Interactions of Bacillus licheniformis ATCC 10716 and Normal Flora of Human Skin

To determine whether antibiotic production might be ecologically advantageous in the survival of Bacillus species on human skin, we applied spores of a bacitracin-producing strain of Bacillus licheniformis (ATCC 10716) to the forearms of 11 volunteers. Three additional strains of B. licheniformis which did not synthesize antibiotics, including a mutant of ATCC 10716, were used in subsequent control trials. Samples of flora were taken from inoculated and control (opposite forearm) sites during the colonization period, generally 3 weeks. Although population densities were unaltered, changes in the carriage, composition, and bacitracin sensitivity of resident flora were related with the presence of ATCC 10716 only, which suggests that microbial interactions are important in bacillus colonization and in maintenance of normal flora. Interactions were examined in vitro by comparing growth curves of representative skin bacteria, including isolates of Staphylococcus epidermidis, Staphylococcus saprophyticus, Micrococcus luteus, and a large-colony diphtheroid, grown individually, in mixed culture with each other, and together in presence of each test strain of B. licheniformis. We observed some diminution of growth of M. luteus and the diphtheroid in the first mixed culture, and the diphtheroid was completely retarded in common culture with ATCC 10716. Lesser antibiotic effects were seen on the cocci, whose rank of sensitivity was similar to that in vivo. The growth of the diphtheroid was enhanced in mixed culture with those strains of bacilli which lack antibiotic activity.     

Amino acid control mechanism for bacitracin formation byBacillus licheniformis

Several amino acids affected the amount of bacitracin produced by Bacillus licheniformis ATCC 10716 and the high-yielding mutant B. licheniformis AL. The possibility of an amino acid control mechanism for bacitracin biosynthesis is discussed. The two strains showed a different response to the addition of some amino acids. This indicate that they may have differences in their amino acid control mechanism.     

Distribution and variation of bacitracin synthetase gene sequences in laboratory stock strains of Bacillus licheniformis

Distribution and variation of bacitracin synthetase gene (bac) sequences in 22 laboratory stock strains of Bacillus licheniformis were studied by Southern hybridization of bac gene probes from B. licheniformis ATCC 10716 to genomic PstI or HindIII restriction fragments. Eleven strains gave hybridization signals. These hybridization patterns were classed into two types. Eight strains showed similar patterns to that of ATCC 10716 and all, except one, produced bacitracin. The three strains showed fairly different hybridization patterns from that of ATCC 10716, and one (ATCC 33632) of them produced bacitracin. None of the remaining 11 strains, including ATCC 14580 (type strain), gave any hybridization signals. All strains carrying bac gene sequences were erythromycin resistant. With one exception, all strains without bac gene sequences were erythromycin sensitive. These results show that B. licheniformis strains are divided into two groups with respect to presence of bac gene sequences and erythromycin resistance. Received: 5 September 2001 / Accepted: 19 October 2001     

Phenotypic variations in re-lysogenization ofBacillus licheniformis with temperate phage

A bacitracin-producing strainBacillus licheniformis ATCC 10716 harbors two types of inducible phages (LP52 and DLP 10716). 156 strains re-lysogenized with phage LP52 were independently isolated from a cured strain UM12 ofB. licheniformis. Those strains were divided into 12 groups based on colony morphology and pigment production. Some of the re-lysogenized strains grew faster than UM12 and others produced more bacitracin than the cured strain. For example, the production of bacitracin by one of the relysogenized strains, L89, was enhanced by about 70% in comparison with UM12. The phenotypic variations observed with re-lysogenized strains might be due to the re-insertion of the phage genome at different sites of the chromosome in addition to the pleiotropic effect assumed.Abbreviations ATCC American Type Culture Collection - DNA Deoxyribonucleic acid - MC Mitomycin C - OD Optical density - PFU Plaque forming unit - rpm Revolutions per minute - UOD Unit of optical density - UV Ultraviolet Definition Specific growth rate (h^-1) - t time (h) - X cell concentration (g/l)     

Distribution and characteristics of Bacillus bacteria associated with hydrobionts and the waters of the Peter the Great Bay, Sea of Japan

Bacilli of the species Bacillus subtilis, B. pumilus, B. mycoides, B. marinus and B. licheniformis (a total of 53 strains) were isolated from 15 invertebrate species and the water of the Vostok Bay, Peter the Great Bay, Sea of Japan. Bacilli were most often isolated from bivalves (22.7%) and sea cucumbers (18.9%); they occurred less frequently in sea urchins and starfish (13.2 and 7.5%, respectively). Most of bacilli strains were isolated from invertebrates inhabiting silted sediments. No Bacillus spp. strains were isolated from invertebrates inhabiting stony and sandy environments. The species diversity of bacilli isolated from marine objects under study was low. Almost all bacterial isolates were resistant to lincomycin. Unlike B. pumilus, B. subtilis isolates were mostly resistant to benzylpenicillin and ampicillin. Antibiotic sensitivity of B. licheniformis strains was variable (two strains were resistant to benzylpenicillin and oxacillin, while one was sensitive). A significant fraction of isolated bacilli contained pigments. Pigmented strains were more often isolated from seawater samples, while colorless ones predominated within hydrobionts. B. subtilis colonies had the broadest range of colors. In the Bacillus strains obtained, DNase, RNase, phosphatase, elastolytic, chitinase, and agarolytic activity was detected. Bacilli strains with hydrolytic activity occurred in invertebrates more often than in seawater.     

Genetic mapping of the bacitracin synthetase gene(s) in Bacillus licheniformis

The map position of a mutation in the bacitracin synthetase gene(s) in Bacillus licheniformis ATCC 10716 was determined by transduction with phage SP-15. Results indicate that it is linked to the lys and trp loci and is distinct from the known sporulation loci on the chromosome of Bacillus licheniformis. The defect(s) of the enzyme complex were analysed in terms of its ability to bind covalently 14C-labelled amino acid precursors of the bacitracin molecule.     

Siderophores of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis

Three Bacillus anthracis Sterne strains (USAMRIID, 7702, and 34F2) and Bacillus cereus ATCC 14579 excrete two catecholate siderophores, petrobactin (which contains 3,4-dihydroxybenzoyl moieties) and bacillibactin (which contains 2,3-dihydroxybenzoyl moieties). However, the insecticidal organism Bacillus thuringiensis ATCC 33679 makes only bacillibactin. Analyses of siderophore production by previously isolated [Cendrowski et al., Mol. Microbiol. 52 (2004) 407-417] B. anthracis mutant strains revealed that the B. anthracis bacACEBF operon codes for bacillibactin production and the asbAB gene region is required for petrobactin assembly. The two catecholate moieties also were synthesized by separate routes. PCR amplification identified both asbA and asbB genes in the petrobactin producing strains whereas B. thuringiensis ATCC 33679 retained only asbA. Petrobactin synthesis is not limited to the cluster of B. anthracis strains within the B. cereus sensu lato group (in which B. cereus, B. anthracis, and B. thuringiensis are classified), although petrobactin might be prevalent in strains with pathogenic potential for vertebrates.     

Participation of temperate phages LP52 and BL20 in the control of bacitracin synthesis in Bacillus licheniformis

The synthesis of the antibiotic bacitracin in lysogenic and nonlysogenic strains of Bacillus licheniformis 1001 and ATCC10716 has been studied. The antibiotic activity was shown to be about 20% less in lysogens, as compared to nonlysogens. However, the level of bacitracin production was completely restored when temperate bacteriophages BL20 and LP52 were reintroduced into the nonlysogenic strains by virtue of genetic transformation with DNA from lysogenic strains or by transduction with LP52. This may indicate that both phages take part in control of the synthesis of bacitracin. For the time being, the mechanism of regulation is not known. It is likely to be either direct (provided that prophage DNA contains "bacitracin" genes), or indirect.     

Physical mapping of LP51 and LP52 prophages of lysogenic strains of Bacillus licheniformis

Summary The physical maps of the LP51 and LP52 prophages in lysogenic strains of Bacillus licheniformis were constructed on the basis of data obtained by hybridization of phage DNA probes with Southern blots of restricted DNA of the lysogens. The data were compatible with the Campbell model for chromosomal integration; the attP site was mapped at 58.7–61.8 map units of the genomes of both phages. Identification of prophage-host DNA junction fragments indicated the presence of a unique attB site on the bacterial chromosome; the set of junction fragments in the strain B. licheniformis ATCC 10716 was identical to that of ATCC 11946, but different from ATCC 8187. Both the LP51 and LP52 phages used the same integration sites. Upon reinfection with either phage, the cured strains UM12 and UM18 (i.e. 10716 and 11946 cured of LP52 or LP51, respectively) turned out to be integration deficient. In surface cultures the reinfected bacteria could be maintained in the lysogenic state without, however, integrating the phage genome; when these bacteria were passaged in submerged cultures, several modes of anomalous integration were observed, and the phage segregated into a variety of forms, discernible by virulence and plaque morphology. In liquid cultures of UM12(LP51) or UM12(LP52) lytic forms finally predominated, while most lysogenized UM18 were converted into defective lysogens which contained a defective prophage in a stably integrated form.     

Detection of molecular diversity in Bacillus atrophaeus by amplified fragment length polymorphism analysis

Phenotypically, Bacillus atrophaeus is indistinguishable from the type strain of Bacillus subtilis except by virtue of pigment production on certain media. Several pigmented variants of B. subtilis have been reclassified as B. atrophaeus, but several remain ambiguous in regard to their taxonomic placement. In this study, we examined strains within the American Type Culture Collection originally deposited as Bacillus globigii, B. subtilis var. niger, or Bacillus niger using 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis to determine the level of molecular diversity among these strains and their relationship with closely related taxa. The 16S rRNA gene sequences revealed little variation with one base substitution between the B. atrophaeus type strain ATCC 49337 and the other pigmented bacilli. AFLP analysis produced high-quality DNA fingerprints with sufficient polymorphism to reveal strain-level variation. Cluster analysis of Dice similarity coefficients revealed that three strains, ATCC 31028, ATCC 49760, and ATCC 49822, are much more closely related to B. atrophaeus than to B. subtilis and should be reclassified as B. atrophaeus. A very closely related cluster of B. atrophaeus strains was also observed; this cluster was genetically distinct from the type strain. The level of variation between the two groups was approximately the same as the level of variation observed between members of the two B. subtilis subspecies, subtilis and spizizenii. It is proposed that the cluster of strains typified by ATCC 9372 be designated a new subspecies, B. atrophaeus subsp. globigii.     

Genetic polymorphism by RAPD-PCR and phenotypic characteristics of isolated thermotolerant Bacillus strains from hot spring sources

The polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPD) assay, morphological, physiological, biochemical and antimicrobial susceptibility test methods have been evaluated for use in the taxonomy of isolated thermotolerant Bacillus from Jordanian hot springs, with specific reference to strains Geobacillus stearothermophilus (ATCC 12980), Bacillus circulans (ATCC 4513) and Bacillus sphaericus (ATCC 14577). A RAPD assay has been optimized and is able to discriminate between numerous thermotolerant Bacillus strains. RAPD-PCR was found to give reproducible thermotolerant Bacillus strains classification of DNA fingerprints for 14 strains including 3 reference strains. A study of 14 isolates and 3 reference strains, analyzing 53 phenotypic characters, resulted in their allocation to five major clusters at 60% similarity. Whereas at 80% similarity, twelve taxonomically distinct groups were evident.     

Biosurfactant production and surface translocation are regulated by PlcR in Bacillus cereus ATCC 14579 under low-nutrient conditions

Bacillus cereus ATCC 14579 can respond to nutrient changes by adopting different forms of surface translocation. The B. cereus ATCC 14579 DeltaplcR mutant, but not the wild type, formed dendritic (branched) patterns on EPS [a low-nutrient medium that contains 7.0 g K(2)HPO(4), 3.0 g KH(2)PO(4), 0.1 g MgSO(4).7H(2)O, 0.1 g (NH(4))(2)SO(4), 0.01 g CaCl(2), 0.001 g FeSO(4), 0.1 g NaCl, 1.0 g glucose, and 125 mg yeast extract per liter] containing 0.7% agar. The dendritic patterns formed by sliding translocation of nonflagellated cells are enhanced under low-nutrient conditions and require sufficient production of a biosurfactant, which appears to be repressed by PlcR. The wild-type and complemented strains failed to slide on the surface of EPS agar because of the production of low levels of biosurfactant. Precoating EPS agar surfaces with surfactin (a biosurfactant produced by Bacillus subtilis) or biosurfactant purified from the DeltaplcR mutant rescued the ability of the wild-type and complemented strains to slide. When grown on a nutrient-rich medium like Luria-Bertani agar, both the wild-type and DeltaplcR mutant strains produced flagella. The wild type was hyperflagellated and elongated and exhibited swarming behavior, while the DeltaplcR mutant was multiflagellated and the cells often formed long chains but did not swarm. Thin-layer chromatography and mass spectrometry analyses suggested that the biosurfactant purified from the DeltaplcR mutant was a lipopeptide and had a mass of 1,278.1722 (m/z). This biosurfactant has hemolytic activity and inhibited the growth of several gram-positive bacteria.     

Distribution and characteristics of <Emphasis Type="Italic">Bacillus</Emphasis> bacteria associated with hydrobionts and the waters of the Peter the Great Bay,Sea of Japan

Bacilli of the species Bacillus subtilis, B. pumilus, B. mycoides, B. marinus and B. licheniformis (a total of 53 strains) were isolated from 15 invertebrate species and the water of the Vostok Bay, Peter the Great Bay, Sea of Japan. Bacilli were most often isolated from bivalves (22.7%) and sea cucumbers (18.9%); they occurred less frequently in sea urchins and starfish (13.2 and 7.5%, respectively). Most of bacilli strains were isolated from invertebrates inhabiting silted sediments. No Bacillus spp. strains were isolated from invertebrates inhabiting stony and sandy environments. The species diversity of bacilli isolated from marine objects under study was low. Almost all bacterial isolates were resistant to lincomycin. Unlike B. pumilus, B. subtilis isolates were mostly resistant to benzylpenicillin and ampicillin. Antibiotic sensitivity of B. licheniformis strains was variable (two strains were resistant to benzylpenicillin and oxacillin, while one was sensitive). A significant fraction of isolated bacilli contained pigments. Pigmented strains were more often isolated from seawater samples, while colorless ones predominated within hydrobionts. B. subtilis colonies had the broadest range of colors. In the Bacillus strains obtained, DNase, RNase, phosphatase, elastolytic, chitinase, and agarolytic activity was detected. Bacilli strains with hydrolytic activity occurred in invertebrates more often than in seawater.     

Longevity of Microbial Biocontrol Agents in a Planting Mix Amended with Glomus intraradices

Commercial sources of the biological control agents Bacillus subtilis , Trichoderma harzianum and Streptomyces griseoviridis and experimental single isolates of Serratia plymuthica , a Pseudomonas fluorescens parent and its lacZY mutant were evaluated for their survival and compatibility with the mycorrhizal fungus, Glomus intraradices , in a commercial planting mix. The control treatments were the unamended mix and mix amended with G. intraradices alone. All were applied to 128-cell Speedling styrofoam flats and planted with the tomato cultivar 'Sunny'. At four to five intervals during the growing period (6.5-8 weeks), each organism was quantified by dilution plating and G. intraradices infection (%) was determined at the end of each test. The number of Trichoderma isolates increased slightly within 2 weeks after application and then stabilized through the end of the test. Serratia and Streptomyces isolates declined throughout the test from about 7.75 log colony-forming units (CFU) g - 1 to numbers at the end (6.2 log CFU g - 1) similar to the controls. Bacillus isolates declined about 1 log CFU g - 1 in the first week but then stabilized. G. intraradices had no influence on numbers of these four genera. The Pseudomonas parent and its lacZY mutant declined about 1 log CFU g - 1 during the test with the mutant yielding higher CFU for each sampling period. Propagules of both Pseudomonas isolates were greater when mixed with G. intraradices than when alone. In these experiments, Bacillus and Trichoderma were the best survivors in a mix for potential use as biocontrol agents for tomato transplants.     

Enzyme-bound intermediates in the biosynthesis of bacitracin.

1. Bacitracin synthetase, a three-component enzyme complex which catalyzes synthesis of the dodecapeptide bacitracin A, has been prepared from Bacillus licheniformis strains ATCC 10716, AL and SB 319. During synthesis of bacitracin, the amino acids (smaller amounts) and peptides are covalently bound to the enzyme complex. The nature of the bindings suggest that the amino acids and peptides are thioester linked. 2. The peptides, identified by thin-layer chromatography after performic acid liberation were Ile-Cys, Ile-Cys-Leu, Ile-Cys-Leu-Glu, Ile-Cys-Leu-Glu, Ile-Cys-Leu-Glu-Ile, Ile-Cys-Leu-Glu-Ile-Lys-Orn, Ile-Cys-Leu-Glu-Ile-Ile-Orn-Ile, Ile-Cys-L-EU-Glu-Ile-Lys-Orn-Ile-Phe, Ile-Cys-Leu-Glu-Ile-L-YS-Orn-Ile-Phe-His-Phe-His and Ile-Cys-Leu-Glu-Ile-Lys-Orn-Ile-Phe-His-Asp. 3. The labelled peptides covalently bound to bacitracin synthetase were intermediates in bacitracin synthesis. 4. Chain growth is initiated on one enzyme component (A) by the addition of isoleucine and cysteine. The sequential addition of the other amino acids proceeds in the C-terminal direction until the pentapeptide is formed. Further addition of amino acids and production of bacitracin are obtained by adding the other enzyme components (B and C) to the incubation mixture.     

Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates

We examined 154 Norwegian B. cereus and B. thuringiensis soil isolates (collected from five different locations), 8 B. cereus and 2 B. thuringiensis reference strains, and 2 Bacillus anthracis strains by using fluorescent amplified fragment length polymorphism (AFLP). We employed a novel fragment identification approach based on a hierarchical agglomerative clustering routine that identifies fragments in an automated fashion. No method is free of error, and we identified the major sources so that experiments can be designed to minimize its effect. Phylogenetic analysis of the fluorescent AFLP results reveals five genetic groups in these group 1 bacilli. The ATCC reference strains were restricted to two of the genetic groups, clearly not representative of the diversity in these bacteria. Both B. anthracis strains analyzed were closely related and affiliated with a B. cereus milk isolate (ATCC 4342) and a B. cereus human pathogenic strain (periodontitis). Across the entire study, pathogenic strains, including B. anthracis, were more closely related to one another than to the environmental isolates. Eight strains representing the five distinct phylogenetic clusters were further analyzed by comparison of their 16S rRNA gene sequences to confirm the phylogenetic status of these groups. This analysis was consistent with the AFLP analysis, although of much lower resolution. The innovation of automated genotype analysis by using a replicated and statistical approach to fragment identification will allow very large sample analyses in the future.     

Isolation and Identification of Psychrophilic Species of Bacillus from Milk

Forty isolates from 97 raw milk samples (heated to 80 C for 10 min and stored at 4 to 7 C for 3 to 4 weeks) were sporeforming, aerobic, gram-positive or gram-variable, rod-shaped bacteria. Fifteen isolates that were identified had characteristics similar to species of Bacillus, except that they had lower growth temperature ranges, were gram-variable, and were somewhat different in sugar fermentations. Four isolates grew well within 2 weeks at 0 C, but they grew faster at 20 to 25 C. These psychrophilic sporeforming bacteria, the importance of which is discussed, are considered to be variant strains of mesophilic bacilli adapted to low temperatures.     

Effects of Freon-113 on the survival of bacteria

An initial investigation was made of the bactericidal and sporicidal activity of the dry cleaning solvent Freon-113 (1,1,2-trichlorotrifluoroethane) under various conditions. Representative bacterial strains selected for this study were Escherichia coli (ATCC 11229), Staphylococcus aureus (ATCC 6538) and spores of Bacillus globigii (Bacillus subtilis var. niger). Various conditions were studied, including temperature, moisture, detergents and organic load. The results of this study indicate that there is the potential for survival of significant levels of micro-organisms in Freon-113 within the conditions evaluated. However, the bactericidal efficiency of this solvent increases significantly against vegetative forms at elevated temperature and, to a greater extent, upon the addition of detergents. Organic material present in the form of soiled fabric was observed to depress this efficacy whereas bacterial spores were virtually unaffected under all conditions evaluated.     

Regeneration of protoplasts of Bacillus subtilis 168 and closely related strains

Regeneration of protoplasts to bacilli was attempted in several strains of Bacillus closely related to Bacillus subtilis 168. On DM3 and similar media using succinate as osmotic support, only B. subtilis 168 and Bacillus natto ATCC 15245 were able to regenerate. Media containing mannitol as osmotic support, and agar as gelling agent gave rise to L-form colonies with Bacillus licheniformis NCTC 6346. Many of the L-form colonies were able to regenerate to the bacillary form when plated on the mannitol medium solidified with gelatin. All of the Bacillus species tested were able to regenerate on the latter medium at rates sufficient to allow protoplast transformation and fusion experiments.     

Effects of Freon-113 on the survival of bacteria

An initial investigation was made of the bactericidal and sporicidal activity of the dry cleaning solvent Freon-113 (1,1,2-trichlorotrifluoroethane) under various conditions. Representative bacterial strains selected for this study were Escherichia coli (ATCC 11229), Staphylococcus aureus (ATCC 6538) and spores of Bacillus globigii ( Bacillus subtilis var. niger ). Various conditions were studied, including temperature, moisture, detergents and organic load. The results of this study indicate that there is the potential for survival of significant levels of micro-organisms in Freon-113 within the conditions evaluated. However, the bactericidal efficiency of this solvent increases significantly against vegetative forms at elevated temperature and, to a greater extent, upon the addition of detergents. Organic material present in the form of soiled fabric was observed to depress this efficacy whereas bacterial spores were virtually unaffected under all conditions evaluated.