A complex four-gene operon containing essential cell division gene pbpB in Bacillus subtilis

Abstract

We have cloned and sequenced the promoter-proximal region of the Bacillus subtilis operon containing the pbpB gene, encoding essential penicillin-binding protein PBP2B. The first two genes in the operon, designated yllB and yllC, are significantly similar to genes of unknown function similarly positioned upstream of pbpB in Escherichia coli. Both B. subtilis genes are shown to be nonessential. The third B. subtilis gene, yllD, is essential, as is the correspondingly positioned ftsL gene of E. coli. The predicted product of yllD is similar to FtsL in size and distribution of charged residues but is not significantly related in primary amino acid sequence. The major promoter for the cluster lies upstream of the first gene, yllB, but at least one minor promoter lies within the yllC gene. The operon is transcribed throughout growth at a low level.Vegetative cells of Bacillus subtilis, like those of most other rod-shaped bacteria, grow by lengthwise extention and divide by ingrowth of a centrally positioned septum. A number of genes involved in formation of the septum have been identified, mainly by the isolation and characterization of temperature-sensitive mutants that form nonseptate filaments at the nonpermissive temperature (5). In Escherichia coli several of these fts genes are clustered in the 2-min region of the chromosome, along with many other genes involved in division and cell wall synthesis (DCW) (26). Apparent homologs of most genes in the cluster are grouped together on the B. subtilis chromosome. Alignment of the two DCW regions reveals that the linear order of genes, over 25 kbp or so, is generally conserved, although some open reading frames (ORFs) occur in only one of the organisms (8).At the upstream margin of the known sequence in B. subtilis, a likely homolog of the E. coli pbpB (ftsI) gene was found (25). In E. coli, this gene encodes the penicillin-binding protein PBP3, which is essential for synthesis of septal peptidoglycan (19). In B. subtilis the equivalent gene is duplicated. The downstream copy, called spoVD, encodes a protein specialized for synthesis of modified cell wall material that forms the spore cortex (3). The upstream copy was previously shown to encode PBP2B, which seems to be the functional homolog of PBP3 (4, 25). Here we extend the sequence of the B. subtilis DCW cluster upstream of pbpB and describe the remainder of the pbpB operon. The operon contains four genes, which we argue are probably all functionally homologous to equivalently positioned genes in E. coli. The third gene in the operon is shown to be essential for cell viability and therefore is probably functionally equivalent to the E. coli ftsL (mraR) gene (12, 24), even though it shows negligible primary sequence conservation. The first two genes in the operon, which are very similar to the equivalent (but uncharacterized) genes in E. coli, are shown to be dispensable in B. subtilis. MATERIALS AND METHODSBacterial strains. The bacterial strains, phages, and plasmids used in this study are listed in Table 1. The i...

Morphological changes and proteome response of Corynebacterium glutamicum to a partial depletion of FtsI

“…Therefore, it can be concluded that ftsI is mainly cotranscribed along with the mraZ, mraW, ftsL and murE genes of the operon probably from the Pmra promoter, as described in E. coli and B. subtilis (Hara et al, 1997), and probably also from a minor promoter (PftsI) as in B. subtilis (Daniel et al, 1996). This result also suggests that the second GTG (at position 2 293 165) most probably is the start codon of ftsI.…”

“…Furthermore, the localization of GFP-FtsI at the mid-cell is strongly indicative of the involvement of FtsI in C. glutamicum cell division, most likely in the biosynthesis of septum peptidoglycan as described for other bacteria. ftsI seems to be an essential gene in C. glutamicum ftsI has been shown to be an essential gene in E. coli (Begg et al, 1992) and in B. subtilis (Daniel et al, 1996). Therefore, in order to determine whether ftsI was also necessary for the viability of C. glutamicum, we performed gene disruption experiments using the suicide plasmid pKInt1 (Table 1); all attempts to inactivate the ftsI gene using internal fragments were unsuccessful, similar to our earlier studies involving murC, another essential gene in C. glutamicum (Ramos et al, 2004).…”

“…The availability of the complete genome sequence of C. glutamicum (GenBank accession nos NC_003450 and BX927154) has enabled us to study the expression profile/ regulation of the cell division gene ftsI, and perform proteomic studies in the organism. FtsI homologues have been described in different bacteria, such as Escherichia coli (Begg et al, 1992;Botta & Park, 1981) or Bacillus subtilis (Daniel et al, 1996;Marston et al, 1998). FtsI (also called penicillin-binding protein 3, PBP3) is a well-characterized protein that has been reported to be expressed in very low amounts in the cell (about 100 molecules) (Dougherty et al, 1996).…”

A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA

“…1 and Table 2). Both ftsL and pbpB are essential for cell division (34). FtsL is an unstable protein (35,36); a decrease in ftsL mRNA levels quickly causes a decrease in FtsL protein and inhibits cell division (37).…”

d-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling

“…Likewise, strains of Bacillus subtilis lacking PBP1, PPB2c, PBP2d, and PBP4 (either alone or in various combinations) display little to no change in peptidoglycan cross-linking ratios (16). Genetic deletion of PBP2b in B. subtilis is lethal, suggesting that it may play a primary role in TP activity (17). Together, these examples highlight the limitations of using genetic manipulations alone to track PBP enzymatic activity.…”