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Stable maintenance of the low-copy-number plasmid pBsph in Bacillus sphaericus requires a partitioning ( par ) system that consists of a filament-forming protein, B. sphaericus TubZ (TubZ-Bs); a centromere-binding protein, TubR-Bs; and a centromere-like DNA site, tubC , composed of three blocks (I, II, and III) of 12-bp degenerate repeats. Previous studies have shown that mini-pBsph replicons encoding the TubZ system are segregationally highly unstable, whereas the native pBsph is stably maintained. However, the mechanism underlying the stability discrepancy between pBsph and its minireplicon is poorly understood. Here orf187 (encoding TubX), a gene downstream of tubZ -Bs, was found to play a role in plasmid stabilization. Null mutation or overexpression of tubX resulted in a defect in pBsph stability and a significant decrease in the level of tubRZ -Bs expression, and the TubX-null phenotype was suppressed by ectopic expression of a wild-type copy of tubX and additional tubRZ -Bs. An electrophoresis mobility shift assay (EMSA) and a DNase I footprinting assay revealed that the TubX protein bound directly to five 8-bp degenerate repeats located in the par promoter region and that TubX competed with TubR-Bs for binding to the par promoter. Further studies demonstrated that TubX significantly stimulated the transcription of the par operon in the absence of tubR -Bs, and a higher level of gene activation was observed when tubR -Bs was present. These results suggested that TubX positively regulates tubRZ -Bs transcription by interfering with TubR-Bs-mediated repression and binding directly to the tubRZ -Bs promoter region.

Bacillus sphaericus strain C3-41 is an aerobic, mesophilic, spore-forming bacterium that has been used with great success in mosquito control programs worldwide. Genome sequencing revealed that the complete genome of this entomopathogenic bacterium is composed of a chromosomal replicon of 4,639,821 bp and a plasmid replicon of 177,642 bp, containing 4,786 and 186 potential protein-coding sequences, respectively. Comparison of the genome with other published sequences indicated that the B. sphaericus C3-41 chromosome is most similar to that of Bacillus sp. strain NRRL B-14905, a marine species that, like B. sphaericus , is unable to metabolize polysaccharides. The lack of key enzymes and sugar transport systems in the two bacteria appears to be the main reason for this inability, and the abundance of proteolytic enzymes and transport systems may endow these bacteria with exclusive metabolic pathways for a wide variety of organic compounds and amino acids. The genes shared between B. sphaericus C3-41 and Bacillus sp. strain NRRL B-14905, including mobile genetic elements, membrane-associated proteins, and transport systems, demonstrated that these two species are a biologically and phylogenetically divergent group. Knowledge of the genome sequence of B. sphaericus C3-41 thus increases our understanding of the bacilli and may also offer prospects for future genetic improvement of this important biological control agent.

In Bacillus sphaericus and other Bacillus spp., d -amino acid transaminase has been considered solely responsible for biosynthesis of d -glutamate, an essential component of cell wall peptidoglycan, in contrast to the glutamate racemase employed by many other bacteria. We report here the cloning of the dat gene encoding d -amino acid transaminase and the glr gene encoding a glutamate racemase from B. sphaericus ATCC 10208. The glr gene encodes a 28.8-kDa protein with 40 to 50% sequence identity to the glutamate racemases of Lactobacillus , Pediococcus , and Staphylococcus species. The dat gene encodes a 31.4-kDa peptide with 67% primary sequence homology to the d -amino acid transaminase of the thermophilic Bacillus sp. strain YM1.

After ingestion of the parasporal crystals of Bacillus sphaericus, mosquito larvae process the 42-kilodalton (kDa) toxin to a protein of 39 kDa, which has an increased toxicity (A. H. Broadwell and P. Baumann, Appl. Environ. Microbiol. 53:1333-1337, 1987). A similar activation is performed by trypsin and chymotrypsin. Using site-directed mutagenesis, we have constructed derivatives of the 42-kDa toxin with a deletion of 10 amino acids at the N terminus and deletions of 7, 17, or 20 amino acids at the C terminus. Toxicity for mosquito larvae was retained upon deletion of 7 or 17 amino acids but was lost upon deletion of 20 amino acids. Evidence is presented indicating that the protein containing deletions of 10 amino acids at the N terminus and 17 amino acids at the C terminus (corresponding to potential chymotrypsin cleavage sites) is similar to the 39-kDa protein produced in mosquito larvae or by digestion with chymotrypsin. Digestion with trypsin appears to generate a protein lacking 16 or 19 amino acids from the N terminus and 7 amino acids from the C terminus. As is the case with the recombinant-made 42-kDa protein, toxicity of its derivatives is dependent on the presence of a 51-kDa protein which is a component of the parasporal crystal of B. sphaericus 2362.

After site-directed mutagenesis, the genes coding for the 42- and 51-kilodalton (kDa) mosquitocidal proteins of Bacillus sphaericus 2362 were placed under the regulation of the aprE (subtilisin) promoter of the Bacillus subtilis vector pUE (a derivative of pUB18). The levels of expression of the gene products in B. subtilis DB104 and B. sphaericus 718 were assessed by bioassays with larvae of Culex pipiens and by Western immunoblots. The results indicated that a higher amount of protein was produced in B. subtilis DB104. Electron microscopic examination of B. subtilis DB104 and B. sphaericus 718 containing the 42- and 51-kDa proteins indicated that amorphous inclusions accumulated in the former species and that crystals identical in appearance to that found in B. sphaericus 2362 were produced in the latter. Strains producing only the 42- or the 51-kDa protein were not toxic to larvae of C. pipiens. A mixture of both strains, a single strain producing both proteins, or a fusion of the 51- and the 42-kDa proteins was toxic. The amount of B. subtilis DB104 containing the 42- and the 51-kDa proteins necessary to kill 50% of the larvae of C. pipiens was 5.6 ng (dry weight) of cells per ml. This value was significantly lower than that for B. sphaericus 2362 (14 ng [dry weight] per ml). Larvae consuming purified amorphous inclusions containing the 42-kDa protein degraded this protein this protein to primarily 39- and 24-kDa peptides, whereas inclusions with the 51-kDa protein were primarily degraded to a protein of 44 kDa. Past studies involving purified proteins from B. sphaericus 2362 indicate an associate of toxicity with the 39-kDa peptide. The results presented here suggest that the 44-kDa degradation product of the 51-kDa protein may also be required for toxicity.

A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene.

Clones expressing regions of the 100-kDa Bacillus sphaericus SSII-1 mosquitocidal toxin (Mtx) as fusion proteins with glutathione S-transferase were constructed, and the toxin-derived peptides were purified. The in vitro ADP-ribosylation activities of these peptides and their effects on larvae and cells in culture were studied. Mtx25 (amino acids 30 to 493) was found to ADP-ribosylate two proteins with molecular masses of 38 and 42 kDa, respectively, in Culex quinquefasciatus (G7) cell extracts, in addition to ADP-ribosylating itself. Mtx21 (amino acids 30 to 870; or a combination of Mtx25 and Mtx26 (amino acids 259 to 870) caused mortality in C. quinquefasciatus larvae. Mtx25, Mtx26, or Mtx24 (amino acids 30 to 276) alone and Mtx24 in combination with Mtx26 were not toxic to larvae. Mtx21 and Mtx26 produced marked morphological changes in G7 cells and to a lesser extent in Aedes aegypti cells but had no effect on Anopheles gambiae or HeLa cells. Thus, a domain in the N-terminal region of the Mtx protein is sufficient for ADP-ribosylation of C. quinquefasciatus cell protein, and a domain in the C-terminal region is sufficient for toxicity to cultured C. quinquefasciatus cells; however, both regions are necessary for toxicity to mosquito larvae.

The protein surface layers on the cell walls of mosquito-pathogenic and nonpathogenic Bacillus sphaericus strains were studied by structural, biochemical, and serological methods. The surface structure of two representative insect-pathogenic strains had the form of a delicate linear array with a repeat interval of 5 nm. This was distinctly different from the tetragonal array of the P-1 strain in spacing and arrangement. The surface layers were composed of acidic glycoproteins with molecular weights ranging from approximately 133,000 to 155,000. Peptide mapping and serological analysis of the surface proteins revealed eight distinct groups among the pathogens. These groups were very similar to the groupings determined by flagellar-antigen serotyping and bacteriophage typing.

The level of three peptidoglycan hydrolases was determined in the mother cell compartment and forespores of Bacillus sphaericus. Vegetative and sporulating cells contained in LD-carboxypeptidase active only on the vegetative cell wall peptidoglycan, and we have previously shown that sporulation is accompanied by the production of two new enzymes active only on the spore cortex peptidoglycan. These gamma-D-glutamyl-meso-diaminopimelate endopeptidase and a meso-diaminopimelate-D-alanine dipeptidase. The LD-carboxypeptidase activity appeared to be located in the membranes of both the mother cells and forespores. Endopeptidase activity was located in the integument fraction of the forespores, and the dipeptidase activity was only found in the forespore cytoplasm. These different locations comply with the probable different functions of these enzymes.

The outermost layer of the cell wall of Bacillus sphaericus strain P-1 is a tetragonally arrayed structure (T-layer) which is assembled from a single polypeptide. No turnover of T-layer was detected during growth of cultures. In contrast, the turnover of peptidoglycan was between 20 and 25% per generation. The sites of deposition of new T-layer on the cell surface were identified by the indirect fluorescent antibody technique, which labeled old T-layer, and by the reverse technique, which labeled new T-layer. These experiments demonstrated that the major area of T-layer deposition was a band at the site of an incipient cell division. This band subsequently split and covered the new pole of each progeny cell. Little or no T-layer was inserted into existing poles. In addition, multiple bands of new T-layer, which probably accommodate cell elongation, were inserted along the lateral surface of the cell.

Tetragonal layer protein (T-layer) isolated from Bacillus sphaericus NTCC 9602 (wild type) or 9602 Lmw (variant) bonded specifically to the sacculi (peptidoglycan) of either cell type. Only uncleaved T-layer subunits were capable of specific recognition of the B. sphaericus sacculi; other Bacillus strains and gram-positive bacterial sacculi would not adsorb B. sphaericus strain 9602 T-layer. The peptidogylcan did not function as a template since isolated T-layer subunits self-assembled into characteristic pattern. Upon reassociation with sacculi, T-layer assemblies were randomly oriented patches compared with more continuous strictly oriented pattern on cells or fresh cell walls. T-layer associated with the sacculus was less susceptible to conditions that dissociated in vitro-assembled T-layer. Mild proteolysis of both wild-type and variant T-layer subunits by a variety of enzymes reduced the molecular weight by 18,000 in all cases, indicating that one region of the molecule was particularly susceptible to cleavage. Subunits from which the minor fragment had been cleaved upon aging retained the capacity to assemble in vitro, but would no longer adsorb to sacculi. Thus, the ability of T-layer to form networks was separate from its ability to bind cell walls, and the 18,000-dalton piece of the T-layer polypeptide was necessary for attachment to the cell wall.

The muramic lactam content of spores of Bacillus sphaericus mutants defective in meso-diaminopimelic acid synthesis increases almost linearly with an increase of meso-diaminopimelic acid concentration in the medium. Since muramic lactam content is a measure of cortex content, the amount of cortex in spores of the mutants can be easily varied by changing the meso-diaminopimelic acid concentration in the medium. Characteristic properties were tested in spores containing different amounts of cortex. Critical amounts of cortex were associated with different spore properties. Refractility and dipicolinic acid accumulation in the spores both required about 20% of the maximum cortex content (although refractility is independent of dipicolinic acid content). For xylene octanol resistance, about 25% of the maximum cortex content was required.

The distribution of bacterial L-ornithine: alpha-ketoglutarate delta-aminotransferase (L-ornithine:2-oxo-acid aminotransferase [EC 2.6.1.13]) was investigated, and Bacillus sphaericus (IFO 3525) was found to have the highest activity of the enzyme, which was inducibly formed by addition of L-ornithine or L-arginine to the medium. L-Ornithine:alpha-ketoglutarate delta-aminotransferase, purified to homogeneity and crystallized from B. sphaericus, had a molecular weight of about 80,000 and consisted of two subunits identical in molecular weight (41,000) and in amino-terminal residue (threonine). The enzyme exhibited absorption maxima at 278,343, and 425 nm and contained 1 mol of pyridoxal 5'-phosphate per mol of enzyme. The formyl group of pyridoxal 5'-phosphate was bound through an aldimine linkage to the epsilon-amino group of a lysine residue of the protein. The enzyme-bound pyridoxal 5'-phosphate, absorbing at 425 nm, was released by incubation with phenylhydrazine to yield the catalytically inactive form. The inactive enzyme, which was reactivated by addition of pyridoxal 5'-phosphate, still had a 343-nm peak and contained 1 mol of a vitamin B6 compound. The holoenzyme showed positive circular dichroic bands at 340 and 425 nm, whereas the inactive form had no band at 425 nm. The enzyme was highly specific for L-ornithine and alpha-ketoglutarate and catalyzed delta-transamination between them to produce L-glutamate and L-glutamate-gamma-semialdehyde, which as spontaneously converted to delta 1-pyrroline-5-carboxylate. The enzyme activity was significantly affected by nonsubstrate amino acids, amines, and carbonyl reagents.

The tetragonally arranged cell wall layer (T-layer) of Bacillus sphaericus NTCC 9602 was isolated and characterized. Parallel studies were made on a spontaneous variant of the wild-type strain which had a T-layer subunit of altered molecular weight. A purification method for the T-layers was devised which involved separation of the cell walls from the cytoplasmic contents, urea dissociation of the T-layer from the cell walls, removal of soluble contaminants by differential centrifugation, and finally selective adsorption of uncleaved subunits to sacculi. The purified subunits retained the capacity to form an assembly in vitro with the same lattice parameters as that observed on whole cells or cell walls and could readsorb to the cell walls from which they had been extracted. Both the wild-type and the variant subunits behaved as single, homogeneous polypeptide chains. Carbohydrate assay and isoelectric point determinations revealed that both subunit types were acidic glycoproteins. Values obtained for thebuoyant density, isoelectric point, and extinction coefficient differed minimally; major differences were observed in the molecular weight and the characterisitc width of cylinders formed by in vitro-assembled T-layer of the wild-type and variant. Assembled T-layer was subject to alkaline or acid dissociation and in acid titration dissociated at its isoelectric point.

An LL-oligopeptidase was characterized in the cell cytoplasm of sporulating Bacillus sphaericus 9602. Its activity showed a threefold increase throughout sporulation. The enzyme has lytic activity on various LL-dipeptides, especially on dipeptides with N-terminal L-alanine. Lytic activity was also found on some tripeptides and larger peptides which contain the sequence L-Ala-L-Ala. The role of this oligopeptidase in relation to sporulation may be to supply the cell with L-alanine for the biosynthesis of the peptide chains of the spore cortex.

The nucleotide sequences of a 3,479-base-pair HindIII DNA fragment from Bacillus sphaericus 2362 and a 2,940-base-pair fragment from strain 2297 were determined; only minor differences were detected between them. Each contained two open reading frames coding for proteins of 51.4 and 41.9 kilodaltons. Both proteins were required for toxicity to larvae of the mosquito Culex pipiens.

In synchronously sporulating cells of Bacillus sphaericus 9602, the specific activities of those enzymes specifically required for the synthesis of the UDP- N -acetyl-muramyl-pentapeptide precursor of vegetative cell wall peptidoglycan decay by 50% after the end of exponential cell division, probably as a consequence of dilution by newly synthesized protein. The meso -diaminopimelate ligase is the only new activity whose synthesis is required for synthesis of the nucleotide-pentapeptide precursor of spore cortex peptidoglycan. The addition of d -Ala- d -Ala to the nucleotide tripeptide is catalyzed by an enzyme present in both vegetative and sporulating cells, which apparently does not discriminate between lysine- and diaminopimelate-containing acceptors. The activities of the l -Ala and d -Ala- d -Ala ligases and of the d -Ala- d -Ala synthetase increases in parallel with the appearance of the diaminopimelate ligase, indicating coordinate derepression and suggesting operon-like organization of the appropriate structural genes.

A high activity of meso-alpha-epsilon-diaminopimelate dehydrogenase was found in extracts of Bacillus sphaericus, Brevibacterium sp., Corynebacterium glutamicum, and Proteus vulgaris among bacteria tested. B. sphaericus IFO 3525, in which the enzyme is most abundant, was chosen to study the enzyme reaction. The enzyme was not induced by the addition of meso-alpha-epsilon-diaminopimelate to the growth medium. The reaction product was isolated and identified as alpha-amino-epsilon-ketopimelate by a comparison of the properties of its 2,4-dinitrophenylhydrazone with those of an authentic sample in silica gel thin-layer chromatography, absorption, infrared and proton nuclear magnetic resonance spectrometry, and elemental analyses. The alpha-amino-epsilon-ketopimelate formed enzymatically was decarboxylated by H2O2 to yield L-alpha-aminoadipate. This suggests that the amino group with D-configuration in the substrate is oxidatively deaminated; the enzyme is a D-amino acid dehydrogenase. L-alpha-Amino-epsilon-ketopimelate undergoes spontaneous dehydration to the cyclic delta1-piperideine-2,6-dicarboxylate. The enzyme reaction is reversible, and meso-alpha-epsilon-diaminopimelate was formed in the reductive amination of L-alpha-epsilon-ketopimelate.

The cell wall peptidoglycan of vegetative cells of Bacillus sphaericus 9602 contains l -lysine and d -isoasparagine and is devoid of diaminopimelic acid (Dap), whereas the peptidoglycan of its spore cortex is devoid of l -lysine and d -isoasparagine and contains meso -Dap. These two structures have a common biosynthetic precursor, uridine-diphospho- N -acetylmuramyl- l - alanyl- d -glutamic acid, which accepts either l -lysine or meso -Dap, the latter reaction being the first unique to the synthesis of the spore cortex peptidoglycan. l -lysine-adding activity decays at the end of vegetative growth to a level which is maintained until Dap-adding activity appears, when it declines rapidly again. Dap-adding activity is not detectable in refractile spores, in vegetative cells, or in sporulating cells until about 4 hr after the end of vegetative growth, when it increases rapidly for about 1.5 hr in a process dependent on continued protein and ribonucleic acid (RNA) synthesis. This process apparently involves transcription and translation during this period of a “sporulation-specific” gene whose product is essential for and unique to sporulation. It is closely followed by the acquirement of refractility. Another sporulation-specific gene, that for dipicolinate synthase, is apparently transcribed and translated in an overlapping period commencing about 0.5 hr later, although dipicolinate does not accumulate rapidly until 1.5 hr later, when about 75% of the cells are already refractile. Inhibition of protein synthesis with chloramphenicol or of RNA synthesis with streptolydigin inhibited accumulation of these enzymes in sporulating cells; this inhibition could be reversed by washing out the antibiotics after 1.5 hr. Sporulation recommenced with an unaltered sequence of events but with poorer synchrony. There was no evidence for a messenger RNA for either enzyme of lifetime greater than a small fraction of the period of enzyme accumulation, although dilution with 10 volumes of fresh medium failed to prevent synthesis of Dap-adding enzyme in cells which had become terminally swollen, a process preceding enzyme synthesis by about 1.5 hr. The synthesis of this enzyme in B. sphaericus is apparently dependent on programmed transcription of the appropriate gene.

Baker, Edgar E. (Boston University School of Medicine, Boston, Mass.), and Roberta E. Whiteside . Preparation and properties of a Vi antigen-degrading enzyme. J. Bacteriol . 89: 1217–1224. 1965.—Vi antigen can be hydrolyzed by an inducible enzyme produced by a microorganism isolated from soil. The organism can be identified as Bacillus sphaericus . The enzyme in the culture supernatant fluid can be concentrated and partially purified by precipitation with ammonium sulfate at 50% of saturation. The partially purified enzyme is relatively stable. Incubation of the enzyme with Vi antigen causes a rapid loss of serological activity, a decrease in viscosity, the loss of ability to precipitate albumin at p H 4.0, and the production of reducing sugar and hexosamine. Optimal conditions for enzyme action on Vi antigen are p H 8.4 and 40 C. The reaction rate is independent of the substrate concentration. Intact protein and calcium activates the Vi antigen-enzyme system. The enzyme is not inhibited by its reaction products.

Bacillus sphaericus strain P-1 has previously been shown to have a tetragonally arrayed (T layer) protein which forms the outer layer of the cell wall. The T layer was quantitatively extracted from whole cells by 6 M urea, and the T layer subunits were purified by electrophoresis of the extracts on acrylamide gels containing 0.1% sodium dodecyl sulfate or 6 M urea. Using ethylene diacrylate cross-linked gels, the T layer was found to make up 16% of the total cellular protein. A virulent bacteriophage which is inactivated by purified T layer was isolated from soil. Twenty-four phage-resistant mutants were isolated, of which 17 had T layer subunits of increased mobility on sodium dodecyl sulfate acrylamide gels. No mutants devoid of T layer were found. Mutants were grouped into six classes according to the molecular weight of their T layer subunits. These ranged from that of the wild type, 150,000 down to 86,000. Two mutants from different classes were examined in detail. Cells of the mutant strains did not adsorb phage nor did cell walls isolated from these mutants inactivate phage. The amino acid composition of the T layers from mutants differed little from that of the wild-type T layer.

The three-dimensional structure of the regular surface layer of Bacillus sphaericus P-1 (T-layer) was determined to a resolution of ca. 2.5 nm by electron microscopy and image analysis. The T-layer has P4 symmetry, a lattice constant of 13 +/- 0.2 nm, and a thickness of ca. 8 nm. The reconstruction revealed three distinct domains: a major, a minor, and an arm domain. In the z-direction, the domains are arranged in two planes creating two different surface reliefs.

Particulate preparations from sporulating cells of Bacillus sphaericus 9602 contained an endopeptidase activity that hydrolyzed the γ- d -glutamyl-( l ) meso -diaminopimelic acid linkages found in the spore cortical peptidoglycan of this organism. Diaminopimelic acid did not occur in the vegetative cell wall peptidoglycan, and the γ- d -glutamyl- l -lysine linkages found in this polymer were not hydrolyzed by the endopeptidase. The endopeptidase hydrolyzed (X)- l -alanyl-γ- d -glutamyl-( l ) meso -diaminopimelyl( l )- d -alanyl- d -alanine only after removal of the terminal d -alanine residue. The preparations contained an acyl- d -alanyl- d -alanine carboxypeptidase I activity which converted such pentapeptides into substrates for the endopeptidase and which was inhibited 50% by 4 × 10 −7 M benzylpenicillin. This activity also hydrolyzed the analogous pentapeptide substrates containing l -lysine. The preparations also contained an acyl- l -lysyl- d -alanine carboxypeptidase II activity that was not active on the meso -diaminopimelic acid-containing analogue. Neither this activity nor the endopeptidase was inhibited by 10 −3 M benzylpenicillin. The specificities of the carboxypeptidases were consistent with the exclusive presence of l -lysine C-termini in the vegetative peptidoglycan and of meso -diaminopimelyl- d -alanine C-termini in the spore cortical peptidoglycan of B. sphaericus 9602.

Two major low-molecular weight, acid-soluble proteins (termed A and B proteins) were purified from Bacillus sphaericus spores and had properties similar to those of the analogous proteins from spores of other Bacillus species. These proteins were accumulated late in sporulation, when the developing spores became resistant to UV light, and were degraded during spore germination by a spore protease. A mutant of B. sphaericus unable to make spore cortex because of a block in diaminopimelic acid (DAP) biosynthesis accumulated and maintained levels of the A and B proteins similar to those in the DAP+ parent or the DAP- strain in which cortex formation was restored by growth with DAP. In addition, the DAP- strain grown without DAP acquired a level of UV light resistance identical to that of wild-type spores and at the time of appearance of the A and B proteins. These findings indicate that formation of little, if any, spore cortex is required for acquisition of UV light resistance or maintenance of high levels of A and B proteins. The data provide further support for a role of the A and B proteins in the spore's UV light resistance.

Sporulating cells of Bacillus sphaericus 9602 containing fully engulfed forespores at different stages of maturity were broken by ultrasonic disruption, followed by grinding with alumina. In this way soluble enzymes derived mainly from the sporangial or from the forespore cytoplasms were obtained. Diaminopimelate ligase activity is required exclusively for cortical peptidoglycan synthesis, is absent during vegetative growth, and is synthesized during forespore maturation. It is found exclusively in the sporangial cytoplasm. L-lysine ligase is required for vegetative cell wall peptidoglycan synthesis but not for cortex synthesis. It is found in both fractions, but it has a fourfold higher specific activity in the forespore cytoplasm. Other enzymes that are required for synthesis of the nucleotide-pentapeptide precursors of both cortical and vegetative cell wall peptidoglycans are found in similar specific activities in both compartments. Mature spores, free of any residual sporangial material, have specific activities of all of these enzymes and of L-lysine ligase similar to those in forespores and in vegetative cells and are devoid of diaminopimelate ligase activity. Thus, the differential expression of at least one gene required for spore cortex synthesis in B. sphaericus occurs exclusively in the sporangial cytoplasm.

Spore cortex of conditional cortexless mutants of Bacillus sphaericus 9602 was not detectable by electron microscopy unless the medium was supplemented with meso-alpha,epsilon-diaminopimelic acid during sporulation. Other spore structures appeared normal. Spore shape was quite irregular in the absence of meso-alpha,epsilon-diaminopimelic acid.