Effect of bacteria on the inactivation and adsorption on clay minerals of reovirus

This investigation studied the antiviral activity of, and the utilization of viruses as substrates by, bacteria. Reovirus type 3 and bacterial species representative of those endemic to sewage, aquatic, and terrestrial habitats were used in the model systems. Culture supernatants from Bacillus subtilis maintained for 5 days in a minimal salts medium displayed antiviral activity, but supernatants from Escherichia coli or Serratia marcescens did not. Both live and toluene-killed cells reduced the inactivation of reovirus during 4 days of incubation at 23 +/- 2 degrees C. This protective effect was more pronounced with killed than with live cells of B. subtilis, confirming the presence of an antiviral component(s) in this species and indicating that the component(s) was metabolic in origin. When reovirus was presented to these bacteria as a sole source of carbon, some growth (determined spectrophotometrically) of B. subtilis and S. marcescens occurred with reovirus concentrations of 3.1 X 10(6) and 8.2 X 10(6) mean tissue culture infective dose-fifty X mL-1, respectively. Growth of S. marcescens did not occur with a reovirus concentration of 8.0 X 10(4) mean tissue culture infective dose-fifty X mL-1, nor did that of E. coli with any virus concentration used in this study. Adsorption of reovirus on kaolinite was enhanced by the culture supernatant from S. marcescens and on montmorillonite, albeit to a lesser extent, by that from E. coli. The effect of culture supernatants from B. subtilis on the adsorption of reovirus on clay minerals could not be determined, as a result of the antiviral component produced by these cells. The virus was not adsorbed on the bacteria.     

Purification of a RecA protein analogue from Bacillus subtilis

We have identified in Bacillus subtilis an analogue of the Escherichia coli RecA protein. Its activities suggest that it has a corresponding role in general genetic recombination and in regulation of SOS (DNA repair) functions. The B. subtilis protein (B. subtilis Rec) has a Mr of 42,000 and cross-reacts with antisera raised against E. coli RecA protein. Its level is significantly reduced in the recombination-deficient recE4 mutant. B. subtilis Rec is induced 10- to 20-fold in rec+ strains following treatment with mitomycin C, whereas it is not induced in the recombination-deficient mutants recE4, recE45, and recA1. We have purified B. subtilis Rec about 2000-fold to near homogeneity and we describe its activities. It catalyzes DNA-dependent hydrolysis of dATP at a rate comparable to that of E. coli RecA protein. However, B. subtilis Rec has a negligible ATPase activity, although ATP effectively inhibits dATP hydrolysis. In the presence of dATP, B. subtilis Rec catalyzes DNA strand transfer, assayed by the conversion of phi X174 linear duplex DNA and homologous circular single-stranded DNA to replicative form II (circular double-stranded DNA with a discontinuity in one strand). ATP does not support strand transfer by this protein. B. subtilis Rec catalyzes proteolytic cleavage of E. coli LexA repressor in a reaction that requires single-stranded DNA and nucleoside triphosphate. This result suggests that an SOS regulatory system like the E. coli system is present in B. subtilis. The B. subtilis enzyme does not promote any detectable cleavage of the E. coli bacteriophage lambda repressor.     

Plasmid transformation in Bacillus subtilis NB22, an antifungal-antibiotic iturin producer

A transformation system with plasmids was developed for Bacillus subtilis NB22, an antibiotic iturin producing strain. Treatment of B. subtilis NB22 with 4 M KCl was effective for the induction of competence, followed by uptake of plasmid DNA in the presence of polyethylene glycol. The efficiency of transformation of this bacterium with pC194 and pUB110 was 4.1 X 10(3) and 1.5 X 10(3) transformants per micrograms DNA, respectively and the transformation frequency was 3.3 X 10(-3) and 7.2 X 10(-4), transformants per viable cell, respectively. This method was much faster and three orders of magnitude more efficient in transformation efficiency than protoplast transformation methods.     

Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems.

Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 10(4) CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml.     

Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems

Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 10(4) CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml.     

Expression of Bacillus circulans Teri-42 xylanase gene in Bacillus subtilis

The xylanase gene of Bacillus circulans Teri-42 was cloned in both B. subtilis and Escherichia coli. The enzyme activity was almost 87% higher in B. subtilis (pBA7) than in E. coli (pAQ4). No cellulase activity was detected in the clones, B. subtilis (pBA7) and E. coli (pAQ4). Approximately 1120 U (80%) of the xylanase was secreted extracellularly by the clone B. subtilis (pBA7) as compared to 79 U (88%) excreted in E. coli (pAQ4). In B. subtilis (pBA7) the optimal xylanase activity was at pH 7.0 and 50 degrees C, which was the same as that of the parent B. circulans Teri-42. The recombinant xylanase in B. subtilis was more stable at higher temperatures than the parent B. circulans Teri-42. Purification of xylanase from the clone B. subtilis (pBA7) showed a 71 kDa polypeptide similar to that observed in B. circulans Teri-42.     

Secretion of Bacillus subtilis levansucrase: a possible two-step mechanism

The rate of exocellular levansucrase synthesis in an overproducing (sacUh) strain of Bacillus subtilis was shown to be directly proportional to the amount of two different transient forms of this enzyme located within the membrane fraction of the cells. The apparent Mr of the larger membrane form was 53,000, and that of the smaller form 50,000; the half-life time of each form was estimated in vivo to be 4-6 s and 32-42 s, respectively. Ethanol treatment of the cells lead to the accumulation of the 53,000-Mr form which may represent 1.5% of total membrane proteins. This latter form, partially purified, was transformed in vitro into the 50,000-Mr form by the action of the Escherichia coli leader peptidase. These enzyme forms were quite different from the exocellular levansucrase since they showed a weak affinity for hydroxyapatite and needed complexed iron to display enzyme activity. Assuming the membrane forms were precursors of exocellular levansucrase, we propose a two-step mechanism for the secretion process of levansucrase. The number of exoprotein synthesis/secretion sites in a B. subtilis cell is estimated to 2.5 X 10(4).     

Construction of a marker rescue system in Bacillus subtilis for detection of horizontal gene transfer in food

A marker rescue system based on the repair of the kanamycin resistance gene nptII was constructed for use in Gram-positive bacteria and established in Bacillus subtilis 168. Marker rescue was detected in vitro using different types of donor DNA containing intact nptII. The efficiency of marker rescue using chromosomal DNA of E. coli Sure as well as plasmids pMR2 or pSR8-30 ranged from 3.8 x 10(-8) to 1.5 x 10(-9) transformants per nptII gene. Low efficiencies of ca. 10(-12) were obtained with PCR fragments of 792 bp obtained from chromosomal DNA of E. coli Sure or DNA from a transgenic potato. B. subtilis developed competence during growth in milk and chocolate milk, and marker rescue transformation was detected with frequencies of ca. 10(-6) and 10(-8), respectively, using chromosomal DNA of E. coli Sure as donor DNA. Although the copy number of nptII genes of the plant DNA exceeded that of chromosomal E. coli DNA in the marker rescue experiments, a transfer of DNA from the transgenic plant to B. subtilis was detectable neither in vitro nor in situ.     

Comparison of the relative mutagenicities of O-alkylthymines site-specifically incorporated into phi X174 DNA

The relative mutagenicities of O-alkylthymine-DNA adducts were analyzed in vivo by site-specific mutagenesis. Purified DNA polymerases were used to incorporate O4-methyl (Me)-, O4-ethyl (Et)-, O4-isopropyl (iPr)-, or O2-Me-dTTP onto the 3' terminus of a synthetic oligonucleotide (15-mer) hybridized to phi X174 am3 DNA. The product oligonucleotides were further extended in the presence of unmodified dNTPs to yield 21-mers containing single O-alkylthymine adducts opposite the adenine residue of the bacteriophage amber codon. Polyacrylamide gel electrophoresis and nearest-neighbor analyses confirmed the identities and nucleotide positions of the adducts. Transfection and replication of the site-specifically alkylated DNAs in ada- Escherichia coli (defective in the alkyltransferase capable of repairing O4-alkylthymine-DNA adducts) yielded mutant progeny phage with reversion frequencies of: O4-Me-dThd (19.5 X 10(-6) ) greater than O4-Et-dThd (7.5 X 10(-6) ) greater than O4-iPr-dThd (3.0 X 10(-6) ) greater than or equal to O2-Me-dThd (1.0 X 10(-6) ) approximately equal to dThd (2.0 X 10(-6) ). None of the adducts produced mutations above background following replication in ada+ E. coli. DNA sequence analyses of 40 independently isolated mutant phage derived from the O4-Me- or O4-Et-dThd-containing DNAs showed that all mutants contained guanine residues opposite the original site of the alkylthymines. These data are consistent with a mechanism of mutagenesis involving the formation of O4-alkyl-T.G base pairs during DNA replication in E. coli and suggest that the formation of A.T----G.C transition mutations is characteristic of mutagenesis by O4-Me- and O4-Et-dThds in vivo.     

Evidence that the clindamycin-erythromycin resistance gene of Bacteroides plasmid pBF4 is on a transposable element.

We constructed a shuttle vector, pE5-2, which can replicate in both Bacteroides spp. and Escherichia coli. pE5-2 contains a cryptic Bacteroides plasmid (pB8-51), a 3.8-kilobase (kb) EcoRI-D fragment from the 41-kb Bacteroides fragilis plasmid pBF4, and RSF1010, an IncQ E. coli plasmid. pE5-2 was mobilized by R751, an IncP E. coli plasmid, between E. coli strains with a frequency of 5 X 10(-2) to 3.8 X 10(-1) transconjugants per recipient. R751 also mobilized pE5-2 from E. coli donors to Bacteroides uniformis 0061RT and Bacteroides thetaiotaomicron 5482 with a frequency of 0.9 X 10(-6) to 2.5 X 10(-6). The Bacteroides transconjugants contained only pE5-2 and were resistant to clindamycin and erythromycin. Thus, the gene for clindamycin and erythromycin resistance must be located within the Eco RI-D fragment of BF4. A second recombinant plasmid, pSS-2, which contained 33 kb of pBF4 (including the EcoRI-D fragment and contiguous regions) could also be mobilized by R751 between E. coli strains. In some transconjugants, a 5.5-kb (+/- 0.3 kb) segment of the pBF4 portion of pSS2 was inserted into one of several sites on R751. In some other transconjugants this same 5.5-kb segment was integrated into the E. coli chromosome. This segment could transfer a second time onto R751. Transfer was RecA independent. The transferred segment included the entire EcoRI-D fragment, and thus the clindamycin-erythromycin resistance determinant, from pBF4.     

Bactericidal effect of solar water disinfection under real sunlight conditions

Batch solar disinfection (SODIS) inactivation kinetics are reported for suspensions in water of Campylobacter jejuni, Yersinia enterocolitica, enteropathogenic Escherichia coli, Staphylococcus epidermidis, and endospores of Bacillus subtilis, exposed to strong natural sunlight in Spain and Bolivia. The exposure time required for complete inactivation (at least 4-log-unit reduction and below the limit of detection, 17 CFU/ml) under conditions of strong natural sunlight (maximum global irradiance, approximately 1,050 W m(-2) +/- 10 W m(-2)) was as follows: C. jejuni, 20 min; S. epidermidis, 45 min; enteropathogenic E. coli, 90 min; Y. enterocolitica, 150 min. Following incomplete inactivation of B. subtilis endospores after the first day, reexposure of these samples on the following day found that 4% (standard error, 3%) of the endospores remained viable after a cumulative exposure time of 16 h of strong natural sunlight. SODIS is shown to be effective against the vegetative cells of a number of emerging waterborne pathogens; however, bacterial species which are spore forming may survive this intervention process.     

Effect of SOS repair in enterobacteria on their interaction with the complement system

The interaction of E. coli (serovar 0124) and its rec A-mutants with serum complement resulting in the alternative pathway activation was studied. Bacteria VT1240 (original smooth strain), VT1241 (rough mutant) and VT 2240 (recA56 mutant) were shown to be complement-sensitive when treated with 1.5 X 10(8)--1.9 X 10(8) cells per ml of normal human serum, while the cells with SOS-activated system (recA441 mutant, strain VT3251) retained their viability. An alternative pathway of complement activation was minimal with E. coli VT1241, while VT3251 demonstrated intermediate activity. To decrease the level of complement components (AH50) and factor B (BH50) by 50%, 3.5 X 10(6)--4.5 X 10(6) cells of VT1240 and VT2240 strains were required. R-mutants and recA441 mutants caused a 50% reduction in AH50, when used in the amount of 6.4 X 10(7) and 2.6 X 10(7), respectively, the same degree of BH50 decrease was achieved with the amounts used equal to 1.1 X 10(8) and 4.3 X 10(6), respectively. C3 conversions caused by 4 X 10(8) cells in I ml of the normal human serum in the four strains tested accounted for 5-15%.     

Tn10-derived transposons active in Bacillus subtilis.

Small derivatives of the Escherichia coli transposon Tn10, comprising IS10 ends and a chloramphenicol resistance gene, were introduced in Bacillus subtilis on a thermosensitive plasmid, pE194. In the presence of the Tn10 transposase gene fused to signals functional in B. subtilis, these derivatives transposed with a frequency of 10(-6) per element per generation. They had no highly preferred insertion site or region, as judged by restriction analysis of the chromosomal DNA, and generated auxotrophic and sporulation-deficient mutants with a frequency of about 1%. These results suggest that Tn10 derivatives might be a useful genetic tool in B. subtilis and possibly other gram-positive microorganisms.     

Thiolation and 2-methylthio- modification of Bacillus subtilis transfer ribonucleic acids.

Six thionucleosides found in Bacillus subtilis transfer ribonucleic acids were investigated: N6-(delta 2-isopentenyl)-2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, 2-methylthioadenosine, N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine, and one unknown (X1). The presence of N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine was demonstrated based on the affinity of the transfer ribonucleic acid containing it for an immunoadsorbent made with the antibody directed toward N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine. The existance of N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine in two species of lysine transfer ribonucleic acids was also confirmed by high-resolution mass spectrometry. Four of these thionucleosides--N6-(delta 2-isopenenyl)-2-methylthioadenosine, 2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, and the unknown designated X1--occurred only in specific areas in the elution profile of an RPC-5 column and probably affect the chromatographic properties of the transfer ribonucleic acids containing them. In contrast with Escherichia coli, where 4-thiouridine is the most frequent type of sulfur-containing modification, approximately one-third of the sulfur groups in B. subtilis transfer ribonucleic acid are present as thiomethyl groups on the 2 position of an adenosine or modified adenosine residue.     

Methylation of ribosomal proteins in bacteria: evidence of conserved modification of the eubacterial 50S subunit.

Methylation of the 50S ribosomal proteins from Bacillus stearothermophilus, Bacillus subtilis, Alteromonas espejiana, and Halobacterium cutirubrum was measured after the cells were grown in the presence of [1-14C]methionine or [methyl-3H]methionine or both. Two-dimensional polyacrylamide gel electrophoretic analysis revealed, in general, similar relative electrophoretic mobilities of the methylated proteins from each eubacterium studied. Proteins known to be structurally and functionally homologous in several microorganisms were all methylated. Thus, the following group of proteins, which appear to be involved in peptidyltransferase or in polyphenylalanine-synthesizing activity in B. stearothermophilus (P.E. Auron and S. R. Fahnestock, J. Biol. Chem. 256:10105-10110, 1981), were methylated (possible Escherichia coli methylated homologs are indicated in parentheses): BTL5(EL5), BTL6(EL3), BTL8(EL10), BTL11(EL11), BTL13(EL7L12) and BTL20b(EL16). In addition, the pentameric ribosomal complex BTL13 X BTL8, analogous to the complex EL7L12 X EL10 of E. coli, contained methylated proteins. Analysis of the methylated amino acids in the most heavily methylated proteins, BSL11 from B. subtilis and BTL11 from B. stearothermophilus, showed the presence of epsilon-N-trimethyllysine as the major methylated amino acid in both proteins, in agreement with known data for E. coli. In addition, BSL11 appeared to contain trimethylalanine, a characteristic, modified amino acid previously described only in EL11 from E. coli. These results and those previously obtained from other bacteria indicate a high degree of conservation for ribosomal protein methylation and suggest an important, albeit unknown, role for the modification of these components in eubacterial ribosomes.     

Synthesis of ethyl (<Emphasis Type="Italic">S</Emphasis>)-4-chloro-3-hydroxybutanoate using <Emphasis Type="Italic">fabG</Emphasis>-homologues

This paper is a report on the successful application of bioinformatics to enzyme screening. The synthesis of ethyl ( S)-4-chloro-3-hydroxybutanoate (ECHB) by asymmetric reduction of ethyl 4-chloroacetoacetate (ECAA) using fabG-homologues was studied. beta-Ketoacyl-acyl carrier protein reductases from both Escherichia coli and Bacillus subtilis, which are components of type II fatty acid synthase, could reduce ECAA to ( S)-ECHB with 94-98% ee. Furthermore, acetoacetyl-CoA reductases (ARs) from both Ralstonia eutropha and Zoogloea ramigera, whose genes are significantly similar to fabG genes and play a physiological role in the biosynthesis of poly-beta-3-hydroxybutyrate, could also catalyze the asymmetric reduction of ECAA to ( S)-ECHB with >99% ee. ( S)-ECHB was synthesized to 48.7 g/l with an optical purity of 99.8% ee, using recombinant E. coli cells coexpressing AR from R. eutropha and glucose dehydrogenase from B. subtilis for the regeneration of NADPH.     

Transfer of transposon Tn916 from Bacillus subtilis to Thermus aquaticus

Broad host range conjugating transposon Tn916 has been introduced into the extreme thermophile Thermus by transposon transformation and transposition into the Bacillus subtilis chromosome followed by broth mating with Thermus aquaticus ATCC27634. Tetracycline resistant Thermus transconjugants were obtained at a frequency of 1.4 X 10(-7) per donor and 1.2 X 10(-7) per recipient. Transposon transfer from Thermus to Bacillus subtilis was also demonstrated in similar broth matings. Transfer characteristics were consistent with the conjugation mechanism described for Tn916 in mesophiles.     

Antibacterial effects of water-soluble low-molecular-weight chitosans on different microorganisms

Low-molecular-weight chitosans with a viscosity-average molecular weight (Mv) of 5 to 27 kDa and equal degree of deacetylation (DD, 85%) were highly active against Pseudomonas aureofaciens, Enterobacter agglomerans, Bacillus subtilis, and Bifidobacterium bifidum 791, causing death of 80 to 100% of cells. An exception to this tendency was Escherichia coli, for which the rate of cell death, induced by the 5-kDa chitosan, was 38%. The antibacterial effect was manifested as early as 10 min after incubation of 12-kDa chitosan with B. subtilis or E. coli cells. Candida krusei was almost insensitive to the above crab chitosans. However, Candida krusei was highly sensitive to chitosans with Mv 5, 6, 12, 15.7, and 27 kDa: the minimum inhibitory concentration (MIC) varied from 0.06 to 0.005%. Chitosans with M, 5, 12, and 15.7 kDa exerted an antibacterial effect on Staphylococcus aureus. Chitosans with Mv 5, 15.7, and 27 kDa had no effect on Bifidobacterium bifidum ATCC 14893. The antibacterial effect of the 4-kDa chitosan on E. coli and B. bifidum 791 increased with DD in the range 55-85%.     

Characterization of a region of the Enterococcus faecalis plasmid pAM beta 1 which enhances the segregational stability of pAM beta 1-derived cloning vectors in Bacillus subtilis.

The nucleotide sequence of a 2.13-kb EcoRI-HindIII, pAM beta 1-derived fragment, isolated from the gram-positive cloning vector pHV1431, has been determined and shown to encode two ORFs. ORF H encodes for a protein of 23,930 Da which exhibits substantial homology to bacterial site-specific recombinases, particularly the resolvases of the gram-positive transposons Tn917 (30.3% identity) and Tn552 (31.6% identity) and the clostridial plasmid pIP404 (27.1% identity). The second ORF (I) is incomplete and encodes a polypeptide which has significant homology with Escherichia coli topoisomerase I (26.0% identity). Insertion of either the entire 2.13-kb EcoRI-HindIII fragment or a 0.73-kb EcoRI-DraI subfragment encoding only the resolvase into the pAM beta 1-based cloning vector pMTL500E causes a significant enhancement of segregational stability (from 6.5 X 10(-2) to 3.0-4.0 X 10(-3) plasmid loss per cell per generation). Improved segregational stability is mirrored by a reduction in plasmid polymerization. The introduction of a stop codon into the resolvase coding region negates its ability to promote segregational stability. It is proposed that the identified determinant stabilizes pAM beta 1-based vectors in Bacillus subtilis by maintaining the plasmid population in the monomeric state, thereby reducing the chances of producing plasmid-free segregants.