging to the metallo-β-lactamase superfamily; and yycK, an HtrA-like serine protease (25). While many low-G+C Gram-positive genomes lack yycH, yycI, or yycK orthologs, a walJ (yycJ) ortholog is always found downstream of walRK (22).

The cellular role of WalJ has not been well established. In B. subtilis, only mild phenotypes, including an increased frequency of colonies that could not sporulate and a change in colony morphology, have been reported for ΔyycJ mutants (68). A role for WalJ in cell wall metabolism has been postulated based on the finding that the walJ ortholog in Streptococcus pneumoniae, walJSpn (vicX), is essential when expression of the walRK two-component system is limiting (54). Additional support for this hypothesis comes from the strong cooccurrence of walJ and walRK and the predicted hydrolase activity of the protein (22). In Streptococcus mutans, a mutation in vicX has been reported to affect several phenotypes, including competence and biofilm formation (63).

The metallo-β-lactamase superfamily to which walJ is predicted to belong is a large and diverse family of hydrolases that are found in bacteria, archaea, and eukaryotes. One branch of the superfamily includes the well-studied bacterial enzymes that confer resistance to β-lactam antibiotics by cleaving the amide bond of the β-lactam ring (71). Other members of the enzyme superfamily act on diverse substrates and participate in a variety of cellular processes, including RNA processing, DNA repair, and V(D)J recombination (5, 18, 21). These enzymes share common sequence domains and a highly conserved metal-binding motif (HXHXDH) at the active site. Most metallo-β-lactamase superfamily enzymes utilize two zinc ions at the catalytic center, although some are known to use iron instead (2, 21).

Here we report that WalJBsu affects the coordination of cell division with DNA replication in B. subtilis. ΔwalJBsu mutants divide over unsegregated chromosomes more frequently than wild-type cells, and this phenotype is exacerbated when DNA replication is perturbed. Our data suggest that WalJBsu plays a role in cell wall metabolism, since ΔwalJBsu cells are more sensitive to certain cephalosporin antibiotics and WalJBsu is required for normal growth when walRK-dependent expression is limited. The cellular role of walJBsu orthologs may be at least partially conserved within the low-G+C Gram-positive bacteria, as the antibiotic sensitivity phenotype is also observed in Streptococcus pneumoniae. These results support the hypothesis, previously described in the literature (47), that cell wall metabolism can affect the regulation of cell division in B. subtilis.

MATERIALS AND METHODS

Media.

B. subtilis strains were grown in LB medium (48), Difco nutrient sporulation medium, CH and SM media (32), GMD medium (31), or S750-trace defined minimal medium (8). S. pneumoniae strains were cultured statically in BD brain heart infusion (BHI) broth at 37°C in an atmosphere of 5% CO2 or on plates containing TSA II medium plus 5% (wt/vol) sheep blood (BD) (59, 70).

Strains, plasmids, and oligonucleotides.

The strains used in this study are listed in Table Table1,1, and plasmids and oligonucleotides used in this study are listed in Tables S1 and S2 in the supplemental material. Unless otherwise noted, Bacillus subtilis strains used in the experiments are derivatives of strain JH642 and have the common genotype trpC2 pheA1 (14). In the strain genotypes, a double colon followed by a plasmid name indicates integration by single crossover and a double colon followed directly by parentheses indicates integration by double crossover. Antibiotic resistance markers were selected using the drug concentrations indicated as follows: amp, 100 μg ml−1 ampicillin; cat, 5 μg ml−1 chloramphenicol; erm, 0.5 μg ml−1 erythromycin and 12.5 μg ml−1 lincomycin; kan, 5 μg ml−1 kanamycin; spc, 100 μg ml−1 spectinomycin; tet, 12.5 μg ml−1 tetracycline.

TABLE 1.
Strains used in this studya

Routine B. subtilis transformations were performed by preparing competent cells essentially as described by Msadek et al. (51), with GE medium replaced with MD medium (100 mM potassium phosphate, pH 7.4, 4 mM trisodium citrate, 2% [wt/vol] glucose, 11 mg liter−1 ferric ammonium citrate, 0.25% [wt/vol] potassium aspartate, 3 mM MgSO4, 50 μg ml−1 l-tryptophan, and 50 μg ml−1 l-phenylalanine). To transform strains containing the dnaB19 allele, the method of Bott and Wilson (13) was used to increase transformation efficiency.

PCR.

Unless otherwise noted, genomic DNA from B. subtilis strain JH642 was used as the template for PCRs.

Plasmid construction.

We note that the genome of our laboratory strain of B. subtilis, JH642, was recently resequenced (GenBank accession no. ABQM00000000) (66), and the C terminus of walJBsu was found to differ by a single base pair from the original published B. subtilis genome sequence (44). This difference changes the originally published sequence of the WalJBsu C terminus from WLYNFSM to CAV in JH642.

The PtagC-lacZ transcriptional fusion was constructed as follows. The tagC promoter region was amplified using oligonucleotides OBB167 and OBB168. The PCR product was digested with EcoRI and BamHI and ligated into pDG1663 digested with the same enzymes, resulting in pSB220.

To overexpress spoIIIE36 from the Pspac-hy promoter at the thrC locus, we digested plasmid pSB38 (8) with EcoRI and BamHI. The plasmid fragment was gel extracted and ligated into plasmid pDG1664 digested with the same enzymes, yielding pSB223.

To complement the region from walR to walJBsu, we placed a heterologous copy of this region, under the control of its native promoter, at the amyE locus. This genomic region was amplified with oligonucleotides OSB195 and OSB196. This PCR product was digested with SalI and SphI and ligated into pDG1662 digested with the same enzymes to yield plasmid pSB126. To complement walJBsu from the native operon promoter, we deleted walR, walK, yycH, and yycI from plasmid pSB126 by PCR. Oligonucleotides OSB197 and OSB199 were used to carry out a long-distance inverse PCR using pSB126 as the template. This PCR product was digested with NotI and ligated to itself to yield pSB128. The walJBsu H60A mutation was introduced into the walJBsu open reading frame (ORF) of plasmid pSB128 using the QuikChange mutagenesis kit (Stratagene) and primers OSB305 and OSB306, yielding pSB300.

To overexpress walJBsu, we amplified walJBsu using oligonucleotides OSB209 and OSB210. This PCR product was digested with XbaI and BglII and ligated into pDR67 that had been digested with the same enzymes, yielding pSB135. Overexpression of a proteolytically stabilized walJBsuDD mutant (described below) was done similarly, using oligonucleotides OSB209 and OSB251, yielding pSB141.

B. subtilis deletion strains.

The Δnoc::kan and ΔyneA::tet alleles have been described previously (8). walJBsu was deleted in two different ways. To inactivate walJBsu by single crossover, oligonucleotides OSB167 and OSB168 were first used to amplify a region within the walJBsu ORF. The PCR product was digested with EcoRI and BamHI and ligated into pUS19 digested with the same enzymes, yielding plasmid pSB109. We also constructed a mutant with a deletion of the walJBsu ORF linked to a downstream spc marker; this was done to avoid the possibility that the single-crossover deletion construct could recombine back out of the genome and to minimize any impact of a linked marker on the expression of yycK. This construct was generated in several steps. Oligonucleotides OSB215 and OSB216 were used to amplify a region downstream of the transcriptional terminator after yycK (in the rocR gene region). The PCR product was digested with AatII and NdeI and ligated into pUS19 digested with the same enzymes, yielding plasmid pSB142 (a cloning intermediate not listed in Tables Tables1).1). Next, the region extending from the yycK terminator to the last 50 bp of walJBsu was amplified with oligonucleotides OSB217 and OSB218. This product was digested with XmaI and BamHI and ligated into pSB142 digested with the same enzymes, resulting in pSB159 (another cloning intermediate not listed in Tables Tables1).1). Oligonucleotides OSB195 and OSB219 were used to amplify the region from the first 50 bp of walJBsu (introducing an early stop codon into the ORF) to the beginning of the walR operon. This PCR product was digested with SalI and BamHI and ligated into pSB159 digested with the same enzymes, yielding plasmid pSB387. We did not observe any differences between the behaviors of the single-crossover mutant and the full deletion strain.

Construction of S. pneumoniae strains.

S. pneumoniae strains were constructed by the Janus method of allele replacement used previously (59, 67, 70). All amplicons were constructed by fusion PCR. A ΔwalKSpn::[Kanr-RpsL+] amplicon was transformed into strain IU1781 (D39 rpsL1, resistant to 150 μg ml−1 streptomycin), resulting in strain IU1885 that is resistant to 250 μg ml−1 kanamycin and sensitive to streptomycin. A markerless amplicon containing a deletion of walJSpn was made using oligonucleotide pairs SR027-SR033 and SR034-SR028 (S. pneumoniae strain D39 template). This was introduced into strain IU1885, resulting in a ΔwalJSpn strain (IU3176) that is resistant to streptomycin and sensitive to kanamycin. The IU3176 strain contains the first 18 nucleotides (nt) and the last 24 nt of the walJSpn ORF. To construct the ΔwalJSpn complementation strain (IU4321), a CEP::Pc[Kanr-RpsL+] amplicon was introduced into strain IU3176, resulting in IU4240 (an intermediate strain not listed in Table Table1)1) that is sensitive to streptomycin and resistant to kanamycin. The CEP::Pfcsk-walJSpn construct was made using oligonucleotides KW116 to KW158 (D39 template, yielding amiF′-spr1702-Pfcsk), KW159 to KW121 (IU3130 template, yielding the walJSpn ORF plus walJSpn terminator plus 26 bp of relB, included for cloning purposes), and KW122-KW123 (D39 template, yielding treR). This construct was transformed into IU4240, resulting in strain IU4321, which is kanamycin sensitive and streptomycin resistant. The CEP locus was previously determined to be suitable for ectopic expression in S. pneumoniae (30).

Microscopy.

Samples were taken for fluorescence microscopy (0.5 to 1.0 ml) at the times indicated in the figures. Cells were pelleted by centrifugation for 1 min at 2,500 × g and resuspended in a small amount (~100 μl) of the culture medium. Cell membranes were visualized using the fluorescent dye FM4-64 (Invitrogen; 5-μg ml−1 final concentration), and DNA was visualized using DAPI (4′,6-diamidino-2-phenylindole; 1 μg ml−1). Samples were immobilized on either 1% agarose pads or on coverslips treated with 0.1% poly-l-lysine solution (Sigma) and visualized using a Zeiss AxioImager M1 microscope fitted with an Orca-ER charge-coupled device (CCD) camera (Hamamatsu). The following Zeiss filter sets were used: 43 (FM4-64), 44 (green fluorescent protein [GFP]), 46 (yellow fluorescent protein [YFP]), 47 (cyan fluorescent protein [CFP]), and 49 (DAPI). Images were collected and processed using Openlab 5 (Improvision).

Fluorescent fusion proteins.

The spoIIIE36-gfp and PtagC-yfp constructs have been described previously (8), as have the GFP-PBP1 and GFP-PBP2b constructs (61). The inducible N-terminal YFP-WalJBsu translational fusion was constructed in two steps. First, we amplified YFP (Venus) from pMR123(8) using oligonucleotides OSB516 and OSB517, which placed the native walJBsu ribosome binding site (RBS) in front of YFP. This PCR product was digested with HindIII and BamHI and ligated into pDR67 that had been cut with HindIII and BglII; this yielded plasmid pSB401 (a cloning intermediate not listed in Table S1 in the supplemental material). We then amplified the walJBsu gene using OSB514 and OSB515, digested the PCR product with NotI and NheI, and ligated this into pSB401 digested with the same enzymes to yield pSB403. To make the C-terminally stabilized YFP-WalJBsuDD allele, we changed, using the QuikChange mutagenesis kit (Stratagene), the last two codons of the walJBsu ORF of pSB403 to encode amino acids DD with primers OSB529 and OSB530, yielding pSB408. To introduce the metal-binding pocket mutation H60A into walJBsuDD, pSB408 was mutagenized by QuikChange using primers OSB305 and OSB306, yielding pSB409. The constitutively expressed yfp construct was constructed in two steps. First, we amplified YFP (Venus) using oligonucleotides OSB46 and OMR92, digested the PCR product with SacI and SacII, and ligated this into pALMO3 that had been cut with the same enzymes; this yielded plasmid pSB18 (a cloning intermediate not listed in Table S1 in the supplemental material). The constitutive Pspac promoter, lacking Lac repressor operator sites, was placed in front of yfp by annealing primers OEN10 and OEN11 and ligating these into pSB18 that had been cut with EcoRI and HindIII, yielding pSB22.

Spore preparation and germination.

Spores were prepared as described previously (8, 31). Briefly, cells were spread on potato dextrose agar plates and incubated for several days to obtain spores. The spores were resuspended in sterile water, treated with 1 mg ml−1 lysozyme (Sigma) for 1 h at 37°C, and shaken with 2% sodium dodecyl sulfate (SDS) for 30 min. Spores were then repeatedly washed with sterile distilled water (pelleting spores by centrifugation at 4,500 × g for 5 min) until the supernatant remained clear. Spore preparations were inspected by microscopy to ensure that phase-bright spores comprised at least 90% of the visible particles. Spores were stored in sterile distilled water at 4°C.

To germinate spores, purified spores were resuspended in 10 ml sterile deionized water to a final optical density at 600 nm (OD600) of 1. Spores were incubated at 80°C for 45 min and then allowed to cool for 10 min at room temperature. The spores were centrifuged at 4,500 × g for 10 min, resuspended in an equal volume of prewarmed GMD medium, and incubated at 37°C with shaking (the 0-min time point). When indicated, HPUra [6-(p-hydroxyphenylazo)-uracil; 50-μg ml−1 final concentration] was added to block replication elongation 50 min after the spores had been resuspended in GMD medium, prior to the initiation of DNA replication. Samples were collected at the indicated times, and spores were visualized.

β-Galactosidase assays.

β-Galactosidase specific activity [(ΔA420 per min per ml of culture per OD600) × 1,000] was determined as described previously (48) after the cell debris was pelleted.

Lipid isolation and analysis.

Five replicate cultures of each strain were grown in S750-trace medium at 37°C to an OD600 of ~0.4 and then shifted to 45°C for 1 h to block replication initiation in strains containing the dnaB19 mutation. Forty milliliters of cells was spun down (5,000 × g, 5 min at 4°C) and frozen at −80°C until extracted. Lipids were isolated as described previously (11), and the chloroform used included 0.01% butylated hydroxytoluene. Lipid analysis and data analysis were performed by the Kansas Lipidomics Research Center (Kansas State University, Manhattan, KS).

Phylogenetic analysis.

To identify putative orthologs of WalJBsu, we manually examined the phylogenetic tree for the metallo-β-lactamase superfamily domain from Pfam (PF00753, 4,948 sequences; June 2007). B. subtilis has 17 genes encoding members of the superfamily, including WalJBsu. We selected sequences belonging to the largest “branch” of the tree that included B. subtilis WalJBsu but did not include the other 16 paralogs. Out of 133 sequences, a core group of 115 (see Tables S4 in the supplemental material) shared a conserved motif near the N terminus of the protein that was unique within the 4,948 sequences for the metallo-β-lactamase superfamily [(L/I)XSGSXGN].

RESULTS

ΔwalJBsu cells divide over unsegregated chromosomes more frequently than wild-type cells.

We previously described a genetic approach for identifying genes that are required to prevent cells from dividing over unsegregated chromosomes when DNA replication is perturbed (8). In Bacillus subtilis, the SpoIIIE DNA translocase assembles around chromosomal DNA trapped in a division septum and pumps the chromosome directionally into one of the daughter cells (6, 58, 77). SpoIIIE is not essential for growth, and we reasoned that mutants that divided over unsegregated chromosomes at high frequencies would lose viability unless SpoIIIE was available to complete chromosome segregation postseptationally. We therefore designed an agar plate-based screen to identify mutants that required SpoIIIE for viability after the initiation of DNA replication was transiently inhibited several times (8). As a proof of principle, we found that cells lacking the nucleoid occlusion protein Noc, which divide over unsegregated chromosomes more frequently than wild-type cells when DNA replication is blocked (77), required functional SpoIIIE for growth on agar plates only when DNA replication was repeatedly inhibited (8). One gene identified in the screen, sftA, encodes a paralog of SpoIIIE that appears to play a specialized role in promoting the efficient completion of chromosome segregation when cells divide (8, 36, 74).

As a complement to our genetic screen, we took a bioinformatic approach to identify Noc-like proteins based on the hypothesis that there could be physical or genetic interactions between Noc and other proteins involved in preventing aberrant cell divisions. We searched the Stanford Network Browser (http://networks.stanford.edu), a database for inferring functional interactions between bacterial genes based on the coevolution of conserved domains, cooccurrence within sequenced genomes, gene proximity, and a limited number of data sets from whole-genome expression analysis (26, 65).

Among the genes identified as having a potential interaction with Noc using the Network Browser database were yycH and yycI, encoding proteins that control the activity of the essential WalRK (YycFG) two-component system in B. subtilis (68, 69). We first asked if inactivating yycH or yycI conferred a noc-like phenotype in the agar plate assay used for our genetic screen. As previously described (8), SpoIIIE was conditionally inactivated by overexpressing a SpoIIIE mutant, SpoIIIE36, which assembles around DNA trapped in the membrane but has little or no DNA translocase activity (76, 77, 78). We found that overexpressing SpoIIIE36 specifically inhibits the activity of endogenous SpoIIIE (8). The strain used for screening, SB288, also carries a temperature-sensitive allele of dnaB, encoding an essential component of the helicase loader complex, and a mutant allele of flgM, which reduces cell chaining and improves the reproducibility of the plate assay (8). Replicaton initiation was repeatedly blocked by cycling agar plates between permissive and nonpermissive growth temperatures, providing several opportunities for cells to initiate DNA replication and subsequently divide over unsegregated chromosomes. As we predicted, cells lacking the nucleoid occlusion protein Noc, which divide over chromosomes at high frequencies when DNA replication is blocked, did not form patches on agar plates when SpoIIIE was inactivated and DNA replication was repeatedly inhibited but did grow when SpoIIIE was functional and DNA replication was perturbed and when SpoIIIE was inactivated in the absence of replication stress (8). The Noc+ parent strain grew and formed patches under all conditions.

We first asked if inactivating yycH or yycI conferred a noc-like phenotype in the agar plate assay used for our genetic screen (8). We inactivated yycH and yycI by integrating plasmids by single crossover into their respective ORFs and found that both mutant strains required SpoIIIE for viability when DNA replication was repeatedly inhibited, similar to noc mutants (data not shown). However, complementation analysis revealed that the phenotype was due to a polar effect on the downstream walJBsu gene rather than to disruption of yycH or yycI (data not shown). Disrupting walJBsu was sufficient to confer a synthetic growth defect when SpoIIIE was inactivated and the initiation of DNA replication was transiently inhibited several times (Fig. (Fig.1),1), and expressing walJBsu from a heterologous locus complemented the growth phenotype (Fig. (Fig.1).1). Disrupting the last gene in the operon downstream of walJBsu, yycK, had no effect (data not shown).

FIG. 1.
Strains lacking noc or walJBsu require functional SpoIIIE for viability when replication initiation is repeatedly inhibited. Fresh single colonies were patched onto two LB plates, one of which contained 1 mM IPTG to induce expression of the nonfunctional ...

Since the walJBsu mutants behaved like noc mutants in the plate assay, we next examined whether walJBsu mutants exhibited increased frequencies of nucleoid bisection by using fluorescence microscopy (Fig. (Fig.2).2). The strains used in this assay were flgM+ and expressed a SpoIIIE36-GFP translational fusion from the endogenous spoIIIE locus, which both prevented the translocation of trapped chromosomes during the replication block and aided in the identification of sites where chromosomal DNA had been trapped. A nucleoid was scored as bisected if one of the following criteria were met: (i) a division septum (visualized with the membrane dye FM4-64) clearly overlapped with both DAPI-stained DNA and a focus of SpoIIIE36-GFP; (ii) in the absence of a focus of SpoIIIE36-GFP, the DAPI-stained DNA was unambiguously compact and clearly seen on both sides of a flat septum (the morphology after cytokinesis and before autolysin-dependent cell separation begins); and (iii) the DAPI signal spanned the rounded ends of two cells that had begun to separate (a SpoIIIE36-GFP signal was occasionally present in these cases).

FIG. 2.
walJBsu mutants divide over chromosomal DNA at higher frequencies than wild-type cells. (A) Cell division and nucleoid bisection were monitored by fluorescence microscopy. Two cultures for each strain were grown in LB medium at 32°C until early ...

We measured the effect of deleting walJBsu on nucleoid bisection under multiple growth conditions. When replication initiation was inhibited in the dnaB19 strains, the nucleoids tended to become compact and we observed a significant increase in the frequency of nucleoid bisections in the ΔwalJBsu mutant compared to that of a walJBsu+ strain (Fig. (Fig.2B).2B). This bisection phenotype was complemented by heterologous expression of walJBsu from its native promoter. The frequency of nucleoid bisections in a ΔyycK mutant was not significantly different from that of the wild type (data not shown), confirming that the observed phenotype was due to the deletion in walJBsu. In comparison, the frequency of nucleotide bisection was increased ~3- to 4-fold in the Δnoc strain. walJBsu does not appear to function in the same linear pathway as noc, as the bisection frequency of the Δnoc ΔwalJBsu mutant was more severe than for either single mutant (Fig. (Fig.2B;2B; 95% confidence intervals do not overlap).

The nucleoid bisection phenotype of ΔwalJBsu mutants could also be observed in the absence of any specific disruption to DNA replication. There was a significant increase in the nucleoid bisection frequency of a ΔwalJBsu mutant in a dnaB+ background (~3-fold higher in the dnaB+ spoIIIE36-gfp ΔwalJBsu strain than in the dnaB+ spoIIIE36-gfp walJBsu+ strain) (Fig. (Fig.2B).2B). This phenotype could again be complemented by heterologously expressing a second copy of walJBsu. These data indicate that the nucleoid bisection phenotype of a ΔwalJBsu mutant is not dependent on replication stress but is instead exacerbated by it.

WalJBsu affects the regulation of cell division.

To clarify whether the increased nucleoid bisection frequencies observed in the ΔwalJBsu mutant were due to changes in the timing or frequency of cell division, we monitored synchronously growing populations of cells in a time course assay. Relatively synchronous populations of cells were obtained by germinating spores as previously described (31; E. Harry, personal communication). Heat-activated spores were induced to germinate by diluting them into germination medium, and the outgrowth of cells was observed by fluorescence microscopy. Under the conditions used, most cells initiate the first round of DNA replication approximately 75 to 90 min after germination and subsequently divide approximately 130 to 145 min after germination. In the absence of a replication block, outgrowing spores of the ΔwalJBsu mutant divided slightly more frequently than wild-type cells at 130 min, but by 145 min, cells of both strains had divided at similar frequencies (Fig. (Fig.3A).3A). When DNA replication was blocked using the drug HPUra, which inhibits replication elongation by trapping B. subtilis DNA polymerase IIIC (PolC) in a stable ternary complex (16, 28), cell division was largely inhibited in wild-type cells. Fewer than 3% of wild-type cells divided by 4 h after germination (Fig. (Fig.3B).3B). In contrast, cells lacking walJBsu divided at significantly higher frequencies than treated wild-type cells (~4-fold) (Fig. (Fig.3B)3B) but with delayed timing compared to untreated cels (Fig. 3A and B). Among the cells that had divided during HPUra treatment, 13% of the walJBsu mutant cells formed a division septum that appeared to colocalize with chromosomal DNA, which may reflect nucleoid bisection events, whereas division septa were observed only in nucleoid-free areas of wild-type cells. (Nucleoid bisection could not be scored with certainty using the localization of SpoIIIE36-GFP as a marker, since functional SpoIIIE is required for sporulation.) These data suggest that WalJBsu is required for cells to delay cell division when DNA replication is blocked and that the effect is on division site utilization rather than the overall timing of cell division.

FIG. 3.
The frequency of cell division is affected in walJBsu mutants. (A) Fraction of outgrowing wild-type or walJBsu mutant spores that have divided at least once by the indicated times after spores were induced to germinate by resuspension in GMD medium at ...

SOS induction is observed in a subpopulation of cells lacking walJBsu.

We wondered whether the nucleoid bisection events occurring in ΔwalJBsu mutants would result in induction of the SOS response. We first measured the SOS response using a transcriptional fusion of the lacZ gene to the LexA-repressed tagC (dinC) promoter. The transcriptional fusion was integrated by double crossover at the amyE locus and was therefore unaffected by transcription of the WalRK-regulated tagAB genes. The ΔwalJBsu cells had ~5-fold higher SOS induction than wild-type cells (Fig. (Fig.4A),4A), and expressing the native walJBsu from a heterologous locus complemented this phenotype. The level of LacZ activity was significantly lower than that observed when cells are exposed to a DNA-damaging agent such as mitomycin C, suggesting that either all cells have a very low level of SOS induction or the response is strongly induced only in a subpopulation of cells. SOS induction was also observed in walJBsu mutants using a lacZ transcriptional fusion to the LexA-repressed yneA promoter (see Fig. S1 in the supplemental material).

FIG. 4.
Deleting walJBsu induces the SOS response in a subpopulation of cells. (A) Measurement of SOS induction using an SOS-inducible LacZ reporter. Strains containing a PtagC-lacZ fusion were grown in LB medium at 37°C to an OD600 of ~1, and ...

To determine the pattern of SOS induction in single cells, we monitored the expression of an SOS-inducible PyneA-yfp transcriptional fusion by fluorescence microscopy (Fig. 4B and C). In exponentially growing cells, a small (~3%) fraction of ΔwalJBsu cells showed YFP expression. This represented a significant increase in SOS induction over wild-type cells but was ~5-fold less than what we observed when the SOS response was specifically induced with ciprofloxacin (Fig. (Fig.4C).4C). Many cells in the YFP+ subpopulation were elongated; however, we also observed SOS induction in cells of average length, and many elongated cells had no visible YFP signal. The SOS induction was not a consequence of nucleoid bisection, as cells expressing the PtagC-yfp reporter did not have a bisected chromosome. In addition, we have not observed SOS induction in other strains that bisect their chromosomes at similar frequencies (8).

walJBsu mutants are slightly elongated in a yneA-independent manner.

We observed that the median length of ΔwalJBsu cells was ~10% longer than that of wild-type cells (Fig. (Fig.5)5) (the cell length distributions were significantly different from that of the wild type; Mann-Whitney test). One possible explanation for this phenotype was expression of the SOS-induced cell division inhibitor YneA (39), which is responsible for filamentation of cells experiencing DNA damage. The median cell length of a ΔwalJBsu ΔyneA strain was slightly shorter than the ΔwalJBsu strain, but the distribution was still significantly different from that of the wild type (Mann-Whitney test) (Fig. (Fig.5).5). The difference between the ΔwalJBsu and ΔwalJBsu ΔyneA strain distributions was not statistically significant (P = 0.09; Mann-Whitney test), leading us to conclude that ΔwalJBsu cells are not elongated due to SOS-induced filamentation. Overexpression of ΔwalJBsu had no significant effect on cell length (strain SB494, Pspac-walJBsuDD; data not shown).

FIG. 5.
walJBsu mutants are slightly elongated. Cells were grown in LB medium at 37°C to mid-exponential phase (OD600, ~0.5 to 0.6), and samples were collected for microscopy. Cell lengths were measured from FM4-64-stained fields using the measurement ...

Since deleting walJBsu caused some cells to divide before completing chromosome segregation, we hypothesized that WalJBsu could affect the regulation of timing of cell division. Mutations in other genes affecting cell division, such as noc, can be synthetically lethal with mutations in other division inhibitors (75), so we asked if walJBsu exhibited any synthetic phenotypes. We were unable to find any obvious synthetically lethal combinations, as ΔwalJBsu ΔezrA and ΔwalJBsu ΔminCD mutants were viable and did not exhibit any obvious changes in colony morphology compared to the wild type. In addition, deleting walJBsu did not suppress the growth defect conferred by overexpressing MinCD (strain SB595, Pspac-hy-minCD ΔwalJBsu).

A predicted hydrolase active-site residue is required for WalJBsu activity.

WalJBsu is predicted to be a 264-amino-acid (aa) protein belonging to the metallo-β-lactamase superfamily. No transmembrane regions could be identified by the TMHMM algorithm (43). A functional YFP-WalJBsu fusion exhibited diffuse cytoplasmic localization during exponential growth in LB medium (strain SB1295; Pspac-yfp-walJBsu). Expression of a proteolytically stabilized form of YFP-WalJBsu (strain SB1306; Pspac-yfp-walJBsuDD) (see below) yielded similar results, except that polar YFP puncta were occasionally present; these might represent the formation of inclusion bodies (data not shown). In Streptococcus pneumoniae, the WalJ ortholog WalJSpn appears to localize to cell membranes (72).

The metallo-β-lactamase superfamily domain (Pfam PF00753) comprises approximately 80% of the total protein sequence and contains the signature HXHXDH metal-binding motif found in all metallo-β-lactamases (2). Mutations in this sequence have been shown to disrupt the function of other metallo-β-lactamases (15, 49), so we asked if altering the central histidine residue (H60A) in the HXHXDH motif would affect the activity of WalJBsu. When this mutation was introduced into the wild-type allele, we found that walJBsuH60A was unable to complement the plate growth defect (Fig. (Fig.11).

To determine if the H60A mutation affected protein expression levels instead of activity, we determined protein levels by immunoblotting. Protein levels of the YFP-WalJBsu fusion were low even when overexpressed from the Pspac promoter, suggesting that WalJBsu may be proteolytically unstable (see Fig. S2 in the supplemental material). The C-termnal sequence of WalJBsu is CAV, which is similar to the nonpolar C-terminal sequences recognized by ClpXP and other AAA+-ATPase-associated proteases (41). Substituting or adding a pair of aspartic acid residues at the C terminus of protease substrates recognized via their C-termini frequently increases protein stability. We found that adding two aspartic acid residues to the C terminus of YFP-WalJBsu greatly increased protein levels, consistent with the idea that WalJBsu is targeted for proteolysis by one or more proteases via its nonpolar C-terminal sequence (Fig. S2). In this context, the H60A mutation did not have any effect on protein expression (Fig. S2). These data suggest that the metal-binding motif is at least partially required for the folding or activity of WalJBsu and supports the hypothesis that WalJBsu could function as a hydrolase.

WalJBsu has no obvious effect on lipid composition.

We hypothesized that WalJBsu might be directly or indirectly involved in lipid or peptidoglycan metabolism. To see if cellular lipids were altered in a ΔwalJBsu mutant, the lipid compositions of the mutant and a wild-type reference were analyzed by mass spectrometry at the Kansas Lipidomics Research Center. Given that ΔwalJBsu mutants have elevated SOS induction levels, we conducted these experiments in the PY79 strain background, which lacks the SOS-inducible SBβ prophage found in our usual JH642 strain background to avoid any possible complications with prophage-induced lysis. Total cellular lipids were extracted from exponentially growing wild-type and mutant strains containing wild-type dnaB alleles (SB792 and SB794). We did not find a significant difference in the total mass fractions of lipid species between the wild-type and mutant strains (see Fig. S3 in the supplemental material) nor any significant differences in any particular lipid species. These data indicate that the SOS susceptibility and cell division phenotypes of walJBsu mutants are not due to altered membrane lipid composition.

walJBsu mutants are sensitive to cephalosporin antibiotics.

To increase our understanding of the cellular function of WalJBsu, we screened ΔwalJBsu mutants for drug sensitivities using Biolog Phenotype MicroArrays (12). walJBsu+ and ΔwalJBsu strains (SB792 versus SB794) were grown in a defined minimal medium in the presence of a wide range of different antibiotics and other chemical compounds; by comparing the growth kinetics of the two strains in the presence of these compounds, we sought to identify other relevant phenotypes of the ΔwalJBsu mutant. Significant growth effects were observed only for a few cationic compounds (such as domiphen bromide) and certain cephalosporins (beta-lactam antibiotics that block the final transpeptidation steps of peptidoglycan synthesis) (see Table S3 in the supplemental material). The observed set of drug sensitivity phenotypes was consistent with the hypothesis that ΔwalJBsu mutants have a defect in cell wall metabolism.

The Biolog results were independently confirmed by plating out dilution series of wild-type and ΔwalJBsu mutants on LB agar plates containing different concentrations of antibiotic (Fig. (Fig.66 and see Fig. SA4 in the supplemental material). ΔwalJBsu strains were sensitive to the cephalosporins cefotaxime, cefuroxime, and cefmetazole but had little to no sensitivity to cefoxitin or cefoperazone. ΔwalJBsu mutants also showed an increased sensitivity to moxalactam, a beta-lactam closely related to the cephalosporins, but not to the penicillin-like beta-lactam carbenicillin. Overexpression of walJBsu did not significantly impact cephalosporin sensitivity (see Fig. SA5 in the supplemental material). Thus, cells lacking walJBsu have an increased sensitivity to certain cephalosporin antibiotics.

FIG. 6.
walJBsu mutants have increased sensitivity to certain cell wall antibiotics. Strains were grown in LB medium to an OD600 of ~2. Tenfold serial dilutions of each culture were pinned onto LB agar plates containing antibiotics at the indicated concentrations. ...

Cephalosporins are thought to inhibit the extracellular pencillin-binding proteins (PBPs) that catalyze the final transpeptidation reactions cross-linking adjacent peptidoglycan strands. To identify the step in cell wall synthesis that is most affected by WalJBsu, we also tested the sensitivity of ΔwalJBsu mutants to drugs that act on other aspects of cell wall biosynthesis: phosphomycin (which blocks the synthesis of UDP-MurNAc peptidoglycan precursors), tunicamycin, bacitracin (both interfering with the lipid II carrier cycle), and vancomycin (which block transglycosylase activity). ΔwalJBsu mutants were not more sensitive to any of these four drugs (data not shown), suggesting that the WalJBsu-dependent effect on the cell wall may be somewhat localized to the final peptidoglycan transpeptidation steps. Unlike Streptococcus mutans (63), the B. subtilis ΔwalJBsu strain was not sensitive to paraquat-induced oxidative stress.

Since WalJBsu orthologs are found in most low-G+C Gram-positive bacteria, we asked if the antibiotic sensitivity of a B. subtilis ΔwalJBsu mutant could be replicated in other species. To begin to address this question, we tested the cephalosporin sensitivity of a Streptococcus pneumoniae mutant lacking the walJBsu ortholog, walJSpn, by disc diffusion assays. Compared to the wild type, the S. pneumoniae ΔwalJSpn mutant was more sensitive to cefoperazone, cefotaxime, cefoxitin, and cefuroxime (Fig. (Fig.77 and see Fig. S6 in the supplemental material). The antibiotic sensitivity of the mutant could be complemented by heterologous expression of walJSpn, confirming that the phenotype was due to inactivation of walJSpn. We did not observe any change in sensitivity to the penicillin-like antibiotic amdinocillin (Fig. S6), suggesting that the S. pneumoniae ΔwalJSpn mutant was, like the B. subtilis ΔwalJBsu mutant, specifically sensitive to certain cephalosporin antibiotics.

FIG. 7.
Streptococcus pneumoniae walJ mutants also exhibit cephalosporin sensitivity. S. pneumoniae strains were grown statically at 37°C in an atmosphere of 5% CO2 as described previously (59, 70). At an OD620 of ~0.400, sterile cotton ...

WalJBsu is required for normal growth when WalRK levels are limiting.

Previous studies of the Streptococcus pneumoniae ortholog of WalJBsu, WalJSpn (VicX), revealed that WalJ was conditionally required for growth when expression of the WalRKSpn TCS was reduced (54). To see if B. subtilis has a similar requirement for WalJBsu when walRKBsu expression is reduced, we constructed a strain in which expression of the endogenous walRKBsu operon was controlled from the isopropyl-beta-d-thiogalactopyranoside (IPTG)-induced promoter Pspac. This strain grew normally in the presence of IPTG but could not grow in the absence of the inducer. We then deleted the native walJBsu locus from the Pspac-walRKBsu strain and reintroduced either the native walR-walK-yycH-yycI-walJBsu operon, walJBsu alone, or a vector control at the heterologous amyE locus.

We grew the three strains to mid-exponential phase in the presence of IPTG and then depleted walRKBsu expression by washing out the inducer. After 2 h of walRKBsu depletion, we split the cultures, supplemented them with either 0 μM, 5 μM, or 100 μM IPTG, and monitored their growth. All three strains grew essentially identically when walRKBsu expression was induced with 100 μM IPTG (Fig. (Fig.8).8). In the absence of walRKBsu expression from the native locus, only the positive-control strain containing the heterologous copy of the walR-walK-yycH-yycI-walJBsu operon grew normally; the strain expressing walJBsu alone and the vector control both failed to grow. We note that after an extended period of growth with no IPTG, cells did begin to grow, but this was found to correlate with the loss of the single-crossover construct used to modify walRKBsu expression (data not shown). However, in the presence of limiting (5 μM) IPTG, the strain expressing a heterologous copy of walJBsu alone was now able to grow as well as the positive control, whereas the vector control could not (Fig. (Fig.8).8). This result indicates that WalJBsu is conditionally required in B. subtilis when walRKBsu-dependent gene expression is misregulated.

FIG. 8.
WalJBsu is required for growth when walRK-dependent expression is limited. Expression of the WalRK TCS was controlled from an IPTG-inducible promoter in strains lacking the endogenous copy of walJBsu. To test the role of walJBsu when WalRK-dependent signaling ...

Previous studies found that WalJBsu does not play a role in regulating WalRKBsu-dependent gene expression (68, 69). To confirm that the ΔwalJBsu mutation did not affect WalRKBsu-dependent signaling when DNA replication was perturbed, we first measured expression levels of the walRKBsu-dependent yocH gene following disruption of DNA replication initiation. Cells were grown to mid-exponential phase, at which point replication initiation was blocked using dnaB19. We measured yocH transcript levels by an RNase protection assay but did not observe any difference in yocH expression in a ΔwalJBsu mutant either in the presence or absence of a replication block (data not shown). Microarray analysis also showed that no known walRKBsu-dependent genes were differentially expressed in a ΔwalJBsu strain compared to the wild type (data not shown). Thus, the phenotypes of a ΔwalJBsu mutant are not likely to result from altered expression of a walRKBsu-regulated gene.

Given that WalRK regulates genes involved in cell wall metabolism, we asked whether the assembly of PBPs at the septum was affected in walJBsu mutants. We did not find any significant change in the septal localization of either a GFP-PBP1 or GFP-PBP2b fusion in strains lacking walJBsu (SB1275, GFP-PBP1 ΔwalJBsu; or SB1276, GFP-PBP2b ΔwalJBsu) (60, 61).

Phylogenetic analysis.

A final link between WalJ and cell wall metabolism is suggested by phylogenetic analysis. Orthologs of walJBsu are conserved in many low-G+C Gram-positive bacteria. Orthologs of walJBsu in species belonging to the Bacilli taxon (including Bacillus, Lactobacillus, Streptococcus, Staphylococcus, and Listeria spp.) lie downstream of walRK orthologs, and to date those have been found to be coexpressed with the walRK genes as part of an operon (the gene neighborhoods in Clostridia spp. are different; see below). Although BLAST searches with the WalJBsu amino acid sequence recovered matches only from low-G+C Gram-positive bacteria, we found evidence for the existence of more distantly related WalJBsu orthologs in other bacterial species by using an alternative approach (see Materials and Methods). We identified putative orthologs in widely distributed taxa, including Pseudomonas, Burkholderia, Ralstonia, and Chlamydia species (see Table S4 in the supplemental material).

Interestingly, most of the putative orthologs in these additional taxa lie downstream of genes involved in peptidoglycan biosynthesis: murA (some Clostridia spp.) or the dapA-nlpB locus (encoding dihydrodipicolinate synthase and a likely ortholog of Escherichia coli NlpB, a lipoprotein involved in membrane biogenesis; Pseudomonas, Burkholderia spp., and Ralstonia spp.). These neighboring genes are oriented and positioned so that they could be coexpressed with the metallo-β-lactamase-encoding gene as part of the same operon. Furthermore, we identified a putative ortholog of walJBsu in Chlamydia spp. downstream of the gene encoding FtsK. The intergenic region between the two genes is 2 to 5 bp, suggesting that their expression may be translationally coupled. FtsK, like SpoIIIE and SftA in B. subtilis, is a DNA translocase that plays a key role in promoting the efficient completion of chromosome segregation in bacteria (7, 74). As shown above, a B. subtilis walJBsu mutant requires spoIIIE for viability when DNA replication is perturbed (Fig. (Fig.1).1). In many cases, the gene neighborhoods include genes often associated with mobile genetic elements, such as transposase or tRNA genes, suggesting that the walJBsu orthologs could have spread by horizontal gene transfer. However, the Gram-negative and Gram-positive sequences cluster separately when aligned, so any transfer events are unlikely to have been recent.

The observations from the phylogenetic analysis are only suggestive, but they motivate the hypothesis that WalJBsu and a widely distributed family of orthologs play a conserved role in cell wall metabolism.

DISCUSSION

In this study, we present data suggesting that the orthologous WalJ proteins from B. subtilis and S. pneumoniae likely play similar roles in cell wall metabolism. In addition to a possible role in cell wall metabolism, our data indicate that B. subtilis WalJBsu is required for accurate coordination of cell division with DNA replication. ΔwalJBsu mutants require SpoIIIE for viability when replication is blocked (Fig. (Fig.1),1), and we found that these mutants divide over unsegregated chromosomes more frequently than wild-type cells (Fig. (Fig.2).2). Experiments in synchronously growing cells showed that walJBsu affects the frequency, but not the timing, of cell division when DNA replication elongation is inhibited (Fig. (Fig.3).3). During unperturbed growth, ΔwalJBsu mutants are slightly longer than wild-type cells, and the SOS response is induced in a subpopulation of cells (Fig. (Fig.44 and and5;5; see Fig. S1 in the supplemental material). SOS induction appears to occur independently of nucleoid bisection events in these cells, suggesting that walJBsu mutants encounter problems with some aspect of chromosome replication, repair, or segregation more frequently than wild-type cells. WalJBsu has been hypothesized to play a role in cell wall metabolism due to its association with the WalRK two-component system (22), and a functional interaction between WalJ and WalRK-dependent gene expression is supported by our observation that WalJ is required for cell viability in both S. pneumoniae and B. subtilis when the expression levels of WalRK are limiting (Fig. (Fig.8)8) (54). The sensitivity of B. subtilis and S. pneumoniae strains lacking walJ to specific cephalosporin antibiotics further suggests that WalJ may play a role in cell wall metabolism (Fig. (Fig.66 and and7;7; see Fig. S4 and S6 in the supplemental material).

WalJBsu belongs to the metallo-β-lactamase enzyme superfamily. Since a mutation in a conserved residue of WalJBsu required for the enzymatic activity of other superfamily members, His60, confers a walJ null phenotype (Fig. (Fig.11 and and6)6) without affecting protein levels (Fig. S2), it may be that the key role of WalJBsu linked to cell cycle coordination and cell wall metabolism is as a hydrolase. WalJBsu might be partially redundant with enzymes that produce or recycle a compound required for growth, such as a cell wall precursor, or might degrade a compound that inhibits growth or division. Thus, the essential role of WalJBsu when WalRK levels are limiting (Fig. (Fig.8)8) may be to compensate for changes in metabolite pools that result from changes in the expression of enzyme-encoding genes transcriptionally regulated by WalRK.

It remains possible that the key function of WalJBsu linked to cell cycle coordination and cell wall metabolism is distinct from any activity it might have as a hydrolase. WalJBsu might positively or negatively regulate the activity of another protein, such as a signaling kinase, transcription factor, enzyme, or scaffolding protein. In that case, the H60A mutation might disrupt the regulation or localization of WalJBsu, perhaps by perturbing the binding site for a small molecule effector.

As the PBP enzymes targeted by cephalosporins are found outside the cell, it does not seem likely that the sensitivity of ΔwalJBsu mutants reflects a role for WalJBsu in hydrolyzing these compounds. Even if WalJBsu can act directly on cephalosporins, it must have an endogenous substrate as well, since the nucleoid bisection, SOS induction, and WalRK-dependent phenotypes were observed in the absence of beta-lactams. A model to explain the cephalosporin sensitivity of the B. subtilis mutant is that the strain lacking ΔwalJBsu had reduced cellular pools of a specific peptidoglycan precursor. Cephalosporins, like other beta-lactam antibiotics, mimic the natural substrates of PBPs and inactivate the final transpeptidation steps in peptidoglycan synthesis. If WalJBsu contributes to the production of a compound involved in cell wall metabolism, pools of a particular PBP substrate may be reduced in ΔwalJBsu mutants; this in turn could reduce competition for the enzyme active site and increase cephalosporin sensitivity.

If WalJBsu does play a role in peptidoglycan turnover, the cephalosporin sensitivity phenotype could be explained by a model where a murein turnover product affects the expression of other genes involved in beta-lactam resistance. Precedence for such a model is found in E. coli, where specific muropeptide degradation products transported into the cytoplasm regulate the activity of the AmpR transcriptional regulator. AmpR, in turn, regulates expression of the AmpC beta-lactamase, which confers antibiotic resistance (35, 45). In the ΔwalJBsu mutant, perturbation of peptidoglycan turnover could potentially change the abundance of a molecule that acts via a similar system to alter the regulation of a beta-lactamase that confers resistance to specific cephalosporins.

While our data are consistent with a role for WalJBsu in peptidoglycan metabolism, another possibility is that WalJBsu instead affects the levels or composition of teichoic acids in the cell wall. Mutations in Staphylococcus aureus that resulted in altered teichoic acids (lacking d-alanyl ester groups) were found to increase the negative surface charge of the cell; this corresponded to an enhanced sensitivity to range of cationic antibiotic compounds, including the cephalosporin cefazolin (56, 57). The observed sensitivity of ΔwalJBsu mutants to cephalosporins and other cationic compounds could be explained by an analogous mechanism. Future biochemical studies will be required to see if this is indeed the case.

Mutations affecting cell wall metabolism can impact cell division in both Gram-positive and Gram-negative bacteria. For example, a B. subtilis strain lacking all four class A PBPs formed division septa over unsegregated chromosomes in a manner similar to what we have observed with a ΔwalJBsu mutant (47). Streptococcus pneumoniae mutants lacking PBP3 (dacA), a dd-carboxypeptidase that modifies the substrates of some high-molecular-weight PBPs, or Pmp23, a peptidoglycan hydrolase, also form aberrant division septa (4, 50, 55, 62). Similarly, loss of the dd-carboxypeptidase PBP5 (dacA) in E. coli also results in abnormal cell shapes that suggest a role for dacA in establishing proper division placement (52, 53). The mechanistic connection linking these changes in cell wall structure to cell division is not currently clear. Perhaps the degree of cross-linking or other chemical aspects of cell wall structure at the division site subtly affect the localization, regulation, or activity of the divisome.

WalJ is widely conserved among the low-G+C Gram-positive bacteria, but reported phenotypes of ΔwalJ mutants vary from organism to organism, which may reflect pleotropic effects of this gene in different organisms. While this study and previous work found no obvious role for WalJBsu in the WalRK signaling pathway of B. subtilis, there is evidence that the walJ ortholog in Streptococcus mutans, walJspn (vicX), does play a role in WalRK signaling. walJSpn (vicX) mutants were reported to have several phenotypes, including altered biofilm formation, genetic competence, and sensitivity to paraquat (63). In constrast, S. aureus mutants lacking the walJ ortholog were not found to have any obvious growth, morphological, or oxidative stress phenotypes (22). The mechanism through which walJ affects the oxidative stress tolerance of S. mutans is not clear (63), and we also do not completely understand why the SOS response is induced only in a subpopulation of B. subtilis ΔwalJBsu mutants. The cells which showed a strong SOS response did not necessarily contain bisected chromosomes, indicating that the weak SOS and prophage induction does not explain the bisection phenotype. Previous studies do not report antibiotic sensitivity data, so it is possible that walJ mutants in other low-G+C Gram-positive bacteria share the cephalosporin sensitivity phenotype observed in B. subtilis and S. pneumoniae.

An important next step in understanding the cell division defect of the ΔwalJBsu strain will be to identify the native substrate(s) of WalJBsu. Our attempts to purify a soluble form of WalJBsu in E. coli have not been successful, but eventual purification of this protein could perhaps help to identify potential target substrates. Biochemical characterization of the peptidoglycan or teichoic acid composition of ΔwalJBsu mutants may prove to be a fruitful approach, although such analyses may be difficult if the relevant activity of WalJBsu is restricted to the septum.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Balaji Srinivasan, Serafim Batzoglou, and their colleagues for assistance with the Stanford Network Browser database, George Wright and Neal Brown for generously providing HPUra, Katherine Cunningham for assistance with cloning and protein expression, Elizabeth Harry for the spore outgrowth protocols, Rob Britton for assistance with microarrays, Angela Kirik for help with lipid isolations, and Mary Roth (Kansas Lipidomics Research Center) for assistance with the lipid profiling. We thank the other members of the Burkholder laboratory, Richard Wheeler and Simon Foster, and generous colleagues for many helpful discussions.

S.J.B. was supported in part by an NSF predoctoral fellowship and an ARCS fellowship. K.J.W. was a predoctoral trainee supported by NIH Genetics, Cellular, and Molecular Sciences training grant T32GM007757. This project was supported by the National Science Foundation, award ID 0744872 (to W.F.B.), and by the National Institute of Allergy and Infectious Diseases, grant number AI060744 (to M.E.W.).

The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

Footnotes

[down-pointing small open triangle]Published ahead of print on 17 December 2010.

Supplemental material for this article may be found at http://jb.asm.org/.

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