spoVG sequence of Bacillus megaterium and Bacillus subtilis.

We have sequenced the stage V sporulation specific gene spoVG in both Bacillus megaterium and Bacillus subtilis. The open reading frames encode polypeptides of 96 and 97 residues, respectively, and have an 88.6% amino acid identity. Both genes have putative rho-independent terminators. No significant amino acid or nucleotide homology of either gene was found when compared with sequences contained in either the Genbank or EMBL data bases.     

Dihydrodipicolinate reductases from Bacillus cereus and Bacillus megaterium.

Dyhydrodipicolinate reductases were purified 100-fold from crude extracts of B. cereus and B. megaterium and their properties were compared with those of the reductase from B. subtilis. The molecular weights of the reductases of B. cereus and B. megaterium were fount to be 155,000 and 150,000, respectively. These reductases were shown to be free of flavin, unlike the B. subtilis enzyme, which contains flavin. Both NADPH and NADH acted as coenzymes for these two reductases. NADPH being three or four times more effective than NADH. The Km values for NADPH and dihydrodipicolinate were 8 micrometer and 62 micrometer, respectively, with B. cereus reductase, and 13 micrometer and 59 micrometer with B. megaterium reductase. The pH optima of the enzymes from B. cereus and B. megaterium were pH 7.4 and 7.2, respectively. The reductases were inhibited by dipicolinate noncompetitively with respect to dihydrodipicolinate and the Ki values were 85 micrometer and 140 micrometer, respectively. Lysine and diaminopimelate were not inhibitory. The properties of the reductases from B. cereus and B. megaterium were similar, but they differed considerably from those of the B. subtilis enzyme. However, all three Bacillus reductases were markedly inhibited by dipicolinate, unlike the enzyme from E. coli.     

Aminopeptidases of Bacillus subtilis.

Three enzymes with L- and one enzyme with D-aminopeptidase (EC 3.4.11; alpha-aminoacyl peptide hydrolase) activity have been separated from each other and partially purified from Bacillus subtilis 168 W.T., distinguished with respect to their molecular weights and catalytic properties, and studied in relation to the physiology of this bacterium. One L-aminopeptidase, designated aminopeptidase I, has a molecular weight of 210,000 +/- 20,000, is produced early in growth, and hydrolyzes L-alanyl-beta-naphthylamide most rapidly. Another, designated aminopeptidase II, molecular weight 67,000 +/- 10,000, is also produced early in growth and hydrolyzes L-lysyl-beta-naphthylamide most rapidly. A third, aminopeptidase III, molecular weight 228,000 +/- 20,000, is produced predominantly in early stationary phase and most efficiently utilizes L-alpha-aspartyl-beta-naphthylamide as substrate. The synthesis of aminopeptidase III in early stationary phase suggests that selective catabolism of peptides occurs at this time, perhaps related to the cessation of growth or the onset of early sporulation-associated events. A D-aminopeptidase which hydrolyzes the carboxyl-blocked dipeptide D-alanyl-D-alanyl-beta-naphthylamide (as well as D-alanyl-beta-naphthylamide and D-alanyl-D-alanyl-D-alanine) has also been identified, separated from aminopeptidase II, and purified 170-fold. D-Aminopeptidase, molecular weight 220,000 +/- 20,000, is localized predominantly in the cell wall and periplasm of the organism. This evidence and the variation of the activity during the growth cycle suggest an important function in cell wall or peptide antibiotic metabolism.     

Fatty acids in the genus Bacillus. II. Similarity in the fatty acid compositions of Bacillus thuringiensis, Bacillus anthracis, and Bacillus cereus

The nature and relative abundance of fatty acids produced by two strains each of Bacillus thuringiensis and of B. anthracis were studied by gas-liquid chromatography on a 12,000 theoretical plate polyester column capable of partially resolving iso- and anteiso-fatty acids with the same number of carbon atoms. Unsaturated fatty acids as the bromo derivatives were separated from the saturated acids and resolved in a short SE-30 column by use of programmed-temperature gas chromatography. All four strains produced 16 major fatty acids: 9 branched (i-C₁₂, i-C₁₃, i-C₁₄, i-C₁₅, i-C₁₆, i-C₁₇, a-C₁₃, a-C₁₅, and a-C₁₇), 3 normal (n-C₁₄, n-C₁₅, and n-C₁₆), and 4 monounsaturated (i-C₁₆¹⁼, i-C₁₇¹⁼, a-C₁₇¹⁼, and n-C₁₆¹⁼), in addition to some minor fatty acids. In all cases, 12 branched acids, including saturated and monounsaturated, made up over 70% of the total fatty acids, and iso-C₁₅ acid was most abundant. These fatty acid distribution patterns were very similar to those of B. cereus and B. cereus var. mycoides. There were, however, minor but clear differences between the fatty acid distribution patterns of B. thuringiensis and B. anthracis. B. thuringiensis, like B. cereus, produced higher proportions of i-C₁₃, a-C₁₃, and i-C₁₄ fatty acids than did B. anthracis. This difference between these two species could be useful as a supplemental criterion in their differentiation. Indications are that the enzyme systems for monounsaturated fatty acid synthesis in B. thuringiensis and B. anthracis prefer normal fatty acids as substrates rather than branched-chain fatty acids.     

Bacillus alcalophilus n. sp.

Samenvatting Mede naar aanleiding van de gunstige ervaringen, die Doorenbos in de practijk opdeed met èèn der door ons voorgestelde nieuwe electieve cholera-voedingsbodems (“glycocollplaat”), wordt getracht ook een aankweek-voedingsbodem, electief voor vibrionen, samen te stellen. Hierbij worden echter bezwaren, o.a. van physico-chemischen aard, ondervonden, terwijl bovendien soms uit faeces van normale individuen in enkele der onderzochte vloeibare aankweek-voedingsbodems (sterk alcalisch haemoglobine-carbonaat-peptonwater, sterk alcalisch carbonaat-peptonwater van pH-ca. 10) electief ook een Gram-positieve sporevormende bacterie wordt aangekweekt. Hieruit kan deze bacil dan op de door ons beschreven choleravoedingsbodems en op Dieudonnè-platen worden ge?soleerd. Dadelijk uit faeces gelukt de isolatie dezer microbe op choleravoedingsbodems niet; evenmin door aankweek op peptonwater van “gewone” pH. In de praktijk geeft deze bacil dus geen moeilijkheden bij het onderzoeken van faeces op vibrionen. Kenmerkend voor deze bacil is, dat deze alleen op en in sterk alcalische voedingsbodems groeit, niet bij zwakke alcaliteit of nog lagere pH. Daar dit micro-organisme, voor zoover ons bekend nog niet eerder beschreven is, stellen wij voor hem “Bacillus alcalophilus n. sp.” to noemen. De bacil is beweeglijk en splitst eiwit (gelatine, haemoglobine) in sterk alcalisch milieu. Summary Also in view of the favourable results whichDoorenbos has obtained in practical cases with one of the new elective cholera media (glycocoll-plate) proposed by us, an attempt has been made to compose a culture medium for the elective enrichment of elective vibrions. Difficulties of a physico-chemical nature are, however, met with in doing so moreover in some of the liquid culture media (highly alkaline haemoglobine-carbonate-peptone water, highly alkaline carbonate-peptone water of pH ca. 10) from the faeces of normal individuals which have been examined, a Gram-positive sporeforming bacterium is cultivated. From this the bacillus may then be isolated on the cholera cultivation media described by us and on Dieudonnè plates. Directly from faeces this microbe does not grow on elective cholera culture media, nor by enrichment in peptone water of “ordinary” pH. So in practice this bacillus does not present any difficulty when examining faeces for vibrions. It is typical of this bacillus that it only grows in and on highly alkaline culture media, and not in cases where the medium is of weak alkalinity or still lower pH. As this micro-organism has not, as far as we know, been previously described, we propose to name it “Bacillus alcalophilus n.sp.” The bacillus is motile and digests albumen (gelatine, haemoglobine) in a strong alkaline medium.     

Clearance of Bacillus sphaericus and Bacillus thuringiensis ssp. israelensis from mammals

The maximum recovery period following topical ocular instillation and intraperitoneal injection of two preparations of Bacillus thuringiensis ssp. israelensis de Barjac and two preparations of Bacillus sphaericus 2362 was evaluated in rabbits and mice. B. sphaericus 2362 persisted for 8 wk after administration to the conjunctival cul-de-sac of rabbits; B. thuringiensis ssp. israelensis persisted for 1 wk. Infection was not evident, but both entomopathogens were recovered from flushed and unflushed eyes. High doses of B. sphaericus 2362 (greater than or equal to 10(8) colony-forming units) were toxic to CD-1 mice, and the toxic factor was heat stable. Injection of 10(7) colony-forming units of B. sphaericus 2362 resulted in clearance from the spleens of euthymic and athymic mice. Recovery occurred up to 67 d after injection. Mice failed to remove one preparation of B. thuringiensis ssp. israelensis from their spleen, and a constant number of colony-forming units were recovered for 80 d. B. sphaericus 2362 and B. thuringiensis ssp. israelensis were recovered from heart blood; their disappearance from heart blood coincided with their clearance from the spleen. There was no evidence that either organism was infectious. We conclude that these organisms can be used safely in environments where human exposure might occur.     

Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis.

A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.     

Studies on transfection and transformation of protoplasts of Bacillus larvae, Bacillus subtilis, and Bacillus popilliae.

Protoplasts of Bacillus larvae NRRL b-3555 and Bacillus subtilis RM125 (restrictionless, modificationless mutant) were transfected with DNA from the B. larvae bacteriophage PBL1c in the presence of polyethylene glycol. B. subtilis 168 and Bacillus popilliae NRRL B-2309M protoplasts could not be transfected with PBL1c DNA. Protoplasts of B larvae NRRL B-3555 were transformed with plasmids pC194 and pHV33 in the presence of polyethylene glycol. The frequency of transformation was much higher when the plasmids were isolated from B. larvae NRRL B-3555 transformants than when they were isolated from B. subtilis 168. These results indicate that the restriction-modification systems found in B. larvae NRRL B-3555 and B. subtilis 168 may be different. Conditions for protoplast formation and cell wall regeneration were developed for B. popilliae NRRL B-2309S. However, no transformation occurred with plasmids pC194 and pHV33 (isolated from B. subtilis 168).     

Mating system for transfer of plasmids among Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis.

To facilitate the analysis of genetic determinants carried by large resident plasmids of Bacillus anthracis, a mating system was developed which promotes plasmid transfer among strains of B. anthracis, B. cereus, and B. thuringiensis. Transfer of the selectable tetracycline resistance plasmid pBC16 and other plasmids from B. thuringiensis to B. anthracis and B. cereus recipients occurred during mixed incubation in broth. Two plasmids, pXO11 and pXO12, found in B. thuringiensis were responsible for plasmid mobilization. B. anthracis and B. cereus transcipients inheriting either pXO11 or pXO12 were, in turn, effective donors. Transcipients harboring pXO12 were more efficient donors than those harboring pXO11; transfer frequencies ranged from 10(-4) to 10(-1) and from 10(-8) to 10(-5), respectively. Cell-to-cell contact was necessary for plasmid transfer, and the addition of DNase had no effect. The high frequencies of transfer, along with the fact that cell-free filtrates of donor cultures were ineffective, suggested that transfer was not phage mediated. B. anthracis and B. cereus transcipients which inherited pXO12 also acquired the ability to produce parasporal crystals (Cry+) resembling those produced by B. thuringiensis, indicating that pXO12 carries a gene(s) involved in crystal formation. Transcipients which inherited pXO11 were Cry-. This mating system provides an efficient method for interspecies transfer of a large range of Bacillus plasmids by a conjugation-like process.     

Membrane-bound nucleotidase of Bacillus cereus.

A membrane-bound nucleotidase of Bacillus cereus T was solubilized by digestion with trypsin and subsequently purified more than 300-fold. The purified nucleotidase was most active on ribonucleoside 5'-monophosphates and was slightly less active (40 to 60%) on deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates. In addition to hydrolytic activity, the nucleotidase preparation possessed phosphotransferase activity by which phosphate is transferred from a phosphate donor to the 5' position of nucleosides.     

Deoxyribonucleoside-requiring mutants of Bacillus subtilis.

A number of deoxyribonucleoside-requiring mutants (dns) of Bacillus subtilis were isolated and their growth characteristics and ribonucleotide reductase activities were compared with those of the wild type and of a dna mutant (tsA13). Both tsA13 and dns mutants required the presence of a mixture of deoxyribonucleosides for growth at 45 degrees C but not at 25 degrees C. All the mutant strains tested contained ribonucleotide reductase activity which showed heat sensitivity similar to that of the enzyme from a wild-type strain. The reductase in B. subtilis seemed to reduce ribonucleoside triphosphates in a similar manner to the enzyme in Lactobacillus leichmannii.     

Intracellular proteinase of Bacillus subtilis.

An intracellular serine proteinase was isolated from Bacillus subtilis, strain A-50. The molecular weight of the enzyme is 30000 +/- 1000, its amino acid composition is enriched in glutamic acid residues, the isoelectric point is 4.3, the N-terminal sequence Glu-Leu-Pro-Glu-Gly-Ile-Gln-Val-Ile-Lys-Ala-Pro-Glu-Leu-Xxx-Ala-Gln-Gly-Phe-Lys Gly-Ser-Asx-Ile-Lys-Ile-Ala-Val-Leu-Asx. The enzyme is structuraly homologous with secretory subtilisins.     

Genetic manipulation of Bacillus amyloliquefaciens.

Application of modern gene technology to strain improvement of the industrially important bacterium Bacillus amyloliquefaciens is reported. Several different plasmid constructions carrying the alpha-amylase gene (amyE) from B. amyloliquefaciens were amplified in this species either extrachromosomally or intrachromosomally. The amyE gene cloned on a pUB110-derived high copy plasmid pKTH10 directed the highest yields both in rich laboratory medium and in crude industrial medium. The alpha-amylase activity, when compared with the parental strain, was enhanced up to 20-fold in the pKTH 10 transformant. This strain showed decreased activities for other exoenzymes, such as proteases and beta-glucanase suggesting common limiting resources in the processing of these enzymes. Deletions were made in vitro in genes encoding neutral (nprE), alkaline (aprE) protease and beta-glucanase (bglA). The engineered genes were cloned into the thermosensitive plasmid pE194, and the resulting plasmids were used to replace the corresponding wild type chromosomal genes in B. amyloliquefaciens by integration-excision at non-permissive temperature. The double mutant deficient in the major proteases (delta nprE delta aprE) showed about a 2-fold further enhancement in alpha-amylase production in the industrial medium compared with the relevant wild type backgroud, both when plasmid-free and when transformed with pKTH10; this strain also produced elevated levels of the chromosomally-encoded beta-glucanase; pKTH10 was stably maintained both in the wild type strain and in the delta nprE delta aprE mutant. We suggest that the higher yields in alpha-amylase and beta-glucanase in the delta nprE delta aprE strain are primarily due to improved access to limiting resources, and that decreased proteolytic degradation may have had a secondary role in retaining the high activity obtained.     

Peptidoglycan synthesis in L-phase variants of Bacillus licheniformis and Bacillus subtilis.

Stable L-phase variants isolated from Bacillus licheniformis and Bacillus subtilis, when grown in osmotically stabilized media, do not synthesize peptidoglycan but have been found to accumulate the nucleotide precursors of this polymer. The enzymes involved in the synthesis of these precursors and the later membrane-bound stages of peptidoglycan synthesis have been investigated, and the L-phase variants have been shown to contain lesions, which provide a rational explanation for the absence of peptidoglycan and for the nature of the precursor accumulated. The majority of the L-phase variants contained a single enzymic defect, but two strains were isolated with double lesions. Five out of seven strains examined accumulated uridine 5'-diphosphate (UDP)-MurAc-L-ala-D-glu and were unable to synthesize diaminopimelic acid as a consequence of a defect in aspartate-beta-semialdehyde dehydrogenase activity. Two strains were deficient in UDP-MurAc: L-alanine ligase and accumulated UDP-MurAc. One strain accumulated the complete nucleotide precursor UDP-MurAc-L-ala-D-glu-mA2pm-D-ala-D-ala and was deficient in phospho-N-acetylmuramyl pentapeptide translocase. A second strain also had this lesion, together with defective aspartate-beta-semialdehyde dehydrogenase activity. The other enzymes of peptidoglycan synthesis were present in the L-phase variants, with activities similar to those found in the parent bacilli grown under identical conditions. Membrane preparations from certain of the L-phase variants were also capable of synthesizing the secondary polymers poly(glycerol phosphate) teichoic acid and teichuronic acid and also a polymer of N-acetylglucosamine.