Identification of Bacillus subtilis genes for septum placement and shape determination.

The Bacillus subtilis divIVB1 mutation causes aberrant positioning of the septum during cell division, resulting in the formation of small, anucleate cells known as minicells. We report the cloning of the wild-type allele of divIVB1 and show that the mutation lies within a stretch of DNA containing two open reading frames whose predicted products are in part homologous to the products of the Escherichia coli minicell genes minC and minD. Just upstream of minC and minD, and in the same orientation, are three genes whose products are homologous to the products of the E. coli shape-determining genes mreB, mreC, and mreD. The B. subtilis mreB, mreC, and mreD genes are the site of a conditional mutation (rodB1) that causes the production of aberrantly shaped cells under restrictive conditions. Northern (RNA) hybridization experiments and disruption experiments based on the use of integrational plasmids indicate that the mre and min genes constitute a five-cistron operon. The possible involvement of min gene products in the switch from medial to polar placement of the septum during sporulation is discussed.     

Amplification of the Bacillus subtilis maf gene results in arrested septum formation.

The Bacillus subtilis homolog of the Escherichia coli morphogene orfE (of the mre operon) has been identified. The determinant is located on the chromosome immediately upstream of the mreBCD-minCD (divIVB) operon. The Maf protein shares substantial amino acid sequence identity with the E. coli OrfE protein. Introduction of the B. subtilis maf determinant on a multicopy plasmid into B. subtilis cells results in an inhibition of septation, which leads to extensive filamentation and loss of viability in the transformed cell population. Insertional inactivation of maf indicated that this gene is not essential for cell division.     

Bacteriophage SPO1-induced macromolecular synthesis in minicells of Bacillus subtilis.

SPO1 bacteriophage injects its DNA into minicells produced by Bacillus subtilis CU403 divIVB1. The injected DNA is partially degraded to small trichloracetic acid-precipitable material and trichloroacetic acid-soluble material. The injected DNA is not replicated; however, it serves as a template for RNA and protein synthesis. The RNA produced specifically hybridizes to SPO1 DNA, and the amount of RNA hybridized can be reduced by competition with RNA isolated at all stages of the phage cycle from infected nucleate cells of the B. subtilis CU403 divIVB1. An unrelated phage, SPP1, also induces phage-specific RNA in infected minicells. Translation occurs in SPO1-infected minicells resulting in at least eight proteins which have been separated by gel electrophoresis, and two of these proteins have mobilities similar to proteins found only in infected B. subtilis CU403 divIVB1 nucleate cells. A large proportion of the polypeptide material synthesized in infected minicells is very small and heterogeneous in size.     

New minC mutations suggest different interactions of the same region of division inhibitor MinC with proteins specific for minD and dicB coinhibition pathways.

Proper positioning of division sites in Escherichia coli requires balanced expression of minC, minD, and minE gene products. Previous genetic analysis has shown that either MinD or an apparently unrelated protein, DicB, cooperates with MinC to inhibit division. We have isolated and sequenced minC mutations that suppress division inhibition caused by overproduction of either DicB or MinD proteins. Most missense mutations were located in the amino acid 160 to 200 region of MinC (231 amino acids). Some mutations exhibited preferential resistance to one or the other coinhibitor, suggesting that two distinct proteins, possibly MinD and DicB themselves, interact in slightly different manners with the same region of MinC to promote division inhibition.     

Nucleotide sequence of the immunity region of Bacillus subtilis bacteriophage phi 105: identification of the repressor gene and its mRNA and protein products

A gene involved in the regulation of lysogeny in the temperate Bacillus subtilis phage phi 105 has been identified and isolated. A plasmid, pDC4, was constructed that contains a 740-bp HindIII-PvuII fragment that is derived from the phi 105 immunity region and is capable of rendering B. subtilis immune to infection by phi 105. Three different hybrid plasmids that contain the 740-bp fragment, pAG101 [Cully and Garro, J. Virol. 34 (1980) 789-791], pDC1 and pDC2, were found to synthesize a common 18-kDal polypeptide in B. subtilis minicells and Escherichia coli maxicells. The nucleotide (nt) sequence of this region revealed three open reading frames (ORFs) that predict proteins with Mrs of 16521, 7332, and 5516. In vivo synthesized phi 105 prophage RNA was mapped by primer extension and shown to be transcribed from the DNA strand coding for the Mr 16521 protein. The 5' end of the phi 105 lysogen RNA was mapped to a region that contains conserved sequences for RNA polymerase recognition.     

Genes and their organization in the replication origin region of the bacterial chromosome

Genes and their organization are conserved in the replication origin region of the bacterial chromosome. To determine the extent of the conserved region in Gram-positive and Gram-negative bacteria, which diverged 1.2 billion years ago, we have further sequenced the region upstream from the dnaA genes in Bacillus subtilis and Pseudomonas putida. Fifteen open reading frames (ORFs) and 11 ORFs were identified in the 13.6 kb and the 9.8 kb fragments in B. subtilis and P. putida, respectively. Eight consecutive P. putida genes, except for one small ORF (homologous to gene 9K of Escherichia coli) in between, are homologous in sequence and relative locations to genes in B. subtilis. Altogether, 12 genes and their organization are conserved in B. subtilis and P. putida in the origin region. We found that the conserved region terminated on one side after the orf290 in P. putida (orf282 in B. subtilis). In the B. subtilis chromosome, five additional ORFs were found in between the conserved genes, suggesting that they are added after Gram-positive bacteria were diverged from the Gram-negative bacteria. One of the ORFs is a duplicate of the conserved gene. The third non-translatable region containing multiple repeats of DnaA-box (second in the case of P. putida) was found flanking gidA in both organisms. This result shows clearly that E. coli oriC and flanking genes gidA and gidB have been translocated by the inversion of some 40 kb fragment.     

A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E. coli

The E. coli minicell locus (minB) was shown to code for three gene products (MinC, MinD, and MinE) whose coordinate action is required for proper placement of the division spetum. Studies of the phenotypic effects of expression of the three genes, alone and in all possible combinations, indicated the following: cell poles contain potential division sites that will support additional septation events unless specifically inactivated; the minC and minD gene products act in concert to form a nonspecific inhibitor of septation that is capable of blocking cell division at all potential division sites; and the minE gene codes for a topological specificity factor that, in wild-type cells, prevents the division inhibitor from acting at internal division sites while permitting it to block septation at polar sites.     

Synthesis of cell envelope components by anucleate cells (minicells) of Bacillus subtilis.

Minicells produced by Bacillus subtilis CU403 (divIVB1) are capable of mucopeptide biosynthesis as shown by the incorporation of L-alanine, D-alanine, and N-acetylglucosamine into trichloroacetic acid-precipitable material, which can be degraded to trichloroacetic acid-soluble material by lysozyme digestion. Incorporation of the precursors is sensitive to vancomycin and D-cycloserine and insensitive to chloramphenicol. Penicillin inhibits the incorporation of D- and L-alanine N-acetylglucosamine at concentrations in excess of 10 mug of penicillin per ml; however, minicells are insensitive to penicillin-induced lysis. The material synthesized in minicells from N-acetylglucosamine is not subject to turnover during a subsequent 6-h incubation period. [2-3H]glycerol is converted to a cold trichloroacetic acid-precipitable form by minicells. This synthesis is not inhibited by vancomycin, penicillin, D-cycloserine, or chloramphenicol. Fractionation of the material synthesized from glycerol into hot trichloroacetic acid-soluble material and chloroform/methanol-extractable material indicates that minicells convert glycerol into teichoic acid and lipid.     

Heterospecific expression of the Bacillus subtilis cell shape determination genes mreBCD in Escherichia coli

The divIVB operon of Bacillus subtilis includes the cell shape-associated mre genes, including the membrane-associated proteins MreC and MreD. TnphoA mutagenesis was utilized to analyze a topological model for MreC. MreC has a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large carboxy terminal domain which lies externally to the outer leaflet of the cell membrane. Expression of the B. subtilis MreB protein, or the Mre C and D proteins, results in a morphological conversion of the Escherichia coli host cells from a rod to a roughly spherical cell, morphologically similar to mre-negative mutants of E. coli. Immunolocalization of the MreC protein in B. subtilis revealed that this protein is found at the midcell division site of the bacterial cells, consistent with the postulated role of the Mre proteins in the regulation of septum-specific peptidoglycan synthesis.     

Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.

The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases.     

The divIVA minicell locus of Bacillus subtilis.

The Bacillus subtilis divIVA1 mutation causes misplacement of the septum during cell division, resulting in the formation of small, circular, anucleate minicells. This study reports the cloning and sequence analysis of 2.4 kb of the B. subtilis chromosome including the divIVA locus. Three open reading frames were identified: orf, whose function is unknown; divIVA; and isoleucyl tRNA synthetase (ileS). We identified the point mutation in the divIVA1 mutant allele. Inactivation of divIVA produces a minicell phenotype, whereas overproduction of DivIVA results in a filamentation phenotype. Mutants with mutations at both of the minicell loci of B. subtilis, divIVA and divIVB, possess a minicell phenotype identical to that of the DivIVB- mutant. The DivIVA-mutants, but not the DivIVB- mutants, show a decrease in sporulation efficiency and a delay in the kinetics of endospore formation. The data support a model in which divIVA encodes the topological specificity subunit of the minCD system. The model suggests that DivIVA acts as a pilot protein, directing minCD to the polar septation sites. DivIVA also appears to be the interface between a sporulation component and MinCD, freeing up the polar septation sites for use during the asymmetric septation event of the sporulation process.     

The ams-1 and rne-3071 temperature-sensitive mutations in the ams gene are in close proximity to each other and cause substitutions within a domain that resembles a product of the Escherichia coli mre locus.

Two temperature-sensitive mutations, ams-1 and rne-3071, in the ams (altered mRNA stability) gene have been used extensively to investigate the processing and decay of RNA in Escherichia coli. We have sequenced these temperature-sensitive alleles and found that the mutations are separated by only 6 nucleotides and cause conservative amino acid substitutions next to a possible nucleotide-binding site within the N-terminal domain of the Ams protein. Computer analysis revealed that the region altered by the mutations has extensive sequence similarity to a predicted gene product from the mre (murein pathway cluster e) locus of E. coli, which has been implicated previously in determining bacterial cell shape.     

Replication origin region of the chromosome of alkaliphilic Bacillus halodurans C-125.

An 18.5-kb DNA fragment containing the oriC region of the chromosome of the alkaliphilic Bacillus halodurans C-125 was obtained by PCR and sequenced. Sixteen open reading frames (ORFs) were identified in this region. A sequencing similarity search using the BSORF database found that ORF1 to 13 all had significant similarities to gene products of Bacillus subtilis. Three other ORFs (ORF14-16) of unknown function were positioned down-stream of gyrB instead of rrnO, which is found in the same region in the case of B. subtilis. The ORF organization from gidA to gyrA was the same as that of B. subtilis. The gene organization and the location of the DnaA-box region were also similar to those of the chromosomes of other bacteria, such as Escherichia coli and Pseudomonas putida. There were two DnaA-box clusters (Box-region C and R) with a consensus sequence TTATCCACA on both sides of the dnaA gene but another DnaA box cluster (Box-region L) which is found in the region between thdF and jag in B. subtilis was not found in the corresponding region in the case of alkaliphilic Bacillus halodurans C-125.     

Cloning and expression of the Escherichia coli recA gene in Bacillus subtilis

By means of homopolymer dG-dC tailing, using PstI linearized pBR327 as vector, we constructed small plasmids containing the entire Escherichia coli recA gene. The 1.8-kb inserts were recloned in the Bacillus subtilis expression vector pPL608 in a B. subtilis recE4 strain. Analysis of plasmid-coded proteins showed expression of the E. coli recA gene both in minicells and whole cells of B. subtilis. Expression was under control of the bacteriophage SP02 promoter, which is part of pPL608. A recA-expressing plasmid completely abolished the transformation deficiency of the recE4 mutant as well as its sensitivity to mitomycin C (MC). The expressed recA gene also restored recombination in other B. subtilis strains lacking the recE gene product. These results indicate a high similarity between the functions of the E. coli RecA and B. subtilis RecE proteins.     

Sequence and analysis of pBM02, a novel RCR cryptic plasmid from Lactococcus lactis subsp cremoris P8-2-47

This paper reports the complete nucleotide sequence of the 3.85 kbp plasmid pBM02 from Lactococcus lactis subsp. cremoris P8-2-47. Analysis of the sequence predicted six ORFs larger than 25 amino acids. They all were transcribed from the same strand and organized in two functional cassettes: the replication region and a putative mobilization region. In the replication region, two ORFs specifying proteins homologous to others found in some classes of rolling circle-replicating plasmids were encountered (copG and repB). In fact, single-stranded DNA was detected as a replication intermediate of pBM02. copG and repB, together with some upstream sequences, formed part of the minimal replication unit of the plasmid. Interestingly, pBM02 shared a 212 bp stretch with plasmids of the pWV01 type, in which the whole single-strand origin of replication is included. In the mobilization region, an ORF coding for a mobilization-like protein was present, preceded by a putative oriT sequence homologous to that of plasmid pMV158. The replicon of pBM02 is of the wide-host range type, and functions in both Gram-positive and Gram-negative bacteria, including Lactobacillus casei, Lactobacillus plantarum, Bacillus subtilis, and Escherichia coli.     

Cloning and characterization of a Bacillus subtilis gene encoding a homolog of the 54-kilodalton subunit of mammalian signal recognition particle and Escherichia coli Ffh.

By using a DNA fragment of Escherichia coli ffh as a probe, the Bacillus subtilis ffh gene was cloned. The complete nucleotide sequence of the cloned DNA revealed that it contained three open reading frames (ORFs). Their order in the region, given by the gene product, was suggested to be ORF1-Ffh-S16, according to their similarity to the gene products of E. coli, although ORF1 exhibited no significant identity with any other known proteins. The orf1 and ffh genes are organized into an operon. Genetic mapping of the ffh locus showed that the B. subtilis ffh gene is located near the pyr locus on the chromosome. The gene product of B. subtilis ffh shared 53.9 and 32.6% amino acid identity with E. coli Ffh and the canine 54-kDa subunit of signal recognition particle, respectively. Although there was low amino acid identity with the 54-kDa subunit of mammalian signal recognition particle, three GTP-binding motifs in the NH2-terminal half and amphipathic helical cores in the COOH-terminus were conserved. The depletion of ffh in B. subtilis led to growth arrest and drastic morphological changes. Furthermore, the translocation of beta-lactamase and alpha-amylase under the depleted condition was also defective.     

Analysis of cell division gene ftsZ (sulB) from gram-negative and gram-positive bacteria.

The ftsZ (sulB) gene of Escherichia coli codes for a 40,000-dalton protein that carries out a key step in the cell division pathway. The presence of an ftsZ gene protein in other bacterial species was examined by a combination of Southern blot and Western blot analyses. Southern blot analysis of genomic restriction digests revealed that many bacteria, including species from six members of the family Enterobacteriaceae and from Pseudomonas aeruginosa and Agrobacterium tumefaciens, contained sequences which hybridized with an E. coli ftsZ probe. Genomic DNA from more distantly related bacteria, including Bacillus subtilis, Branhamella catarrhalis, Micrococcus luteus, and Staphylococcus aureus, did not hybridize under minimally stringent conditions. Western blot analysis, with anti-E. coli FtsZ antiserum, revealed that all bacterial species examined contained a major immunoreactive band. Several of the Enterobacteriaceae were transformed with a multicopy plasmid encoding the E. coli ftsZ gene. These transformed strains, Shigella sonnei, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter aerogenes, were shown to overproduce the FtsZ protein and to produce minicells. Analysis of [35S]methionine-labeled minicells revealed that the plasmid-encoded gene products were the major labeled species. This demonstrated that the E. coli ftsZ gene could function in other bacterial species to induce minicells and that these minicells could be used to analyze plasmid-endoced gene products.     

Characterization of the Azorhizobium sesbaniae ORS571 genomic locus encoding NADPH-glutamate synthase.

Sixteen independent Azorhizobium sesbaniae ORS571 vector insertion (Vi) mutants defective in ammonium assimilation (Asm-) were selected; genomic DNA sequences flanking the insertion endpoints were cloned directly. Resulting recombinant plasmids were used to identify, by hybridization, corresponding wild-type DNA sequences from an A. sesbaniae lambda EMBL3 genomic library (lambda Asm phages). All 16 Asm- Vi mutants physically mapped to a single genomic locus. Plasmid subclones of recombinant phage lambda Asm152 were able to complement both Escherichia coli gltB and A. sesbaniae Asm- Vi mutants; NADPH-glutamate synthase activity was detected in all such strains complemented to Asm+. Heterologous and homologous complementations required both A. sesbaniae gltA+ and (inferred) gltB+ genes. Eleven A. sesbaniae Asm- Vi mutants mapped to a 4-kilobase-pair (kbp) DNA region that exhibited homology with Bacillus subtilis gltA+. In E. coli maxicell labeling experiments, this 4-kbp DNA region encoded a 165-kilodalton polypeptide that was inferred to be the product of the A. sesbaniae gltA+ gene (glutaminase NADPH-dependent L-glutamate synthase subunit). Site-directed Tn5-lacZ mutagenesis of a glt plasmid subclone identified a region that bisected this locus into (at least) two cistrons. Because the remaining five A. sesbaniae Asm- mutants mapped to a 1.5-kbp region adjacent to gltA+, these mutants probably define a single gltB+ gene (glutamate dehydrogenase NADPH-dependent L-glutamate synthase subunit); this region did not exhibit homology with the B. subtilis gltB+ gene.