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PubMed2001

Mechanisms of biofilm formation in paper machine by Bacillus species: the role of Deinococcus geothermalis.

Mechanisms for the undesired persistence of Bacillus species in paper machine slimes were investigated. Biofilm formation was measured for industrial Bacillus isolates under paper machine wet-end-simulating conditions (white water, pH 7, agitated at 45 degrees C for 1-2 days). None of the 40 tested strains of seven Bacillus species formed biofilm on polished stainless steel or on polystyrene surfaces as a monoculture. Under the same conditions, Deinococcus geothermalis E50051 covered all test surfaces as a patchy thick biofilm. The paper machine bacilli, however, formed mixed biofilms with D. geothermalis E50051 as revealed by confocal microscopy. Biofilm interactions between the bacilli and the deinococci varied from synergism to antagonism. Synergism in biofilm formation of D. geothermalis E50051 was strongest with Bacillus coagulans D50192, and with the type strains of B. coagulans, B. amyloliquefaciens or B. pumilus. Two B. licheniformis, one B. amyloliquefaciens, one B. pumilus and four B. cereus strains antagonized biofilm production by D. geothermalis. B. licheniformis D50141 and the type strain of B. licheniformis were the strongest antagonists. These bacteria inhibited deinococcal growth by emitting heat-stable, methanol-soluble metabolite(s). We conclude that the persistence of Bacillus species in paper machine slimes relates to their ability to conquer biofilms formed by primary colonizers, such as D. geothermalis.

Bacillus↗
PubMed2002

Identification and properties of type I-signal peptidases of Bacillus amyloliquefaciens.

The use of Bacillus amyloliquefaciens for enzyme production and its exceptional high protein export capacity initiated this study where the presence and function of multiple type I signal peptidase isoforms was investigated. In addition to type I signal peptidases SipS(ba) [Meijer, W.J.J., de Jong, A., Bea, G., Wisman, A., Tjalsma, H., Venema, G., Bron, S. & van Dijl, J.M. (1995) Mol. Microbiol. 17, 621-631] and SipT(ba) [Hoang, V. & Hofemeister, J. (1995) Biochim. Biophys. Acta 1269, 64-68] which were previously identified, here we present evidence for two other Sip-like genes in B. amyloliquefaciens. Same map positions as well as sequence motifs verified that these genes encode homologues of Bacillus subtilis SipV and SipW. SipU-encoding DNA was not found in B. amyloliquefaciens. SipW-encoding DNA was also found for other Bacillus strains representing different phylogenetic groups, but not for Bacillus stearothermophilus and Thermoactinomyces vulgaris. The absence of these genes, however, could have been overlooked due to sequence diversity. Sequence alignments of 23 known Sip-like proteins from Bacillus origin indicated further branching of the P-group signal peptidases into clusters represented by B. subtilis SipV, SipS-SipT-SipU and B. anthracis Sip3-Sip5 proteins, respectively. Each B. amyloliquefaciens sip(ba) gene was expressed in an Escherichia coli LepBts mutant and tested for genetic complementation of the temperature sensitive (TS) phenotype as well as pre-OmpA processing. Although SipS(ba) as well as SipT(ba) efficiently restored processing of pre-OmpA in E. coli, only SipS(ba) supported growth at TS conditions, indicating functional diversity. Changed properties of the sip(ba) gene disruption mutants, including cell autolysis, motility, sporulation, and nuclease activities, seemed to correlate with specificities and/or localization of B. amyloliquefaciens SipS, SipT and SipV isoforms.

Bacillus↗
PubMed2000

Isolation of Bacillus strains from the rhizosphere of cereals and in vitro screening for antagonism against phytopathogenic, food-borne pathogenic and spoilage micro-organisms.

Bacillus strains were isolated from the rhizosphere of cereals in order to be used as natural biocontrol agents (BCAs). They were screened for antagonism in vitro against various test micro-organisms. The isolates showing antagonism were identified to species level. A combination of techniques was employed for the isolation of Bacillus species. Using the direct method, only one of the 25 isolates screened showed antagonistic properties. This strain (IFS-01) was identified by means of API test strips and the ATB Plus computer programme. It proved to be Bacillus subtilis and consequently has been designated as Bacillus subtilis IFS-01. This strain produced either a broad spectrum antimicrobial compound or several compounds with different activities. The fungi and Gram-positive bacteria were more sensitive to the antagonistic isolate than the Gram-negative bacteria. A Bacillus strain producing BCAs which can be used as biopesticides or organic preservatives has been isolated and identified.

Antibiosis↗
PubMed2001

Characterization and growth of Bacillus spp. in heat-treated cream with and without nisin.

AIMS: To study the germination and growth of both inoculated and naturally occurring Bacillus strains in heat-treated cream with and without nisin. METHODS AND RESULTS: In heat-treated cream (90 degrees C for 15 min) stored at 8 degrees C, growth was dominated by naturally occurring Bacillus strains such as Bacillus pumilus and B. licheniformis. Only six of the 52 isolated strains were B. cereus/thuringiensis. All of the B. cereus strains, but none of the other strains, produced enterotoxin when tested with the TECRA and reverse passive latex agglutination kits. Bacterial growth during storage of the cream at 8 or 10 degrees C was completely inhibited by low concentrations of nisin. CONCLUSION: The high number of Bacillus strains surviving the heat treatment represent a risk for heat-treated food that contains cream. The safety of the cream, for instance in "ready-to-eat" products, can be improved by the addition of low concentrations of nisin. SIGNIFICANCE AND IMPACT OF THE STUDY: Spores of several Bacillus species may survive heat treatment of cream, but low concentration of nisin with inhibit germination and growth.

Anti-Bacterial Agents↗
PubMed2002

Adaptation of a neutrophilic dairy-associated Bacillus cereus isolate to alkaline pH.

AIMS: This study identified and studied the response of five Bacillus strains, isolated from alkaline cleaning in place (CIP) solutions, to alkaline conditions. METHODS AND RESULTS: Isolates were identified as B. cereus by 16S rDNA sequencing. External and internal cell pH and buffering capacity data of a representative strain, Bacillus DL5, were compared to B. cereus ATCC 10702. Results indicated that a buffering system was induced when the pH of the growth medium increased to above pH 10, which was effective up to pH 12 and presumably cell wall associated. Volume measurements and confocal scanning laser microscope images of Bacillus DL5 cells showed that cells exhibited more pronounced stress symptoms when exposed to pH 10 than at pHs above 10. Long-term exposure of Bacillus DL5 to pH 10 or 10.5 indicated that cells grew in planktonic form and formed biofilms at both pHs. CONCLUSIONS: Bacillus DL5 was a neutrophile with a growth pH range similar to B. cereus ATCC 10702, but tolerated alkaline pH. This may be a general trait of the B. cereus species rather than a specific phenomenon of isolates from alkaline ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: Other neutrophilic B. cereus isolates may exhibit similar responses to alkaline conditions as the isolates studied here. These results may have important implications for dairy manufacturers.

Adaptation, Physiological↗
PubMed2000

The dppA gene of Bacillus subtilis encodes a new D-aminopeptidase.

Different strains of Bacillus were screened for their ability to hydrolyse D-alanyl-p-nitroanilide. Activity was detected in Bacillus pumilus, Bacillus brevis, Bacillus licheniformis 749I and Bacillus subtilis 168. The last strain was the best producer and was selected for the production and purification of the enzyme. The determination of the N-terminal sequence identified the enzyme as the product of the dppA gene (previously named dciAA) belonging to the dipeptide ABC transport (dpp) operon expressed early during sporulation. Open reading frames (ORFs) encoding putative related proteins were found in the genomes of a variety of Archaea and both sporulating and non-sporulating bacteria. The enzyme behaves as a D-aminopeptidase and represents the prototype of a new peptidase family. Among the tested substrates, the highest activities were found with D-Ala-D-Ala and D-Ala-Gly-Gly. The active enzyme behaves as an octamer of identical 30 kDa subunits. It exhibits a broad pH optimum, extending between pH 9 and 11. It is reversibly inhibited in the presence of Zn2+ chelators, and the sequence comparisons highlight the conservation of potential Zn-binding residues. As it has been shown by others that null mutations in the dpp operon do not inhibit spore formation, the physiological role of DppA is probably an adaptation to nutrient deficiency.

Amino Acid Sequence↗
PubMed2002

Improved polygalacturonase production from Bacillus sp. MG-cp-2 under submerged (SmF) and solid state (SSF) fermentation.

AIMS: To investigate the effect of amino acids, vitamins and surfactants on polygalacturonase production from Bacillus sp. MG-cp-2 under submerged (SmF) and solid state fermentation (SSF). METHODS AND RESULTS: Bacillus sp. MG-cp-2 was isolated from the outer covering of the seeds of Celastrus paniculatus. Out of the various surfactants, amino acids and vitamins, Tween-60, DL-serine and folic acid maximally enhanced polygalacturonase production by 2.7-fold (240.0 U x ml(-1)), 4.0-fold (360.0 U x ml(-1)) and 3.8-fold (342.0 U x ml(-1)) respectively, under submerged fermentation (SmF). In solid state fermentation (SSF), Tween-80, pyridoxine and DL-ornithine monohydrochloride induced highest enzyme production up to 1.73-fold (6956.5 U x g(-1)), 5.3-fold (21224.4 U x g(-1)) and 5.74-fold (23076.9 U x g(-1)), respectively. CONCLUSION: Amino acids and their analogues, vitamins and surfactants effect significantly polygalacturonase production by Bacillus sp. MG-cp-2 when grown under submerged (SmF) and solid state fermentation (SSF) conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides useful information about regulation of polygalacturonase biosynthesis in Bacillus sp. MG-cp-2, which appears to be an interplay of nutritional and physical factors. Alkaline polygalacturonase from Bacillus sp. MG-cp-2 will be extremely useful in the treatment of alkaline pectic waste waters from vegetable and fruit processing industries and in degumming of bast fibres.

Amino Acids↗
PubMed2004

A simple method to isolate biofilm-forming Bacillus subtilis and related species from plant roots.

A novel method was developed to isolate pure cultures of wild-type Bacillus subtilis and related species from plant roots, even roots washed free of adhering soil. The method uses casein digest-mannitol agarose (CM) media that promote rapid dendritic growth (low K+ ion) or profuse surface film formation (high K+ ion) of Bacillus species at 40 degrees C. Inoculation from the tips of surface growth on agarose leads to self-purification and streaking on CM agar plates (hard agar and high K+) leads to characteristic colony morphology. Phenotypic and 16S rDNA analysis revealed that most root isolates obtained by this method are spore-forming Bacillus species, with enrichment for B. subtilis and its close relatives. Of particular interest is the finding that the majority of these Bacillus isolates and the B. subtilis Marburg strain also form adhering biofilms on inert surfaces. Thus the methods presented may be useful in isolation of biofilm-forming Bacillus and investigation of their role on plant roots.

Bacillus subtilis↗
PubMed1994

Osteomyelitis due to Bacillus cereus in an adolescent: case report and review.

Non-anthracis Bacillus species associated with clinical infections are usually dismissed as contaminants or nonpathogens. As opportunists, however, Bacillus organisms can cause significant systemic infections including bacteremia, endophthalmitis, and pneumonia. Osteomyelitis with non-anthracis Bacillus organisms has been described in adults, although to our knowledge it has been described only once in a child. We report a case of chronic osteomyelitis due to Staphylococcus aureus and superinfection with Bacillus cereus in a 13-year-old adolescent. A Bacillus isolate should be considered a true pathogen in children with chronic osteomyelitis who have a poor clinical response to antistaphylococcal therapy.

Adolescent↗
PubMed2006

Experimental basis for a stable plasmid, pLS30, to shuttle between Bacillus subtilis species by conjugational transfer.

The use of Bacillus subtilis 168 as the initial host for molecular cloning and subsequent delivery of the engineered DNA to other Bacillus hosts appears attractive, and would lead to an efficient DNA manipulation system. However, methods of delivery to other Bacillus species are limited due to their inability to develop natural competence. An alternative, unexplored conjugational transfer method drew our attention and a B. subtilis native plasmid, pLS30, isolated from B. subtilis (natto) strain IAM1168 was characterized for this aim. The nucleotide sequence (6,610 bp) contained the mob gene and its recognition sequence, oriT, that features pLS30 as a mobile plasmid between Bacillus species on conjugational transfer. Plasmid pLS3001, a chimera with a pBR322-based plasmid prepared in Escherichia coli to confer an antibiotic resistance marker, showed apparent mobilizing activity in the pLS20-mediated conjugational transfer system recently established. The rep gene and associated palT1-like sequence common to all other pLS plasmids previously sequenced indicated that pLS30 is a typical rolling circle replicating (RCR) type plasmid. Due to the significant stability of pLS30 in IAM1168, application of a mobile plasmid would allow quick propagation to Bacillus species.

Bacillus subtilis↗
PubMed2002

A functional interaction between the putative primosomal protein DnaI and the main replicative DNA helicase DnaB in Bacillus.

In Gram negative Escherichia coli there are two well-characterised primosomal assembly processes, the PriA- and DnaA-mediated cascades. The presence of PriA and DnaA proteins in Gram positive Bacillus spp. supports the assumption that both the PriA- and DnaA-mediated primosomal assembly cascades also operate in these organisms. However, the lack of sequence homology between the rest of the primosomal proteins indicates significant differences between these two bacterial species. Central to the process of primosomal assembly is the loading of the main hexameric replicative helicase (DnaB in E.coli and DnaC in Bacillus subtilis) on the DNA. This loading is achieved by specialised proteins known as 'helicase loaders'. In E.coli DnaT and DnaC are responsible for loading DnaB onto the DNA during primosome assembly, in the PriA- and DnaA-mediated cascades, respectively. In Bacillus the identity of the helicase loader is still not established unequivocally. In this paper we provide evidence for a functional interaction between the primosomal protein DnaI from B.subtilis and the main hexameric replicative helicase DnaB from Bacillus stearothermophilus. Our results are consistent with the putative role of DnaI as the 'helicase loader' in the Gram positive Bacillus spp.

Adenosine Triphosphatases↗
PubMed2002

Bacillus anthracis as an agent of bioterrorism: a review emphasizing surgical treatment.

OBJECTIVE: To familiarize surgeons with the specific complications of cutaneous, gastrointestinal, inhalation, and systemic infection with Bacillus Anthracis, which may require surgical treatment. SUMMARY BACKGROUND DATA: The recent cases of intentional exposure to Bacillus Anthracis in the United States make familiarity with the basic microbiology, clinical manifestations, diagnosis, treatment, and control of this disease essential if mortality and morbidity is to be minimized, particularly following mass exposure. Although the treatment of Bacillus Anthracis infection is primarily medical, there are specific surgical complications with which the surgeon should be familiar. METHODS: A review of the literature was undertaken, utilizing electronic databases on infection with Bacillus Anthracis, as well as consultation with experts in this field. Emphasis was placed on the diagnosis and treatment of complications of infection that might require surgical intervention. RESULTS: Cutaneous anthrax infection results in eschar formation and massive soft tissue edema. When involving the extremities, increased compartment pressure requiring fasciotomy may result. Primary infection of the gastrointestinal tract may result in oropharyngeal edema and respiratory compromise requiring a surgical airway. Direct involvement of the lower gastrointestinal tract can result in intestinal ulceration, necrosis, bleeding, and perforation, which would require surgical exploration and resection of affected segments. Systemic sepsis, most often associated with inhalation anthrax, can cause massive ascites, electrolyte derangements, and profound shock requiring aggressive fluid resuscitation and careful hemodynamic monitoring and respiratory support. Systemic anthrax infection can also lead to gastrointestinal involvement by hematogenous dissemination, resulting in complications and requiring surgical management similar to direct gastrointestinal infection. CONCLUSIONS: Cutaneous, gastrointestinal, inhalation and systemic infection with Bacillus Anthracis can result in complications which would require familiarity with the pathogenesis and manifestations of this disease in order to recognize and treat promptly and successfully by surgical intervention.

Anthrax↗
PubMed2000

Bacillus thermodenitrificans sp. nov., nom. rev.

A polyphasic study was performed on 10 soil isolates of thermophilic denitrifying Bacillus strains from different geographical areas. The presence of two main characteristic bands following amplification of the internal transcribed spacer (ITS) region of rrn operons suggests a close relatedness to 'Bacillus thermodenitrificans'. The isolates cluster around two strains of 'B. thermodenitrificans' in riboprint and fatty acid analyses, though differences occur at the strain level. Subsequent DNA-DNA reassociation studies including the 10 isolates, 'B. thermodenitrificans' DSM 465T and DSM 466, and Bacillus stearothermophilus ATCC 12980T and Bacillus thermoleovorans ATCC 43513T revealed such a high level of genomic relatedness between the isolates and the DSM strains (> 73% similarity) that they must be considered strains of the same taxon. The degree of DNA-DNA similarity between the 12 strains of 'B. thermodenitrificans' and the type strains of the other two phylogenetically neighbouring Bacillus species was significantly lower (21-43% similarity). Based upon phylogenetic, chemotaxonomic and phenotypic evidence, the designation of B. thermodenitrificans sp. nov., nom. rev. is proposed. The type strain of B. thermodenitrificans is DSM 465T.

Bacillus↗
PubMed1986

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.

Bacillus subtilis↗
PubMed1991

Molecular cloning and nucleotide sequence of the gene encoding an endo-1,4-beta-glucanase from Bacillus sp. KSM-330.

The gene encoding an acid endo-1,4-beta-glucanase from Bacillus sp. KSM-330 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The recombinant plasmid contained a 3.1 kb HindIII insert, 1.8 kb of which was sufficient for the expression of endoglucanase activity in E. coli HB101. Nucleotide sequencing of this region (1816 bp) revealed an open reading frame of 1389 bp. The protein deduced from this sequence was composed of 463 amino acids with an Mr of 51882. The deduced amino acid sequence from amino acids 56 through 75 coincided with the amino-terminal sequence of the endoglucanase, Endo-K, purified from culture of Bacillus sp. KSM-330. The deduced amino acid sequence of Endo-K had 30% homology with that of the celA enzyme from Clostridium thermocellum NCIB 10682 and 25% homology with that of the enzyme from Cellulomonas uda CB4. However, the Endo-K protein exhibited no homology with respect to either the nucleotide or the amino acid sequences of other endoglucanases from Bacillus that had been previously characterized. These results indicate that the gene for Endo-K in Bacillus sp. KSM-330 has evolved from an ancestral gene distinct from that of other Bacillus endoglucanases.

Amino Acid Sequence↗
PubMed2004

Bacillus vietnamensis sp. nov., a moderately halotolerant, aerobic, endospore-forming bacterium isolated from Vietnamese fish sauce.

Five strains of Gram-positive, endospore-forming, moderately halotolerant bacteria were studied taxonomically. Four were isolated from Vietnamese fish sauce and one from the Gulf of Mexico. Phylogenetic analysis based on 16S rRNA gene sequences showed that these strains clustered within the radiation of the genus Bacillus but separately from recognized Bacillus species. DNA G+C composition of the isolates ranged from 43 to 44 mol%. Strains 15-1(T) and NRRL B-14850 showed high levels of DNA-DNA relatedness (82-100 %) to each other and to the other strains isolated here; they displayed low levels of DNA-DNA relatedness (<29 %) to the type strains of selected recognized Bacillus species. They grew in 15 % NaCl and optimally in 1 % NaCl, which is characteristic of moderately halotolerant bacteria. The isolates grew at pH 6.5 to 10.0 but not at pH 6.0. Their cell walls contained meso-diaminopimelic acid. The major isoprenoid quinone was MK-7 and the principal cellular fatty acids were anteiso-C(15 : 0), iso-C(15 : 0) and anteiso-C(17 : 0). Based on these results, the strains tested were regarded as members of a novel Bacillus species for which the name Bacillus vietnamensis sp. nov. is proposed. The type strain is 15-1(T) (=JCM 11124(T)=NRIC 0531(T)=NRRL 23890(T)).

Animals↗
PubMed2004

Bacillus indicus sp. nov., an arsenic-resistant bacterium isolated from an aquifer in West Bengal, India.

Strain Sd/3T (=MTCC 4374T=DSM 15820T), an arsenic-resistant bacterium, was isolated from a sand sample obtained from an arsenic-contaminated aquifer in Chakdah district in West Bengal, India (23 degrees 3' N 88 degrees 35' E). The bacterium was Gram-positive, rod-shaped, non-motile, endospore-forming and yellowish-orange pigmented. It possessed all the characteristics that conform to the genus Bacillus, such as it had A4beta murein type (L-orn-D-Asp) peptidoglycan variant, MK-7 as the major menaquinone and iso-C15 : 0 and anteiso-C15 : 0 as the major fatty acids. Based on its chemotaxonomic and phylogenetic characteristics, strain Sd/3T was identified as a species of the genus Bacillus. It exhibited maximum similarity (95%) at the 16S rRNA gene level with Bacillus cohnii; however, DNA-DNA similarity with B. cohnii was 60.7%. Strain Sd/3T also exhibited a number of phenotypic differences from B. cohnii (DSM 6307T). These data suggest that Sd/3T represents a novel species of the genus Bacillus. The name Bacillus indicus sp. nov. is proposed.

Anti-Bacterial Agents↗
PubMed2005

Bacillus cibi sp. nov., isolated from jeotgal, a traditional Korean fermented seafood.

A Gram-variable, motile, endospore-forming, halotolerant bacillus, strain JG-30(T), was isolated from the traditional Korean fermented seafood jeotgal, and was subjected to a polyphasic taxonomic study. This organism grew optimally at 37 degrees C and in the presence of 0-1 % (w/v) NaCl. 16S rRNA gene sequence analysis showed that strain JG-30(T) forms a distinct phylogenetic lineage within the evolutionary radiation encompassed by the genus Bacillus. Strain JG-30(T) was characterized chemotaxonomically as having cell-wall peptidoglycan based on meso-diaminopimelic acid, MK-7 as the predominant menaquinone and iso-C(15 : 0) and iso-C(14 : 0) as the major fatty acids. The DNA G+C content was 45 mol%. Strain JG-30(T) exhibited levels of 16S rRNA gene sequence similarity of less than 95.7 % to Bacillus species with validly published names. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain JG-30(T) (=KCTC 3880(T)=DSM 16189(T)) was classified within the genus Bacillus as a novel species, for which the name Bacillus cibi sp. nov. is proposed.

Bacillus↗