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Bacteria of the genus Bacillus have a hydrolase stereospecific to the D isomer of benzoyl-arginine-p-nitroanilide.

A stereospecific enzyme activity capable of cleaving the amide bond of the synthetic substrate N-benzoyl-D-arginine-p-nitroanilide (D-BAPA) has been found in all aerobic and anaerobic members of the family Bacillaceae tested by us. Cells of nonsporeforming gram-positive or gram-negative bacteria contain a hydrolase activity stereospecific to N-benzoyl-L-arginine-p-nitroanilide. The D-BAPA-hydrolyzing enzymes (D-BAPAases) of mid-logarithmic-phase cells of Bacillus subtilis 168 and B. cereus T were compared. These enzymes had the same molecular weight of approximately 66,000 in gel filtration and the same electrophoretic mobility after electrophoresis on polyacrylamide gels. The D-BAPAases of B. subtilis 168 and B. cereus T differed in the effect of inhibitors on enzymatic activity. While both hydrolases were inhibited by tosyl-L-lysine chloromethyl ketone and tosyl-L-arginine-methyl ester as well as leupeptin, only the D-BAPAase of B. cereus T was inhibited by p-chloromercuribenzene sulfonic acid. The D-BAPAases of B. subtilis and B. cereus T had a Michaelis constant for D-BAPA of 2.9 x 10(-5) M and 1.4 x 10(-4) M, respectively. D-BAPAase is an intracellular enzyme localized in the protoplast (80 to 90% in soluble form in the cytoplasm). The ability to cleave D-BAPA is suggested as an additional chemotaxonomic characteristic of sporeforming bacteria of the genera Bacillus and Clostridium.

Bacillus↗

Characterization of the surface layer glycoprotein of Clostridium symbiosum HB25.

The cell surface of Clostridium symbiosum HB25 is covered by a squarely arranged surface layer (S-layer) glycoprotein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the sodium dodecyl sulfate-soluble whole-cell extract showed the presence of several high-molecular-weight protein bands in a narrow range (approximate Mr, 140,000) which, upon periodic acid-Schiff staining, gave a positive reaction. After proteolytic degradation of the purified S-layer glycoprotein, a single glycopeptide fraction was obtained by gel permeation chromatography. Hydrolysis, treatment with aqueous hydrofluoric acid, and 1H and 13C nuclear magnetic resonance studies showed that the glycoprotein glycan is a high-molecular-weight polymer (approximate Mr, 15,000) of tetrasaccharide repeating units with the component sugars N-acetylgalactosamine (GalNAc), N-acetylmannosamine (ManNAc), and N-acetylbacillosamine (BacNAc; 2-N-acetyl-4-amino-2,4,6-trideoxy glucose) linked by monophosphate diesters. The following structure is proposed: [----6)-alpha-D-ManpNAc-(1----4)-beta-D-GalpNAc-(1----3)-alpha-D-+ ++BacpNAc- (1----4)-alpha-D-GalpNAc-(1----PO3)----]n. The nuclear magnetic resonance data provided evidence for a charge interaction between the free amino group of BacNAc and the phosphate group of adjacent glycan chains. Since polycationic ferritin did not label the cell surface of intact cells, an electrostatic interaction can also be expected in vivo, leading to a charge-neutral outer surface, which is characteristic of all other S layers from members of the family Bacillaceae studied so far.

Amino Acids↗

Permeability and charge-dependent adsorption properties of the S-layer lattice from Bacillus coagulans E38-66.

We investigated the permeability properties of the oblique S-layer lattice from Bacillus coagulans E38-66 after depositing cell wall fragments on a microfiltration membrane, cross-linking the S-layer protein with glutaraldehyde, and degrading the peptidoglycan with lysozyme. Comparative permeability studies on such multilayered S-layer membranes and suspended S-layer vesicles from thermophilic members of the family Bacillaceae with use of the space technique (M. Sára and U. B. Sleytr, J. Bacteriol. 169:4092-4098, 1987) revealed identical molecular exclusion limits (M. Sára and U. B. Sleytr, J. Membr. Sci. 33:27-49, 1987). Examination of the S-layer lattice from B. coagulans E38-66 with the S-layer membrane technique revealed unhindered passage for molecules up to the size of myoglobin (M(r) 17,000). The molecular dimensions of this protein (2.8 by 3.2 by 4.5 nm) correspond approximately to the size of the ovoid-shaped pore previously shown by high-resolution electron microscopy of negatively stained S-layer self-assembly products (D. Pum, M. Sára, and U. B. Sleytr, J. Bacteriol. 171:5296-5303, 1989). Chemical modification of the S-layer protein and comparative labeling, adsorption, and permeability studies clearly demonstrated that (i) in the native state, free amino and carboxyl groups are present on the outer S-layer face and in the interior of the pores and (ii) electrostatic interactions between these groups prevent unspecific adsorption of the S-layer in vivo.

Adsorption↗

Molecular cloning and characterization of the obg gene of Streptomyces griseus in relation to the onset of morphological differentiation.

Morphological differentiation in microorganisms is usually accompanied by a decrease in intracellular GTP pool size, as has been demonstrated in bacillaceae, streptomycetaceae, and yeasts. The obg gene, which codes for a GTP-binding protein belonging to the GTPase superfamily of proteins, was cloned from Streptomyces griseus IFO13189. The gene is located just downstream of the genes for ribosomal proteins L21 and L27, encoded a protein of 478 amino acids (51 kDa), and possessed three consensus motifs which confer GTP-binding ability; Obg protein expressed in Escherichia coli bound GTP, as demonstrated using a UV cross-linking method. Introduction of multiple copies of obg into wild-type S. griseus suppressed aerial mycelium development in cells on solid media. However, no effect on streptomycin production was detected, indicating that Obg is involved in the regulation of the onset of morphological but not physiological differentiation. Multiple copies of obg also suppressed submerged spore formation in liquid culture. Southern hybridization studies indicated that genes homologous to obg exist widely in streptomycetes, and an obg homolog was successfully cloned from S. coelicolor A3(2). We propose that by monitoring the intracellular GTP pool size, the Obg protein is involved in sensing changes in the nutritional environment leading ultimately to morphological differentiation.

Amino Acid Sequence↗

Localization of the active site of the alpha subunit of the Escherichia coli DNA polymerase III holoenzyme.

Using a deletion approach on the alpha subunit of DNA polymerase III from Escherichia coli, we show that there is an N-proximal polymerase domain which is distinct from a more C-proximal tau and beta binding domain. Although deletion of 60 residues from the alpha N terminus abolishes polymerase activity, deletions of 48, 169, and 342 amino acids from the C terminus progressively impair its catalytic efficiency but preserve an active site. Deletion of 342 C-terminal residues reduces k(cat) 46-fold, increases the Km for gapped DNA 5.5-fold, and increases the Km for deoxynucleoside triphosphates (dNTPs) twofold. The 818-residue protein with polymerase activity displays typical Michaelis-Menten behavior, catalyzing a polymerase reaction that is saturable with substrate and linear with time. With the aid of newly acquired sequences of the polymerase III alpha subunit from a variety of organisms, candidates for two key aspartate residues in the active site are identified at amino acids 401 and 403 of the E. coli sequence by inspection of conserved acidic amino acids. The motif Pro-Asp-X-Asp, where X is a hydrophobic amino acid, is shown to be conserved among all known DnaE proteins, including those from Bacillaceae, cyanobacteria, Mycoplasma, and mycobacteria. The E. coli DnaE deletion protein with only the N-terminal 366 amino acids does not have polymerase activity, consistent with the proposed position of the active-site residues.

Amino Acid Sequence↗

Behavior of bacterial spores in aqueous polymer two-phase systems.

Sacks, L. E. (Western Regional Research Laboratory, Albany, California), and Gordon Alderton. Behavior of bacterial spores in aqueous polymer two-phase systems. J. Bacteriol. 82:331-341. 1961.-Free spores of various species of the Bacillaceae enter the upper phase in a two-phase aqueous polymer system containing polyethylene glycol 4000 and potassium phosphate. Vegetative cells appear in the lower phase and subcellular debris is concentrated chiefly at the interface. The high selectivity of the upper phase for spores makes possible the preparation of greatly enriched concentrates of spores directly from complex natural materials, such as soil and feces. Attraction of spores for the upper phase is not diminished by autoclaving and the release of dipicolinic acid. Loss of attraction of spores for the upper phase is associated in time with the shedding of the spore coat, upon outgrowth. The partition behavior of one preparation of Bacillus megaterium showed the presence of two classes of spores.

Bacillus↗

INTERSPECIFIC TRANSFORMATION IN BACILLUS.

Marmur, J. (Brandeis University, Waltham, Mass.), E. Seaman, and J. Levine. Interspecific transformation in Bacillus. J. Bacteriol. 85:461-467. 1963.-Deoxyribonucleic acids (DNA) from various species of the taxonomic group Bacillaceae were examined for base composition, ability to carry out interspecific transformation, and formation of molecular hybrids in vitro. The minimal requirement for genetic compatibility among different species and for DNA interaction (both reflecting base sequence homologies) is the similarity of the guanine plus cytosine contents of the DNA. The close correlation between the ability of DNA to be competent in interspecific transformation and to form hybrid molecules on denaturation and annealing provided a rational approach to the study of genetic relationship among organisms for which no genetic exchange has yet been demonstrated. Any or all of the criteria (base composition of DNA, transformation, molecular hybrid formation) can be used as tools in the taxonomic assessment of closely related microorganisms.

Journal Article↗

Electron Microscopy of Ultrathin Sections of Sporosarcina ureae.

Mazanec, K. (J. E. Purkyne University, Brno, Czechoslovakia), M. Kocur, and T. Martinec. Electron microscopy of ultrathin sections of Sporosarcina ureae. J. Bacteriol. 90:808-816. 1965.-Ultrathin sections of Sporosarcina ureae cells were studied by means of electron microscopy. The cell wall consists of several layers and is 340 A thick. The cytoplasm is of globular structure and includes ribosomelike structures, occasional mesosomes, and inclusions not precisely identifiable. The nuclear area has various shapes and is formed by filaments 10 to 20 A thick which proceed in various directions. Cell division occurs similarly to that of sarcinate. Both synchronic and asynchronic cell division was observed. The spores of S. ureae consist of an outer coat having several layers, a cortex, a spore wall, and cytoplasm. The results of the present investigation substantiate our previous suggestion that S. ureae should be transferred from the family Micrococcaceae to the family Bacillaceae.

Journal Article↗

Ribosyl and deoxyribosyl transfer by bacterial enzyme systems.

The enzymatic transfer of ribose and deoxyribose residues in pyrimidine nucleosides to purines was catalyzed by cell-free extracts of various bacteria. Almost all the strains belonging to Enterobacteriaceae were capable of catalyzing the transfer reactions. The transfer activities were also detected among some bacterial strains of other families: Pseudomonadaceae, Corynebacteriaceae, Micrococcaceae, Bacteriaceae, and Bacillaceae. The rates of the transfer reactions were greatly enhanced in the presence of phosphate ion, and the participation of nucleoside phosphorylases in the reactions was suggested. Uridine phosphorylase, thymidine phosphorylase, and purine nucleoside phosphorylase were purified from cell-free extract of Aerobacter aerogenes IFO 3321. The ribosyl transfer from uridine to hypoxanthine was found to be catalyzed by the coupled reactions of uridine and purine nucleoside phosphorylases and the deoxyribosyl transfer from thymidine to hypoxanthine by the coupled reactions of thymidine and purine nucleoside phosphorylases.

Aeromonas↗

Cytology of spore formation in Clostridium perfringens.

The sequential morphological events in spore formation by Clostridium perfringens type D were observed in Ellner's medium where 80 to 100% of the cells formed spores. Gross structural changes were studied with the light microscope under phase-contrast, and in fixed cells by the use of both nigrosin and Giemsa preparations. Fine structure was examined with the electron microscope in both thin sections and frozen-etched preparations. During the first 3 hr of incubation, the original rod-shaped cells became ellipsoid to ovoid in shape; by 5 to 6 hr, subterminal spores had developed within these enlarged cells. The fine structural sequence was in most respects identical to that in other Bacillaceae, although some stages were illustrated with particular clarity. A unique feature was the development of a convoluted, membranous exosporium which adhered to the outer surface of the two coats and had an unusual fine structure resembling a rectangular array of subunits.

Clostridium perfringens↗

Cytology of spore germination in Clostridium pectinovorum.

The process of spore germination in Clostridium pectinovorum has been followed by phase-contrast and electron microscopy. Unlike most other Bacillaceae, germination of this species takes place within the sporangium. Under phase-contrast, the spore darkens and swells slightly, and then the vegetative rod slips out through the end opposite the collar-like extension of the sporangium. In thin sections, a spore from an early stage in germination consists of a central protoplast, core membrane, germ cell wall, cortex, and two coats. Within a short period, the cortex disintegrates and the young cell develops. It possesses a large fibrillar nucleoplasm and several mesosomes. Subsequently, the young cell elongates, becomes somewhat deformed, and then emerges through a narrow aperture in the inflexible coats of the spore, finally rupturing the sporangium. Free vegetative cells of C. pectinovorum resemble in their structure other gram-positive rods.

Clostridium↗

Taxonomic comparison of the amino termini of microbial cell proteins.

A comparison was made of the distribution of amino terminal end groups in the cellular proteins of a number of microbes. Among the procaryotes, methionine is a highly variable but virtually ubiquitous major protein end group. This is consistent with its possible role as a general amino acid initiator of protein biosynthesis in the procaryotes. Generally, however, alanine is the most abundant of the major end groups, followed in decreasing order by serine, threonine, the acidic amino acids, and occasionally lysine. No other new major end-groups were found. Among 15 representatives of the Enterobacteriaceae, retention of the initiating methionine terminus of the cellular protein varies considerably at a tribal level and is randomized at a familial level. The profiles of the five remaining end groups, however, are strikingly uniform, and are, for example, close to but significantly different from those of the Erwineae. Among the taxonomically more heterogeneous Bacillaceae, end-group profiles vary more and are sometimes unrelated. End-group analysis is thus particularly useful as a molecular criterion of taxonomy in assessing familial homogeneity. Free NH(2) termini in eucaryote cell proteins are fewer, and they have increased acidic amino acid components and no methionine; they are otherwise similar to those of the procaryotes.

Alanine↗

Compartmentalization of gene expression during Bacillus subtilis spore formation.

Gene expression in members of the family Bacillaceae becomes compartmentalized after the distinctive, asymmetrically located sporulation division. It involves complete compartmentalization of the activities of sporulation-specific sigma factors, sigma(F) in the prespore and then sigma(E) in the mother cell, and then later, following engulfment, sigma(G) in the prespore and then sigma(K) in the mother cell. The coupling of the activation of sigma(F) to septation and sigma(G) to engulfment is clear; the mechanisms are not. The sigma factors provide the bare framework of compartment-specific gene expression. Within each sigma regulon are several temporal classes of genes, and for key regulators, timing is critical. There are also complex intercompartmental regulatory signals. The determinants for sigma(F) regulation are assembled before septation, but activation follows septation. Reversal of the anti-sigma(F) activity of SpoIIAB is critical. Only the origin-proximal 30% of a chromosome is present in the prespore when first formed; it takes approximately 15 min for the rest to be transferred. This transient genetic asymmetry is important for prespore-specific sigma(F) activation. Activation of sigma(E) requires sigma(F) activity and occurs by cleavage of a prosequence. It must occur rapidly to prevent the formation of a second septum. sigma(G) is formed only in the prespore. SpoIIAB can block sigma(G) activity, but SpoIIAB control does not explain why sigma(G) is activated only after engulfment. There is mother cell-specific excision of an insertion element in sigK and sigma(E)-directed transcription of sigK, which encodes pro-sigma(K). Activation requires removal of the prosequence following a sigma(G)-directed signal from the prespore.

Bacillus subtilis↗

Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species.

BACKGROUND: Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature. RESULTS: We determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs. CONCLUSIONS: Despite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.

Anti-Bacterial Agents↗

Comparative investigations of airborne culturable microorganisms in selected waste treatment facilities and in neighbouring residential areas.

The evaluation of airborne microorganisms in waste treatment facilities is complicated by different measuring systems, a lack of measuring standards and large variations between individual counts. In the present study, different sectors of the waste management industry were compared by determining median values of airborne culturable microorganisms from numerous parallel counts over a prolonged time period. The samples were taken during the warm season using the six-stage Andersen volumetric sampler in a large composting plant and its immediate vicinity, in an agricultural composting plant, a waste disposal site, and a sorting facility for recyclable materials. Control samples were taken at a site not influenced by the waste management industry in an open and largely uninhabited area. The highest median values for culturable bacteria (37 degrees C) found were 1.1 x 10(5) CFU/m3, for moulds (25 degrees C) 1.4 x 10(5) CFU/m3, and for A. fumigatus (37 degrees C) 1.7 x 10(4) CFU/m3 in the sorting cabins of the sorting facility (p < 0.001). The highest median values for thermophilic bacteria (actinomycetes and bacillaceae, 50 degrees C) were 7.3 x 10(3) CFU/m3 in the large composting facility. In all other facilities as well as in the neighbouring residential areas of all facilities investigated, all median values were significantly lower and corresponded to the naturally occurring levels: approx. 10(2) CFU/m3 for bacteria, approx. 10(3) CFU/m3 for moulds and approx. 10(1) CFU/m3 for A. fumigatus and thermophilic bacteria. Only in the neighbouring residential area of the large composting plant, the median values for thermophilic bacteria were approx. 10(2) CFU/m3, but an additional impact from farms cannot be excluded in this case. These results show permanent increased loads of the investigated microorganisms inside large composting facilities and especially in the sorting cabins for recyclable materials. The increasing number of reports on potential health hazards in these areas require adequate measures on the part of occupational medicine in order to limit the health risk to a minimum. The most important task is the automatization of the sorting process for recyclable materials.

Air Microbiology↗

Bacterial contamination of water wells in Wadi el-Sheikh area, in southern Sinai.

A bacterial assessment of water supplies must be done in Egypt as in order to improve the quality of useable water. Saint Katherine, Southern Sinai, was selected as being a populated area where wells are the only source of water for the local population. During one year, water of seven wells in Wadi El-Shiekh were bactiologically studied (El-Arbaien, Haron, Eid, Zitona, Farhan, Sahab and Gofa). The pouring plate technique was used to estimate the total viable bacterial count (TVB). The most probable number (MPN) technique was used to estimate total coliform (TC) and fecal coliform (FC) bacterial count. A result indicates that Al-Arabaien well has the least count of TVB throughout the year of study, possibly because of to geologic nature, low content of salts, low pH value of the water of this well. Zitona and Eid gave relatively the highest counts of TVB. This might had been due to the way in which these well were used. Intermediate counts were recorded in other wells. TC counts reached their maximum in Gofa and Farhan in Autumn. Percentages of FC among the TC counts were higher also in Autum in five wells (Al-Arbaien, Haron, Zitona, Eid and Sahab). This might had had been due to the increased agricultural activities around these wells. TC counts reached their minimum in Zitona during Winter, Spring and Summer. Five families of bacteria were identified form water samples, namely: Bacillaceae, Streptococcaceae Micrococcaceae, Neisseriaceae and Enterobacteriaceae. The geological condition of each well, some meteorological factors, and inhabitants activities around the well were found to play an important role in variability of TVB, TC, and FC counts. It is suggested that the inhabitants be educated of the best and healthy ways of using water and the good healthy habits.

Bacteria↗

[Presence of potentially pathogenic bacteria and fungi in the cooling-lubricating emulsion used in the aluminum sheet rolling process].

Microbiological tests of aluminium rolling water-oil emulsion reveal high level of contamination: relatively anaerobic bacteria 10(6)-10(8) cells/ml, fungi and yeasts 10(3)-10(4) cells/ml, and also anaerobic bacteria Desulfovibrie sp. reducing sulfates to hydrogen sulfide. In emulsion samples there were present bacterial strains potentially dangerous to health, belonging to the family Enterobacteriaceae, Vibrionaceae, Pseudomonadaceae, Bacillaceae. The fungi and yeasts found in the emulsion were identified as: Aspergillus sp., Penicillium sp., Cladosporium sp., Cephalosporium sp., Candida sp. The emulsion sprayed in aerosol in air of the mill was the infecting source for the workers. The emulsion was protected by a biocid and thus the growth of potentially pathogenic microorganisms was inhibited.

Aluminum↗

The nucleotide sequence of proline tRNAmo5UGG from Bacillus subtilis.

The nucleotide sequence of a proline tRNA from Bacillus subtilis W168 was determined to be pC-G-G-G-A-A-G-U-A-G-C-U-C-A-G- C-U-U-G-G-D-A-G-A-G-C-A-C-A-U-G-G-psi-U-mo5U-G-G-m1G-A-C-C-A-U-G-G -G-m7G-U-C-G-C-A-G-G-T-psi-C-G-A-A-U-C-C-U-G-U-C-U-U-C-C-C-G-A-C-C- AOH, by the analysis of the unlabeled preparation and by post-labeling technique. This tRNAPro contained 5-methoxyuridine (mo5U) which is specifically distributed in bacillaceae at the wobble position of the anticodon.

Anticodon↗