Anne Moir

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 ≈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.

Genes of Bacillus cereus and Bacillus anthracis Encoding Proteins of the Exosporium

The exosporium is the outermost layer of spores of Bacillus cereus and its close relatives Bacillus anthracis and Bacillus thuringiensis. For these pathogens, it represents the surface layer that makes initial contact with the host. To date, only the BclA glycoprotein has been described as a component of the exosporium; this paper defines 10 more tightly associated proteins from the exosporium of B. cereus ATCC 10876, identified by N-terminal sequencing of proteins from purified, washed exosporium. Likely coding sequences were identified from the incomplete genome sequence of B. anthracis or B. cereus ATCC 14579, and the precise corresponding sequence from B. cereus ATCC 10876 was defined by PCR and sequencing. Eight genes encode likely structural components (exsB, exsC, exsD, exsE, exsF, exsG, exsJ, and cotE). Several proteins of the exosporium are related to morphogenetic and outer spore coat proteins of B. subtilis, but most do not have homologues in B. subtilis. ExsE is processed from a larger precursor, and the CotE homologue appears to have been C-terminally truncated. ExsJ contains a domain of GXX collagen-like repeats, like the BclA exosporium protein of B. anthracis. Although most of the exosporium genes are scattered on the genome, bclA and exsF are clustered in a region flanking the rhamnose biosynthesis operon; rhamnose is part of the sugar moiety of spore glycoproteins. Two enzymes, alanine racemase and nucleoside hydrolase, are tightly adsorbed to the exosporium layer; they could metabolize small molecule germinants and may reduce the sensitivity of spores to these, limiting premature germination.

Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore.

The GerAA, -AB, and -AC proteins of the Bacillus subtilis spore are required for the germination response to L-alanine as the sole germinant. They are likely to encode the components of the germination apparatus that respond directly to this germinant, mediating the spores response; multiple homologues of the gerA genes are found in every spore former so far examined. The gerA operon is expressed in the forespore, and the level of expression of the operon appears to be low. The GerA proteins are predicted to be membrane associated. In an attempt to localize GerA proteins, spores of B. subtilis were broken and fractionated to give integument, membrane, and soluble fractions. Using antibodies that detect Ger proteins specifically, as confirmed by the analysis of strains lacking GerA and the related GerB proteins, the GerAA protein and the GerAC+GerBC protein homologues were localized to the membrane fraction of fragmented spores. The spore-specific penicillin-binding protein PBP5*, a marker for the outer forespore membrane, was absent from this fraction. Extraction of spores to remove coat layers did not release the GerAC or AA protein from the spores. Both experimental approaches suggest that GerAA and GerAC proteins are located in the inner spore membrane, which forms a boundary around the cellular compartment of the spore. The results provide support for a model of germination in which, in order to initiate germination, germinant has to permeate the coat and cortex of the spore and bind to a germination receptor located in the inner membrane.

Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination

The location and function of recognized cortex-lytic enzymes of Bacillus subtilis have been explored, and the involvement in germination of a number of related proteins tested. The SleB and CwlJ proteins are cortex-lytic enzymes, partially redundant in function, that are required together for effective cortex hydrolysis during B. subtilis spore germination. Spores were fractionated, and Western blotting of individual fractions suggests that the CwlJ protein is localized exclusively to the outer layers, or integument. The second spore-lytic enzyme, SleB, is localized both in the inner membrane of the spore and in the integument fraction. Neither protein changes location or size as the spore germinates. The ypeB gene is the second gene in a bicistronic operon with sleB. The SleB protein is absent from ypeB mutant spores, suggesting that YpeB is required for its localization or stabilization. In fractions of wild-type spores, the YpeB protein is found in the same locations as SleB - in both the inner membrane and the integument. As the absence of CwlJ protein does not affect the overall RP-HPLC profile of peptidoglycan fragments in germinating spores, this enzymes hydrolytic specificity could not be defined. The effects of inactivation of several homologues of cortex-lytic enzymes of as yet undefined function were examined, by testing null mutants for their germination behaviour by OD(600) fall and by RP-HPLC of peptidoglycan fragments from dormant and germinating spores. The YaaH enzyme is responsible for a likely epimerase modification of peptidoglycan during spore germination, but the loss of this activity does not appear to affect the spores ability to complete germination. Unlike the other cortex-lytic enzymes, the YaaH protein is present in large amounts in the spore germination exudate of B. subtilis. Mutants lacking either YdhD or YvbX, both homologues of YaaH, had no detectable alteration in either dormant or germinating spore peptidoglycan, and germinated normally. The ykvT gene, which encodes a protein of the SleB/CwlJ family, has no apparent association with germination: the gene is expressed in vegetative cells, and mutants lacking YkvT have no detectable phenotype.

Germination of Bacillus cereus spores in response to L-alanine and to inosine: the roles of gerL and gerQ operons

Bacillus cereus 569 (ATCC 10876) endospores germinate in response to inosine or L-alanine, the most rapid germination response being elicited by a combination of these germinants. The gerI operon has already been characterized as a homologue of the gerA spore-germination receptor family of operons found in all Bacillus spp. examined; the primary defect in gerI mutant spores is in the inosine germination response, although spores were also slower to germinate in L-alanine. Additional transposon-insertion mutants, from similar Tn917-LTV1 mutagenesis and enrichment experiments, now define two more operons, also members of the family of gerA homologues, important in L-alanine and inosine germination. Transposon insertions were identified in an alanine-specific germination locus, named gerL, which represents an operon of three genes, termed gerLA, gerLB and gerLC. By examining the residual germination response to L-alanine in gerI and gerL mutants, it was deduced that the GerL proteins contribute most strongly to the L-alanine germination response, and that the GerI proteins, required primarily in inosine germination, mediate only much slower germination responses to alanine. The L-alanine germination responses mediated by GerL and GerI proteins differ in their germination rates, temperature optima and germinant concentration dependence. The gerQ locus, again identified by transposon insertion, is a second inosine-related germinant-receptor operon. GerQ and GerI proteins are both required for the germination response to inosine as sole germinant, but GerQ has no role in L-alanine germination. Although near-identical homologues of gerI and gerL operons are evident in the Bacillus anthracis genome sequence, there is no evidence of a close homologue of gerQ.

Characterization of the exosporium of Bacillus cereus

Exosporium components from endospores of Bacillus cereus ATCC 10876 were purified and separated by gel electrophoresis. Several of the proteins for which N‐terminal sequences were recovered were found to have homologues in protein databases which have been demonstrated to have enzymic activity in other organisms. Amongst these is a zinc metalloprotease, immune inhibitor A, already described in B. thuringiensis. This has been shown to be present in an active 73 kDa form on the exosporium of B. cereus. Other proteins associated with the exosporium include the molecular chaperone GroEL and a homologue of RocA (1‐pyrroline‐5‐carboxylate dehydrogenase (EC 1.5.1.12)) of B. subtilis. Although these are unlikely to represent integral structural proteins of the exosporium, the observation that they are selectively present in the spore surface layer suggests that this layer may have functional significance.

Sigma M, an ECF RNA polymerase sigma factor of Bacillus subtilis 168, is essential for growth and survival in high concentrations of salt.

The Bacillus subtilis 168 genome encodes seven extracytoplasmic function (ECF) RNA polymerase sigma factors of unknown physiological function. The sigM(yhdM ) gene, encoding an ECF sigma factor σM, is essential for growth and survival in nutrient broth (NB) containing 1.4 M NaCl. Strains insertionally inactivated in the sigM gene form aberrantly shaped cells, which swell and lyse spontaneously during growth in NB medium containing increased levels (0.35–0.7 M) of a wide range of different salts. The sigM gene was co‐transcribed with the yhdL and yhdK genes with transcription initiating from two promoters, PA and PM. The transcript from PM was not detected in a sigM mutant, indicating that the expression of sigM was positively autoregulated. Expression of sigM was maximal during exponential growth and was increased by 50% in NB medium containing 0.7 M NaCl. The activity of σM is negatively regulated by the proteins encoded by the yhdL and yhdK genes.

SigM, an Extracytoplasmic Function Sigma Factor of Bacillus subtilis, Is Activated in Response to Cell Wall Antibiotics, Ethanol, Heat, Acid, and Superoxide Stress

The extracytoplasmic function sigma M of Bacillus subtilis is required for normal cell growth under salt stress. It is expressed maximally during exponential growth and is further induced by the addition of 0.7 M NaCl. The promoter region of the sigM operon contains two promoters; one (P(A)) is sigma A dependent, and the other (P(M)) is sigma M dependent. These have been placed separately at the amy locus, directing expression of a lacZ reporter gene. Only the P(M) fusion responded to salt induction. This promoter, which was responsive to the level of active sigma M in the cell, was also induced by 5% ethanol, by vancomycin, bacitracin, or phosphomycin (inhibitors of cell wall biosynthesis; 2 micro g per ml), and by heat shock of 50 degrees C for 10 min. It was very strongly induced by acid (pH 4.3) and 80 micro M paraquat, but after a 15- to 30-min delay. There was no induction by alkali (pH 9), 5 mM H(2)O(2), the detergents 0.1% Triton X-100 and 0.1% Tween 20, or 50 micro M monensin. In addition to their reduced tolerance to salt, null mutants of sigM were unable to grow at pH 4.3 and lysed after exposure to 5% ethanol. Genes regulated by SigM were also tested for their response to pH 4.3, 5% ethanol, and 2 micro g of vancomycin per ml. Expression of the genes may have been activated by increased levels of sigma M, but at least some were also subject to additional controls, as they responded to one type of stress but not another. Expression of yrhJ, which encodes a cytochrome P450/NADPH reductase, was induced in response to acid and vancomycin. yraA expression was acid, ethanol, and vancomycin induced, whereas yjbD showed only ethanol induction. YraA protein was extremely important to acid survival-a mutation in yraA, like a sigM mutation, resulted in the failure of B. subtilis to grow at pH 4.3. Sigma M is therefore involved in maintaining membrane and cell wall integrity in response to several different stresses in exponential growth phase and is activated by such stresses.

Genetic Analysis of Spore Germination Mutants of Bacillus subtilis 168 : the Correlation of Phenotype with Map Location

The isolation and characterization of 29 new germination (Ger) mutants of Bacillus subtilis 168 is described. These were classified, along with previously described mutants, into seven groups according to map location. The mutations in 26 GerA mutants mapped between cysB and thr; detailed mapping of two of these has located them very close to citG. These mutants were deficient in germination in alanine, but responded to the germinative combination of asparagine, glucose, fructose and KCl. One GerB mutant mapped on the origin-proximal side of hisA; it was normal in germination in alanine, but deficient in termination in a mixture of asparagine, glucose, fructose and KCl. Two GerC mutants were linked to lys, but were separable from a temperature-sensitive growth deficiency mapping between lys and trp. The GerC mutants had a similar germination phenotype to the GerA mutants. Three GerD mutants did not germinate in either of the above germinants or in Penassay Broth. They were located on the side of ery distal to cysA. The GerE mutant, which did not germinate in any of the three germinants, was located very close to citF and possessed an altered spore coat. The two GerF mutants were defective in germination in all three germinants and mapped on the origin proximal-side of hisA, but much closer to his than did the GerB mutant. A phosphoglycerate kinase-negative mutant altered in germination mapped between cysB and hisA (GerG). These mutants have established a minimum of seven locations important to germination, and will be useful in the development and appraisal of theories of spore germination.

Mutations in the gerP Locus of Bacillus subtilis and Bacillus cereus Affect Access of Germinants to Their Targets in Spores

The gerP1 transposon insertion mutation of Bacillus cereus is responsible for a defect in the germination response of spores to both L-alanine and inosine. The mutant is blocked at an early stage, before loss of heat resistance or release of dipicolinate, and the efficiency of colony formation on nutrient agar from spores is reduced fivefold. The protein profiles of alkaline-extracted spore coats and the spore cortex composition are unchanged in the mutant. Permeabilization of gerP mutant spores by coat extraction procedures removes the block in early stages of germination, although a consequence of the permeabilization procedure in both wild type and mutant is that late germination events are not complete. The complete hexacistronic operon that includes the site of insertion has been cloned and sequenced. Four small proteins encoded by the operon (GerPA, GerPD, GerPB, and GerPF) are related in sequence. A homologous operon (yisH-yisC) can be found in the Bacillus subtilis genome sequence; null mutations in yisD and yisF, constructed by integrational inactivation, result in a mutant phenotype similar to that seen in B. cereus, though somewhat less extreme and equally repairable by spore permeabilization. Normal rates of germination, as estimated by loss of heat resistance, are also restored to a gerP mutant by the introduction of a cotE mutation, which renders the spore coats permeable to lysozyme. The B. subtilis operon is expressed solely during sporulation, and is sigma K-inducible. We hypothesize that the GerP proteins are important as morphogenetic or structural components of the Bacillus spore, with a role in the establishment of normal spore coat structure and/or permeability, and that failure to synthesize these proteins during spore formation limits the opportunity for small hydrophilic organic molecules, like alanine or inosine, to gain access to their normal target, the germination receptor, in the spore.