Essential Bacillus subtilis genes.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

Chromatographic method for diaminopimelic acid detection in calcareous rocks. Presence of a bacterial biomarker in stromatolites.

The presence in the environment of diaminopimelic acid (DAP), a specific eubacterial marker, can be attributed to that of bacteria. We report a reliable and highly sensitive method for the quantification of DAP in calcareous rocks. It consists of acid hydrolysis of rock powder, purification of DAP by chromatography on Dowex 50W and Spherogel AA-NA+ columns, and quantitative analysis by high-performance liquid chromatography. Addition of tritiated DAP, the internal standard, allows one to follow the relevant fractions throughout the purification procedure and to determine their yield. The analytical step consists in pre-column derivatization with ortho-phthaldialdehyde of purified samples, and separation through a reversed-phase C18 column. Chemical controls, i.e., oxidation of samples to rule out the presence of co-eluting lanthionine and cystathionine, as well as mass spectrometry, confirm the presence of DAP in analyzed samples. Our method allows the separation of meso- from L- and/or D-stereoisomers of DAP, and reveals their presence in the examined rocks, two stromatolites of different age and geographic origin.

A high-performance liquid chromatography method for the detection of diaminopimelic acid in urine.

We describe a quantitative assay for diaminopimelic acid (DAP) in urine. It involves (i) hydrolysis of urine samples, (ii) purification by several different liquid chromatography steps, and (iii) analysis by high-performance liquid chromatography on a reversed-phase C18 column. Tritiated-DAP, the internal standard, allows one to precisely follow the DAP-containing fractions and to determine the yield during purification. Sensitive and relatively accurate quantification of DAP, with a threshold of 50 fmol, is based on ion-pairing properties of eluants and ortho-phthaldialdehyde derivatization. The presence of DAP in relevant fractions was confirmed by combined gas chromatography and mass spectrometry. The DAP concentration in adult human urine pooled over 24 h ranges from 0.69 to 2.01 microM, a result in fair agreement with previously published values obtained by ninhydrin derivatization or gas chromatography.

Study of chromosome rearrangements associated with the trpE26 mutation of Bacillus subtilis.

Chromosome rearrangements involved in the formation of merodiploid strains in the Bacillus subtilis 168-166 system were explained by postulating the existence of intrachromosomal homology regions. This working hypothesis was tested by analysing sequences and restriction patterns of the, as yet uncharacterized, junctions between chromosome segments undergoing rearrangements in parent, 168 trpC2 and 166 trpE26, as well as in derived merodiploid strains. Identification, at the Ia/Ib chromosome junction of both parent strains, of a 1.3 kb segment nearly identical to a segment of prophage SPbeta established the existence of one of the postulated homology sequences. Inspection of relevant junctions revealed that a set of different homology regions, derived from prophage SPbeta, plays a key role in the formation of so-called trpE30, trpE30+, as well as of new class I merodiploids. Analysis of junctions involved in the transfer of the trpE26 mutation, i.e. simultaneous translocation of chromosome segment C and rotation of the terminal relative to the origin moiety of the chromosome, did not confirm the presence of any sequence suitable for homologous recombination. We propose a model involving simultaneous introduction of four donor DNA molecules, each comprising a different relevant junction, and their pairing with the junction regions of the recipient chromosome. The resolution of this structure, resting on homologous recombination, would confer the donor chromosome structure to the recipient, achieving some kind of 'transstamping'. In addition, a rather regular pattern of inverse and direct short sequence repeats in regions flanking the breaking points could be correlated with the initial, X-ray-induced, rearrangement.

Bacillus subtilis contains two small c-type cytochromes with homologous heme domains but different types of membrane anchors.

We demonstrate that the cccB gene, identified in the Bacillus subtilis genome sequence project, is the structural gene for a 10-kDa membrane-bound cytochrome c(551) lipoprotein described for the first time in B. subtilis. Apparently, CccB corresponds to cytochrome c(551) of the thermophilic bacterium Bacillus PS3. The heme domain of B. subtilis cytochrome c(551) is very similar to that of cytochrome c(550), a protein encoded by the cccA gene and anchored to the membrane by a single transmembrane polypeptide segment. Thus, B. subtilis contains two small, very similar, c-type cytochromes with different types of membrane anchors. The cccB gene is cotranscribed with the yvjA gene, and transcription is repressed by glucose. Mutants deleted for cccB or yvjA-cccB show no apparent growth, sporulation, or germination defect. YvjA is not required for the synthesis of cytochrome c(551), and its function remains unknown.

Teichuronic acid operon of Bacillus subtilis 168.

Sequence analysis reveals that the Bacillus subtilis 168 tuaABCDEFGH operon encodes enzymes required for the polymerization of teichuronic acid as well as for the synthesis of one of its precursors, the UDP-glucuronate. Mutants deficient in any of the tua genes, grown in batch cultures under conditions of phosphate limitation, were characterized by reduced amounts of uronate in their cell walls. The teichuronic acid operon belongs to the Pho regulon, as phosphate limitation induces its transcription. Placing the tuaABCDEFGH operon under the control of the inducible Pspac promoter allowed its constitutive expression independently of the phosphate concentration in the medium; the level of uronic acid in cell walls was dependent on the concentration of the inducer. Apparently, owing to an interdependence between teichoic and teichuronic acid incorporation into the cell wall, in examined growth conditions, the balance between the two polymers is maintained in order to insure a constant level of the wall negative charge.

A novel protein kinase that controls carbon catabolite repression in bacteria.

HPr(Ser) kinase is the sensor in a multicomponent phosphorelay system that controls catabolite repression, sugar transport and carbon metabolism in gram-positive bacteria. Unlike most other protein kinases, it recognizes the tertiary structure in its target protein, HPr, a phosphocarrier protein of the bacterial phosphotransferase system and a transcriptional cofactor controlling the phenomenon of catabolite repression. We have identified the gene (ptsK) encoding this serine/threonine protein kinase and characterized the purified protein product. Orthologues of PtsK have been identified only in bacteria. These proteins constitute a novel family unrelated to other previously characterized protein phosphorylating enzymes. The Bacillus subtilis kinase is shown to be allosterically activated by metabolites such as fructose 1,6-bisphosphate and inhibited by inorganic phosphate. In contrast to wild-type B. subtilis, the ptsK mutant is insensitive to transcriptional regulation by catabolite repression. The reported results advance our understanding of phosphorylation-dependent carbon control mechanisms in Gram-positive bacteria.

Introns and intein coding sequence in the ribonucleotide reductase genes of Bacillus subtilis temperate bacteriophage SPbeta.

The two putative ribonucleotide reductase subunits of the Bacillus subtilis bacteriophage SPbeta are encoded by the bnrdE and bnrdF genes that are highly similar to corresponding host paralogs, located on the opposite replication arm. In contrast to their bacterial counterparts, bnrdE and bnrdF each are interrupted by a group I intron, efficiently removed in vivo by mRNA processing. The bnrdF intron contains an ORF encoding a polypeptide similar to homing endonucleases responsible for intron mobility, whereas the bnrdE intron has no obvious trace of coding sequence. The downstream bnrdE exon harbors an intervening sequence not excised at the level of the primary transcript, which encodes an in-frame polypeptide displaying all the features of an intein. Presently, this is the only intein identified in bacteriophages. In addition, bnrdE provides an example of a group I intron and an intein coding sequence within the same gene.

The lytE gene of Bacillus subtilis 168 encodes a cell wall hydrolase.

Bacillus subtilis cell wall-bound protein CWBP33 is encoded by lytE, a gene expressed during the exponential growth phase. Sequence analysis of LytE, a 33-kDa protein, reveals two domains. The N-terminal domain contains a threefold-repeated motif common to several peptidoglycan binding proteins, while the C-terminal domain, probably carrying the catalytic activity, has homology with certain exoproteins. Zymographs unambiguously reveal that the absence of CWBP33, due to inactivation of lytE, is accompanied by the loss of a lytic activity. In lytE mutants, the cell autolysis rate is significantly decreased, although autolysis of corresponding, purified cell walls does not seem to be affected.

The wprA gene of Bacillus subtilis 168, expressed during exponential growth, encodes a cell-wall-associated protease.

The nucleotide sequence of wprA, a protease-encoding gene of Bacillus subtilis 168, is reported. The gene, expressed during the exponential growth phase, belongs to a monocistronic operon. WprA is a 96 kDa polypeptide endowed with a signal peptide, as well as a propeptide. Upon processing and export, it gives rise to two previously identified cell-wall-bound proteins, CWBP23 and 52. Processing of WprA exhibits a novel feature of protein export, whereby removal of the middle part of the molecule accompanies the targeting to the cell wall of its N- and C-terminal parts, which correspond to CWBP23 and 52, respectively. Sequence analyses and enzymic assays reveal that CWBP52 is a serine protease. Growth rate, cell morphology, sporulation and motility of wprA mutants apparently do not differ from those of the parent strain.

Sequence of the 305 degrees-307 degrees region of the Bacillus subtilis chromosome.

The nucleotide sequence of the Bacillus subtilis 168 chromosomal segment located between yvhJ (307 degrees) and secA (305 degrees) was determined. This 20.3 kb region encompasses 23 ORFs, 17 of which have been sequenced previously. Comparison of sequences obtained here with the previously obtained ones revealed seven discrepancies. The products of the sequenced genes are involved in the regulation of degradative enzymes, competence, flagellar motility and protein secretion. Putative functions of newly identified genes are based on sequence homologies.

Overall protein content and induced enzyme components of the periplasm of Bacillus subtilis.

Estimates for the overall protein content of the periplasm of Escherichia coli range from 4 to 16% of cellular protein. A cursory examination of known sources of contamination inherent to the methods employed for measurement leads to the conclusion that even the lower value may represent an overestimate of the periplasmic protein in E. coli. The protoplast supernatant fraction (PSF) of Bacillus subtilis defines operationally a potential periplasm, which, after correction for cytoplasmic contamination, yielded, in B. subtilis strains 168 and W23, calculated values of 9 and 3%, respectively, of cell protein as being periplasmic. 26 Among enzymes typically periplasmic in E. coli, at least two, RNases and a 5'-nucleotidase, were located in the B. subtilis periplasm. Compared to other cell fractions, RNase activity in the periplasm was associated with several protein bands forming a unique profile. Samples from all growth phases of cells cultured under phosphate-limitation and phosphate-excess revealed that a major part of both investigated activities was induced by phosphate depletion and located outside the plasma membrane. The current belief that a periplasm containing soluble enzymes does not exist in gram-positive bacteria is examined in light of the absence of an outer membrane permeability barrier, and of a clearly defined electron-transparent zone located between the plasma membrane and the cell wall of B. subtilis. Previous results of studies of protein secretion, and cell wall permeability, are reinterpreted by assuming that the thick charged cell wall of gram-positive bacteria can act as the outer permeability barrier, and as such be the functional equivalent of the outer membrane of gram-negative organisms.

Effect of the SinR protein on the expression of the Bacillus subtilis 168 lytABC operon.

Transcription of the lytABC operon was determined by extension of primers on RNAs isolated from strains bearing a deficient sinR gene. A SinR null mutant, in which part of the sinR gene was deleted, exhibits a pattern identical to that characteristic of FlaB (SigD) deficient mutants, i.e., loss of the signal corresponding to the SigD-dependent promoter, but not of that recognized by the SigA form of the RNA polymerase. However, strains bearing either flaD1 or flaD2, two different point mutations of gene sinR, were characterized by a complete loss of signals corresponding to both promoters. Thus, modified FlaD1 and FlaD2 proteins behave like a repressor affecting the expression of lytABC more severely than does the absence of SinR, The most obvious interpretation of this observation is a direct interaction between the SinR protein and the promoters recognized by the SigD form of the RNA polymerase.

Mechanism of the long tail-fiber deployment of bacteriophages T-even and its role in adsorption, infection and sedimentation.

Models for the tail-fiber deployment of T-even bacteriophages have been experimentally tested by correlating sedimentation constants, adsorption rates, protease inactivation kinetics, and fiber configurations of individual phages observed by electron microscopy. Neither the collective nor the individualistic model, i.e. coordinated fiber retraction and expansion or oscillation of fibers independently of each other, respectively, could satisfactorily account for the results presented. We propose a new intermediary model, in which the base-plate determines a collective behaviour by fixing the hinge angle, around which individual fibers oscillate freely. The bidisperse, so-called dual sedimentation was shown to occur mainly with nascent high-concentration phage stocks in potassium glutamate containing media. Indeed, when mature intracellular phages are released in 0.5 M potassium glutamate--a condition simulating the intracellular environment--only the fast form appears. Upon storage in the cold or release into 0.5 M chloride, both forms appear. Results confirming that the sedimentation constants of the fast and slow form roughly correspond to those of the monodisperse sedimentation, characteristic of the extreme pH values, i.e. 5 and 8, do not allow to conclude that fiber configuration is the only cause of the bidisperse sedimentation.

A periplasm in Bacillus subtilis.

The possibility of there being a periplasm in Bacillus subtilis, in the distinct cell compartment bounded by the cytoplasmic membrane and the thick cell wall, has been investigated quantitatively and qualitatively. Cytoplasmic, membrane, and protoplast supernatant fractions were obtained from protoplasts which were prepared isotonically from cells grown under phosphate limitation. The contents of the protoplast supernatant fraction represent an operational definition of the periplasm. In addition, this cell fraction includes cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from, or lysis of a fraction of, protoplasts. The latter, measured by assay of enzyme markers and by radiolabeled RNA and protein, was found to represent 7.6% of total cell protein, yielding a mean of 9.8% +/- 4.8% for B. subtilis 168 protein considered periplasmic. Qualitatively, after subjection of all cell fractions to polyacrylamide gel electrophoresis, RNase and DNase, zymographs revealed that (i) each cell fraction had a unique profile of nucleases and (ii) multiple species and a major fraction of both nucleases were concentrated in the periplasm. We conclude that the operationally defined periplasmic fraction corresponds closely, both quantitatively and qualitatively, to the contents of the periplasm of Escherichia coli. We discuss evidence that the maintenance of the components of this surface compartment in B. subtilis is compatible with the thick negatively charged cell wall acting as an external permeability barrier.

In Bacillus subtilis 168, teichoic acid of the cross-wall may be different from that of the cylinder: a hypothesis based on transcription analysis of tag genes.

Five of the genes known to encode the enzymes for the synthesis of poly(glycerol phosphate), the major teichoic acid of Bacillus subtilis 168, are organized in two divergently transcribed operons, tagAB and tagDEF. lacZ and gus transcriptional fusions to the first genes of these operons revealed that: (i) in media of different richness, higher growth rates were paralleled by lower transcription levels; (ii) upon transition to stationary phase, the transcription per unit mass of both operons increased abruptly by a factor of about two; and (iii) a rise in temperature was accompanied by decreased transcription of tagA and increased transcription of tagD. Mapping of transcription start points revealed two divergent sigma A-controlled promoters. Although tagD and the neighbouring downstream gene tagE are transcribed from the same promoter, the latter was expressed at a much lower level than the former. Moreover, expression of tagE, and of the translationally coupled tagF, did not increase at the onset of the stationary phase, indicating that additional regulatory signals may act in the intergenic tagD-tagE region. Optimal transcription of these operons appears to require the entire regulatory region, suggesting that tag gene expression may, among other factors, be regulated by the three-dimensional configuration of this segment. The biological implications of these results are discussed.

The tagGH operon of Bacillus subtilis 168 encodes a two-component ABC transporter involved in the metabolism of two wall teichoic acids.

We report the nucleotide sequence and the characterization of the Bacillus subtilis tagGH operon. The latter is controlled by a sigma A-dependent promoter and situated in the 308 degrees chromosomal region which contains genes involved in teichoic acid biosynthesis. TagG is a hydrophobic 32.2 kDa protein which resembles integral membrane proteins belonging to polymer-export systems of Gram-negative bacteria. Gene tagH encodes a 59.9 kDa protein whose N-moiety contains the ATP-binding motif and shares extensive homology with a number of ATP-binding proteins, particularly with those associated with the transport of capsular polysaccharides and O-antigens. That the tagGH operon is essential for cell growth was established by the failure to inactivate tagG and the 5'-moiety of tagH by insertional mutagenesis. During limited tagGH expression, cells exhibited a cocoid morphology while their walls contained reduced amounts of phosphate as well as galactosamine. These observations, revealing impaired metabolism of both wall teichoic acids of B. subtilis 168, i.e. poly(glycerol phosphate), and poly(glucose galactosamine phosphate), combined with sequence homologies, suggest that TagG and TagH are involved in the translocation through the cytoplasmic membrane of the latter teichoic acids or their precursors.

Sequence analysis of the 308 degrees to 311 degrees segment of the Bacillus subtilis 168 chromosome, a region devoted to cell wall metabolism, containing non-coding grey holes which reveal chromosomal rearrangements.

The 29.71 kb chromosomal region of Bacillus subtilis 168 extending from 308 degrees to 311 degrees contains 18 ORFs. Functions of most of these ORFs were identified and associated with cell wall metabolism. Sequences of two non-coding regions of 0.7 and 2.2 kb flanking the ggaAB operon involved in the synthesis of poly(3-O-beta-D-glucopyranosyl N-acetylgalactosamine 1-phosphate), a minor teichoic acid, correspond to five degenerate segments of neighbouring protein-coding regions. We discuss the possibility that such grey holes are indicative of a chromosomal rearrangement which could have arisen from horizontal gene transfer.

Specific labeling of diaminopimelate: a radioassay for the determination of the peptidoglycan cross-linking index.

A radioassay for the determination of the peptidoglycan cross-linking index (CLI) was devised. It is based on specific radioactive labeling of diaminopimelic acid (DAP) by diverting [14C]aspartate into the DAP pathway, while inhibiting incorporation of label into other cell wall components. Purified [14C]DAP-labeled cell walls were treated with fluorodinitrobenzene, hydrolyzed, and chromatographed by TLC. The radioactivity in well-separated mono dinitrophenyl-diaminopimelate (DNP-DAP) and DAP spots was counted and the CLI was determined from the ratio of DAP to the total of mono DNP-DAP and DAP counts. The method, suitable for bacteria such as Bacillus subtilis unable to incorporate exogenous DAP, can be applied to other systems. A CLI of 50.8 +/- 1.3% and 55.5 +/- 0.9% was obtained for B. subtilis 168 cells growing exponentially in rich and minimal medium, respectively. Comparison of these to results previously obtained on B. subtilis suggested the existence of a hitherto unreported peptidoglycan endopeptidase activity.

Analysis of Bacillus subtilis tag gene expression using transcriptional fusions.

Five of the genes known to encode the synthesis of poly(glycerol phosphate), the major teichoic acid of Bacillus subtilis 168, are organized in two divergently transcribed operons (a divergon), denoted tagAB and tagDEF. To monitor their expression, the 399 bp intergenic region separating the first structural genes of these operons was fused, in both orientations, to a lacZ reporter gene, allowing measurement of promoter activity under specific physiological conditions. Under all experimental conditions, tagA and tagD appeared coordinately expressed, the level of tagD being always higher than that of tagA. No influence of the chromosomal context was observed. Phosphate limitation was accompanied by reduced tag gene expression. Following the onset of sporulation, expression of tag genes diminished rapidly and was essentially abolished by stage II. During germination, the activity of tag genes was detectable before the rise in culture turbidity associated with spore outgrowth. In contrast to tagC (dinC), the expression of which is DNA-damage-inducible, the induction of SOS functions had no effect on tagA and tagD gene expression. The biological significance of these results is discussed.