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The problem of bacillus species infection with special emphasis on the virulence of Bacillus cereus.

Although Bacillus cereus is an uncommon ocular pathogen, infection with it usually results in loss of the eye. Although previous reports have emphasized endogenous infection, our recent experience indicates the importance of B cereus infection following trauma. Management is hampered by ineffectiveness of current empirical antibiotic regimens. This microorganism is resistant to both the penicillins and the cephalosporins. Although B cereus is susceptible to gentamicin, our studies indicate that gentamicin by itself is inadequate to eradicate the infection. B cereus, however, is susceptible to clindamycin and combined therapy with gentamicin and clindamycin appears to offer the best approach. Early diagnosis is the key to successful treatment. We believe the clinical circumstances likely to lead to B cereus infection, as well as the manifestations of the disease itself, are sufficiently distinctive to alert the ophthalmologist to the possibility of this infection. Prompt recognition of the infection should allow institution of appropriate therapy before permanent structural changes occur.

Abscess↗

An evaluation of the effect of Bacillus cells and Bacillus spores in association with cowpea granules as starter cultures for the fermentation of African oil bean (Pentaclethra macrophylla Bentham) to 'ugba'.

Studies on the improvement of the traditional production of 'ugba', a protein-rich fermented African oil bean seed product, were undertaken, by developing starter cultures of Bacillus subtilis cells and spores in association with cowpea granules. The viability of the cells in association remained stable at 94.5% for 6 months at 30 degrees C and for up to 10 months at 4 degrees C while the viability of the spores in association remained stable at ca. 96% for up to 10 months at both 4 and 30 degrees C. The starter cultures resulted in high increases in protease activity from ca 2.8 mg N/min to about 51.6 +/- 0.4 mg N/min in 48 h and a corresponding increase in amino-nitrogen content of ca 2.0 +/- 0.2 mg N 100 g dry matter (DM) to ca 18.5 +/- 0.3 mg N/100 g (DM) during the same period. Changes in the protease activity of the natural process were gradual and increased from 3.0 mg N/min to 38.0 +/- 0.8 mg N/min after 5 days of fermentation. The maximum amino nitrogen content of 'ugba' produced by the starter cultures (18.5 +/- 0.3 mg N/100 g DM) after 2 days was significantly (p <0.05) higher than the maximum amino nitrogen content (12.5 +/- 0.8 mg N/100 g DM), of 'ugba' obtained by the natural process. 'Ugba' produced by the starter cultures were well accepted and compared favorably with the natural product.

Bacillus subtilis↗

Isolation and partial purification of a novel type II restriction endonuclease Bsu121 I, from Bacillus subtilis. Bsu121I, a type II restriction endonuclease from Bacillus subtilis.

A new type II restriction endonuclease which we designated as Bsu121I has been isolated from gram-positive bacterium Bacillus subtilis strain 121 and partially purified. The restriction endonuclease was isolated from cell extracts using step-wise purification through ammonium sulfate precipitation, followed by phosphocellulose column chromatography. SDS-PAGE profile showed denatured molecular weights (23 and 67 kDa) of the endonuclease. The partially purified enzyme restricted pBR322 DNA into two fragments of 3200 and 1700 bp. The endonuclease activity required Mg(+2) as cofactor like other type II endonucleases.

Bacillus subtilis↗

Highly efficient gene expression of a fibrinolytic enzyme (subtilisin DFE) in Bacillus subtilis mediated by the promoter of alpha-amylase gene from Bacillus amyloliquefaciens.

Subtilisin DFE is a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. The promoter and signal peptide-coding sequence of alpha-amylase gene from B. amyloliquefaciens was cloned and fused to the sequence coding for pro-peptide and mature peptide of subtilisin DFE. This hybrid gene was inserted into the Escherichia coli/Bacillus subtilis shuttle plasmid vector, pSUGV4. Recombinant subtilisin DFE gene was successfully expressed in B. subtilis WB600 with a fibrinolytic activity of 200 urokinase units ml(-1).

Bacillus subtilis↗

Efficient constitutive expression of Bacillus subtilis xylanase A in Escherichia coli DH5alpha under the control of the Bacillus BsXA promoter.

Xylanase A (XynA) is a class G/11 xylanase secreted by Bacillus subtilis. XynA was purified to homogeneity from B. subtilis strain 168 culture supernatants by ethanol precipitation and cation-exchange chromatography. The DNA fragment encoding the XynA together with the BsXA promoter region was amplified by PCR from B. subtilis 168 genomic DNA, and cloned into the plasmid pT7T3 to give the plasmid pT7BsXA. After transformation of Escherichia coli DH5alpha with pT7BsXA, a 19-fold increase in the levels of the secreted XynA was detected in the supernatant as compared with the B. subtilis culture. Correct post-translation modification of the recombinant protein was confirmed by N-terminal amino acid sequencing and MS analyses. The pH- and temperature-dependences of the native and recombinant proteins were identical, indicating that the pT7BsXA may be useful for the constitutive expression of heterologous protein in E. coli.

Amino Acid Sequence↗

Sporulation in Bacillus subtilis. Effect of medium on the form of chromosome replication and on initiation to sporulation in Bacillus subtilis.

Thymine-requiring mutants of Bacillus subtilis and mutants that are temperature-sensitive for initiation of chromosome replication have been used to study the relationship between sporulation and chromosome formation. The DNA synthesis that normally occurs when cells are transferred to sporulation medium is essential for spore induction. This is shown by the fact that thymine-starved cells are unable to form spores and are unable to perform even the earlier steps of sporulation, such as septum formation or synthesis of alkaline phosphatase. The nature of the medium in which the cells are growing while the DNA is being completed is also important because it determines both the shape and the position of the daughter chromosomes. If the cells are in a rich medium, the newly synthesized chromosomes are discrete and compact bodies: the cells are primed for growth, and sporulation cannot be induced by transferring them at this stage to a spore-inducing medium. If DNA synthesis was completed with the cells in a poor medium the daughter chromosomes, by the time DNA synthesis has ceased, are spread in a single filamentous band and the cells are morphologically already in stage I of sporulation.

Alkaline Phosphatase↗

Intracellular serine proteinase of Bacillus subtilis strain Marburg 168. Comparison with the homologous enzyme from Bacillus subtilis strain A-50.

Intracellular serine proteinase was isolated from sporulating cells of Bacillus subtilis Marburg 168 by gramicidin S-Sepharose 4B affinity chromatography. The enzymological characteristics, the amino acid composition and the 19 residues of the N-terminal sequence of the enzyme are reported. The isolated proteinase was closely related to, but not completely identical with, the intracellular serine proteinase of B. subtilis A-50. The divergence between these two intracellular enzymes was less than that between the corresponding extracellular serine proteinases (subtilisins) of types Carlsberg and BPN', produced by these bacterial strains. This may be connected with the more strict selection constraints imposed in intracellular enzymes during evolution.

Amino Acid Sequence↗

Comparison of the wild-type alpha-amylase and its variant enzymes in Bacillus amyloliquefaciens in activity and thermal stability, and insights into engineering the thermal stability of bacillus alpha-amylase.

The starch hydrolysis activity and thermal stability of Bacillus amyloliquefaciens alpha-amylase (wild-type enzyme or WT) and its variant enzymes, designated as M77, M111, and 21B, were compared. All have an optimal pH at around 6, as well as almost the same reaction rates and Km and kcat values. The optimal temperature in the absence of Ca2+ ions is 60 degrees C for WT and M77 and 40 degrees C for M111 and 21B. Those of M111 and 21B rose to 50-60 degrees C upon the addition of 5 mM CaCl2, while those of WT and M77 did not change. The dissociation constants Kd for Ca2+ to WT and M77 are much lower than those of M111 and 21B. Asp233 in WT is replaced by Asn in M111 and 21B, while it is retained in M77, suggesting that Asp233 is involved in the thermal stability of the enzyme through Ca2+ ion binding. These findings provide insight into engineering the thermal stability of B. amyloliquefaciens alpha-amylase, which would be useful for its applications in the baking industry and in glucose manufacturing.

Amino Acid Sequence↗

In vitro transcription of the Bacillus subtilis phage phi 29 DNA by Bacillus subtilis and Escherichia coli RNA polymerases.

The Escherichia coli RNA polymerase bound to phage phi 29 DNA has been visualized by electron microscopy. Thirteen specific binding sites have been observed at 1.7,2.6,5.5,10.4,13.7,25.2,25.7,26.3,33.5,59.5,69.2,91.7 and 99.6 DNA length units and they have been named A1,A1I,A1II,A1III,A1IV,A2,A2I, A3, A4,B1,B1I,C1 and C2, respectively. The binding sites A1,A2,A3,B1,C1 and C2 coincide with those found with Bacillus subtilis RNA polymerase. The transcription of phage phi 29 DNA with B. subtilis or E. coli RNA polymerases has been studied. With the B. subtilis RNA polymerase eight transcripts were found, starting at positions corresponding to the binding sites A1, A1III, A2,A3,B1I,B2,C1 and C2, respectively. With the E. coli RNA polymerase the same transcripts were found and a new one starting at position corresponding to the A4 binding site. The RNAs starting at binding sites A1,A1III,A2,B1I, B2,C1 and C2 are transcribed from right to left, as expected for early RNA. The RNAs which initiate at positions A3 and A4 are transcribed from left to right and probably correspond to late RNAs.

Bacillus subtilis↗

Nucleotide sequence of a Bacillus subtilis promoter recognized by Bacillus subtilis RNA polymerase containing sigma 37.

We report the nucleotide sequence of the promoter for a Bacillus subtilis gene (spoVC) whose transcription is controlled by a 37,000 dalton species of B. subtilis sigma factor known as sigma 37 but not by the principal- sigma factor of 55,000 daltons (sigma 55). Using S1 nuclease mapping we show that the startpoint for sigma 37-directed transcription of the spoVC gene in vitro corresponded closely to the 5' terminus of in vivo synthesized spoVC RNA. The binding site for sigma 37-containing RNA polymerase extended from 43 bp to 51 bp (positions -43 to -51) upstream from the transcription startpoint to 22 bp (position +22) downstream from the startpoint. The nucleotide sequence of the spoVC promoter differed significantly from promoters whose recognition is controlled by sigma 55 but was similar to other sigma 37- controlled promoters in regions known to be important in promoter recognition. Our results are consistent with the hypothesis (Lee and Pero, J. Mol. Biol., in press) that sigma factors work by contacting specific bases in both the -35 and -10 regions of promoters.

Bacillus subtilis↗

Reclassification of Paenibacillus (formerly Bacillus) pulvifaciens (Nakamura 1984) Ash et al. 1994, a later subjective synonym of Paenibacillus (formerly Bacillus) larvae (White 1906) Ash et al. 1994, as a subspecies of P. larvae, with emended descriptions of P. larvae as P. larvae subsp. larvae and P. larvae subsp. pulvifaciens.

A polyphasic taxonomic study of four strains of Paenibacillus larvae and four strains of Paenibacillus pulvifaciens (including duplicates of both type strains) supported the reclassification of both former Bacillus species into one species, P. larvae. Our conclusions were based on morphological and Analytab Products (API) tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins, gas chromatography of methylated fatty acids, pyrolysis mass spectrometry, DNA-DNA binding, and the following genomic fingerprinting methods: amplified ribosomal DNA restriction analysis, random amplified polymorphic DNA analysis, and AFLP analysis. The last method is a novel high-resolution DNA fingerprinting technique based on the selective amplification of restriction fragments. Despite more than 90% DNA relatedness between the strains studied, SDS-PAGE of whole-cell proteins, biochemical tests, and DNA fingerprinting (AFLP) distinguished between the P. larvae and P. pulvifaciens strains at the subspecies level. Taking this evidence along with differences in pathogenicity, we propose to reclassify the honeybee pathogens P. larvae and P. pulvifaciens as P. larvae subsp. larvae and P. larvae subsp. pulvifaciens. An emended description of the species and descriptions of the subspecies are given. The type strains are P. larvae subsp. larvae ATCC 9545 (LMG 9820) and P. larvae subsp. pulvifaciens NRRL B-3685 (LMG 6911 and ATCC 13537).

Bacillus↗

High expression of Bacillus licheniformis alpha-amylase with a Bacillus secretion vector.

A gene coding for the heat-stable alpha-amylase from Bacillus licheniformis ATCC14580 has been expressed with the aid of a B. amyloliquefaciens alpha-amylase-based expression/secretion vector by joining the structural part of the gene to a pool of vectors after the B. amyloliquefaciens alpha-amylase promoter and signal sequence. The recombinant plasmids obtained were stably maintained in B. subtilis and the heat-stable alpha-amylase activity rose several hundred times from the level of the donor. Eight different constructions were further analyzed. Each of them had an intact B. amyloliquefaciens signal sequence, the only difference being in a few nucleotides beyond the C terminus of the signal peptide. This, however, was enough to cause up to fourfold differences in protein yield. Possible reasons for the variation in the production level are discussed.

Bacillus↗

Evaluation of Bacillus subtilis strain IS53 for the production of Bacillus anthracis protective antigen.

An asporogenous strain of Bacillus subtilis, IS53, transformed with plasmid pPA102, produces the protective antigen (PA) component of the tripartite toxin of B. anthracis. Addition of yeast extract was required to support growth and PA production in all the media examined. Protective antigen expression was down-regulated during exponential growth and extracellular proteases caused marked degradation of the mature protein.

Antigens, Bacterial↗

Diarrhoeal enterotoxin production by strains of Bacillus thuringiensis isolated from commercial Bacillus thuringiensis-based insecticides.

Strains of Bacillus cereus and B. thuringiensis were tested by the Tecra VIA kit for the ability to produce a diarrhoeal enterotoxin. The strains of B. thuringiensis were isolated from commercial B. thuringiensis-based insecticides (Bactimos, DiPel, Florbac, FC, Foray 48B, Novodor FC, Turex, VecTobac, XenTari). The production of diarrhoeal enterotoxin varied by a factor of more than 100 among the different strains tested. B. cereus (F4433/73) produced the highest amount of enterotoxin and the B. thuringiensis strain isolated from DiPel the lowest. The products were tested for their content of diarrhoeal enterotoxin and all products, except MVP which does not contain viable B. thuringiensis spores, contained diarrhoeal enterotoxins. The results indicates an potential risk for gastroenteritis outbreak caused by B. thuringiensis.

Bacillus thuringiensis↗

Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. morrisoni isolate PG-14 toxins.

The parasporal bodies of the mosquitocidal isolates of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 were compared with regard to their hemolytic and cytolytic activities and the immunological relatedness of the 28- and 65-kilodalton (kDa) proteins that occur in both subspecies. The alkali-solubilized parasporal bodies of B. thuringiensis subsp. israelensis caused 50% lysis of human erythrocytes at 1.14 micrograms/ml, whereas those of B. thuringiensis subsp. morrisoni caused similar lysis at 1.84 micrograms/ml. Preincubation of solubilized parasporal bodies with dioleolyl phosphatidylcholine significantly inhibited the hemolytic activity of both supspecies. In cytolytic assays against Aedes albopictus cells, the toxin concentrations causing 50% lysis for B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni were 1.87 and 11.98 micrograms/ml, respectively. Polyclonal antibodies raised separately against the 25-kDa protein (a tryptic digest of the 28-kDa protein) or the 65-kDa protein of B. thuringiensis subsp. israelensis cross-reacted, respectively, with the 28- and the 65-kDa proteins of B. thuringiensis subsp. morrisoni. However, neither of these antibodies cross-reacted with the 135-kDa protein of either subspecies. These results indicate that the mosquitocidal and hemolytic properties of B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 are probably due to the biologically related proteins that are present in the parasporal bodies of both subspecies. The lower hemolytic activity of the B. thuringiensis subsp. morrisoni may be due to the presence of lower levels of the 28-kDa protein in that subspecies.(ABSTRACT TRUNCATED AT 250 WORDS)

Aedes↗

Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans.

A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism.

Bacillus↗

Toxin-producing ability among Bacillus spp. outside the Bacillus cereus group.

A total of 333 Bacillus spp. isolated from foods, water, and food plants were examined for the production of possible enterotoxins and emetic toxins using a cytotoxicity assay on Vero cells, the boar spermatozoa motility assay, and a liquid chromatography-mass spectrometry method. Eight strains produced detectable toxins; six strains were cytotoxic, three strains produced putative emetic toxins (different in size from cereulide), and one strain produced both cytotoxin(s) and putative emetic toxin(s). The toxin-producing strains could be assigned to four different species, B. subtilis, B. mojavensis, B. pumilus, or B. fusiformis, by using a polyphasic approach including biochemical, chemotaxonomic, and DNA-based analyses. Four of the strains produced cytotoxins that were concentrated by ammonium sulfate followed by dialysis, and two strains produced cytotoxins that were not concentrated by such a treatment. Two cultures maintained full cytotoxic activity, two cultures reduced their activity, and two cultures lost their activity after boiling. The two most cytotoxic strains (both B. mojavensis) were tested for toxin production at different temperatures. One of these strains produced cytotoxin at growth temperatures ranging from 25 to 42 degrees C, and no reduction in activity was observed even after 24 h of growth at 42 degrees C. The strains that produced putative emetic toxins were tested for the influence of time and temperature on the toxin production. It was shown that they produced putative emetic toxin faster or just as fast at 30 as at 22 degrees C. None of the cytotoxic strains produced B. cereus-like enterotoxins as tested by PCR or by immunological methods.

Animals↗