Obtaining of intrapopulational dissociants of some bacilli and the use of DIR-PCR for their identification

The paper is the first to suggest methods for rapid obtaining and genotypic identification of phenotypic (colonial-morphological) dissociants of bacterial cultures. For revelation of the potential dissociation ability and obtaining of dissociants, the use of bacterial cyst-like refractile cells (CRC) is recommended. These cells are characterized by enhanced variability; upon their first passage, an abrupt increase in the dissociation index is observed as a result of the emergence of cells that form morphologically different types of colonies. The approaches elaborated were tested with Bacillus cereus, B. subtilis, and B. licheniformis, for which colonial-morphological dissociants of various types were obtained after the first passage of CRC (both of those formed in the developmental cycle of bacteria and of those arising as a result of artificial increase of the concentration of anabiosis autoinducers in the cultivation medium). The genomic distinctions between dissociants of B. cereus and B. subtilis were estimated using polymerase chain reaction with a primer system designed based on the analysis of nucleotide sequences of complete prokaryotic genomes available in the GenBank database (DIR-PCR). The application of the suggested method allowed distinctions between the genomes of dissociants of Bacillus cereus and B. subtilis to be revealed, which is in agreement with the hypothesis that suggests reversible intragenomic rearrangements to be the basis of bacterial dissociation into subpopulations.     

Cyclic diguanylate regulation of Bacillus cereus group biofilm formation

Biofilm formation can be considered a bacterial virulence mechanism. In a range of Gram‐negatives, increased levels of the second messenger cyclic diguanylate (c‐di‐GMP) promotes biofilm formation and reduces motility. Other bacterial processes known to be regulated by c‐di‐GMP include cell division, differentiation and virulence. Among Gram‐positive bacteria, where the function of c‐di‐GMP signalling is less well characterized, c‐di‐GMP was reported to regulate swarming motility in Bacillus subtilis while having very limited or no effect on biofilm formation. In contrast, we show that in the Bacillus cereus group c‐di‐GMP signalling is linked to biofilm formation, and to several other phenotypes important to the lifestyle of these bacteria. The Bacillus thuringiensis 407 genome encodes eleven predicted proteins containing domains (GGDEF/EAL) related to c‐di‐GMP synthesis or breakdown, ten of which are conserved through the majority of clades of the B. cereus group, including Bacillus anthracis. Several of the genes were shown to affect biofilm formation, motility, enterotoxin synthesis and/or sporulation. Among these, cdgF appeared to encode a master diguanylate cyclase essential for biofilm formation in an oxygenated environment. Only two cdg genes (cdgA, cdgJ) had orthologs in B. subtilis, highlighting differences in c‐di‐GMP signalling between B. subtilis and B. cereus group bacteria.     

Effect of Carbon Source on the Antimicrobial Activity of the Air Flora

Summary In an attempt to screen for air flora producing new potent antimicrobial substances, Bacillus megaterium NB-3, Bacillus cereus NB-4, Bacillus cereus NB-5, Bacillus subtilis NB-6 and Bacillus circulans NB-7, were isolated and were found to be antagonistic to bacteria and/or fungi. Production of antimicrobial substances by the bacterial strains was greatly influenced by variation of carbon sources. Glycerol strongly enhanced the antimicrobial activity of strains NB-3 and NB-6, whereas glucose increased the antimicrobial activity of strains NB-4 and NB-5. The maximum antibiotic yield of NB-7 was achieved with fructose as a carbon source. Starch (Bacillus megaterium NB-3), maltose (Bacillus cereus NB-5), glycerol (Bacillus circulans NB-7), arabinose, ribose (Bacillus cereus NB-4) and arabinose, fructose, glucose, ribose and sucrose (Bacillus subtilis NB-6) repressed the production of antimicrobial substances by the respective bacterial strains.     

Induction of natural competence in Bacillus cereus ATCC14579

Natural competence is the ability of certain microbes to take up exogenous DNA from the environment and integrate it in their genome. Competence development has been described for a variety of bacteria, but has so far not been shown to occur in Bacillus cereus. However, orthologues of most proteins involved in natural DNA uptake in Bacillus subtilis could be identified in B. cereus. Here, we report that B. cereus ATCC14579 can become naturally competent. When expressing the B. subtilis ComK protein using an IPTG‐inducible system in B. cereus ATCC14579, cells grown in minimal medium displayed natural competence, as either genomic DNA or plasmid DNA was shown to be taken up by the cells and integrated into the genome or stably maintained respectively. This work proves that a sufficient structural system for DNA uptake exists in B. cereus. Bacillus cereus can be employed as a model system to investigate the mechanism of DNA uptake in related bacteria such as Bacillus anthracis and Bacillus thuringiensis. Moreover, natural competence provides an important tool for biotechnology, as it will allow more efficient transformation of B. cereus and related organisms, e.g. to knockout genes in a high‐throughput way.     

Impact of biocontrol bacteria with Rhizophora mucronata plant parts in suppression of Meloidogyne javanica (treub) chitwood on crop plants

Germination of seeds, shoot length, shoot weight, root length and root weight on okra and mung bean showed promising results when bacterial antagonists viz., Bacillus subtilis, B. thuringiensis, B. cereus and Rhizobium meliloti after multiplication on Rhizophora mucronata plant parts were used at 1% w/w. Complete suppression of root knots was observed on okra and mung bean when R. meliloti and Bacillus subtilis after multiplication on R. mucronata plant parts viz., leaves and stem.     

Complete Sequence of the First Chimera Genome Constructed by Cloning the Whole Genome of Synechocystis Strain PCC6803 into the Bacillus subtilis 168 Genome

Genome synthesis of existing or designed genomes is made feasible by the first successful cloning of a cyanobacterium, Synechocystis PCC6803, in Gram-positive, endospore-forming Bacillus subtilis. Whole-genome sequence analysis of the isolate and parental B. subtilis strains provides clues for identifying single nucleotide polymorphisms (SNPs) in the 2 complete bacterial genomes in one cell.     

Erythrophore cell response to food‐associated pathogenic bacteria: implications for detection

Cell‐based biosensors have been proposed for use as function‐based detectors of toxic agents. We report the use of Betta splendens chromatophore cells, specifically erythrophore cells, for detection of food‐associated pathogenic bacteria. Evaluation of erythrophore cell response, using Bacillus spp., has revealed that this response can distinguish pathogenic Bacillus cereus from a non‐pathogenic B. cereus ΔplcR deletion mutant and a non‐pathogenic Bacillus subtilis. Erythrophore cells were exposed to Salmonella enteritidis, Clostridium perfringens and Clostridium botulinum. Each bacterial pathogen elicited a response from erythrophore cells that was distinguished from the corresponding bacterial growth medium, and this observed response was unique for each bacterial pathogen. These findings suggest that erythrophore cell response has potential for use as a biosensor in the detection and toxicity assessment for food‐associated pathogenic bacteria.     

Optimization of inactivation of endospores of Bacillus cereus by antimicrobial lipopeptides from Bacillus subtilis fmbj strains using a response surface method

Bacillus subtilis fmbj can produce a lipopeptide antimicrobial substance, the main components of which are surfactin and fengycin. In this paper, the sensitivity of Bacillus cereus to antimicrobial lipopeptides from B. subtilis fmbj was observed, and the effect of the microstructure of antimicrobial lipopeptide on spores of B. cereus was investigated. At the same time, the optimization of the inactivation of antimicrobial lipopeptides to spores of B. cereus by a response surface methodology was studied. Results showed that B. cereus had high sensitivity to it, whose minimal inhibitory concentration was 156.25 μg/ml. It could result in the death of spores by destroying the structure of resting spores and sprouting spores, as was observed by transmission electron microscopy. The optimization result indicated that spores of B. cereus could be inactivated by 2 orders of magnitude when the temperature was 29.6°C, the action time was 7.6 h, and the concentration was 3.46 mg·ml⁻¹.     

Distribution of ATP-inhibited Ribonuclease in Bacteria

ATP-inhibited ribonuclease was first found in Bacillus amyloliquefaciens cells and was isolated by the authors previously. The distribution of the ATP-inhibited ribonuclease was surveyed on various bacteria. ATP-inhibited ribonuclease was found in only several species of Bacillus genus, i.e., B. subtilis (i), B. amyloliquefaciens (ii), B. cereus (iii), B. cereus var. mycoides (iv), and B. thiiringiensis (v). ATP-inhibited ribonuclease of Bacillus genus was subdivided into two types. The first type of enzyme found in (i) and (ii) cells had optimum pH at 5.7 and was sensitive to 5 mM EDTA. The second type of enzyme found in (iii), (iv), and (v) cells had optimum pH at 6.8 and was partially inhibited by 5 mM EDTA.     

Photo‐inactivation of Bacillus endospores: inter‐specific variability of inactivation efficiency

The aims of this work were to (a) evaluate the susceptibility of endospores of Bacillus cereus, B. licheniformis, B. sphaericus and B. subtilis to photodynamic inactivation using a tricationic porphyrin as photosensitizer, (b) assess the efficiency of adsorption of the photosensitizer in endospore material as a determinant of the susceptibility of endospores of different Bacillus species to photo‐inactivation, (c) determine the value of B. cereus as a model organism for studies of antimicrobial photodynamic inactivation of bacterial endospores. The results of irradiation experiments with endospores of four species of Bacillus showed that B. cereus was the only species for which efficient endospore photo‐inactivation (> 3 log reduction) could be achieved. Endospores of B. licheniformis, B. sphaericus and B. subtilis were virtually resistant to photo‐inactivation with tricationic porphyrin. The amount of porphyrin bound to endospore material was not significantly different between species, regardless of the presence of an exosporium or exosporium‐like outer layer. The sensitivity of endospores to photodynamic inactivation with a tricationic porphyrin is highly variable among different species of the genus Bacillus. The presence of an exosporium in endospores of B. cereus and B. sphaericus, or an exosporium‐like glycoprotein layer in endospores of B. subtilis, did not affect the amount of bound photosensitizer and did not explain the inter‐species variability in susceptibility to photodynamic inactivation. The results imply that the use of B. cereus as a more amenable surrogate of the exosporium‐producing B. anthracis must be carefully considered when testing new photosensitizers for their antimicrobial photo‐inactivation properties.     

Autoregulation of phenotypic dissiciation in Bacillus licheniformis

Regulation of phenotypic variability of Bacillus licheniformis mediated by autoinducers of anabiosis, d1-factors was investigated. These factors are represented by alkylhydroxybenzenes. Colonial morphological variants of B. licheniformis were obtained and described (of R,S,M-types) in the first passage of both vegetative proliferative and resting cells. Resting cells were of different type, spores and cyst-like refractile cells induced by autoinducers of anabiosis. The possibility to manage the spectrum of dissociants by the mean of autoinducers of anabiosis was demonstrated.     

Characterization of corrosive bacterial consortia isolated from petroleum-product-transporting pipelines

Microbiologically influenced corrosion is a problem commonly encountered in facilities in the oil and gas industries. The present study describes bacterial enumeration and identification in diesel and naphtha pipelines located in the northwest and southwest region in India, using traditional cultivation technique and 16S rDNA gene sequencing. Phylogenetic analysis of 16S rRNA sequences of the isolates was carried out, and the samples obtained from the diesel and naphtha-transporting pipelines showed the occurrence of 11 bacterial species namely Serratia marcescens ACE2, Bacillus subtilis AR12, Bacillus cereus ACE4, Pseudomonas aeruginosa AI1, Klebsiella oxytoca ACP, Pseudomonas stutzeri AP2, Bacillus litoralis AN1, Bacillus sp., Bacillus pumilus AR2, Bacillus carboniphilus AR3, and Bacillus megaterium AR4. Sulfate-reducing bacteria were not detected in samples from both pipelines. The dominant bacterial species identified in the petroleum pipeline samples were B. cereus and S. marcescens in the diesel and naphtha pipelines, respectively. Therefore, several types of bacteria may be involved in biocorrosion arising from natural biofilms that develop in industrial facilities. In addition, localized (pitting) corrosion of the pipeline steel in the presence of the consortia was observed by scanning electron microscopy analysis. The potential role of each species in biofilm formation and steel corrosion is discussed.     

High level expression of a recombinant phospholipase C from <Emphasis Type="Italic">Bacillus cereus</Emphasis> in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g⁻¹ wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg⁻¹ protein.     

Antibacterial activity of stevioside towards food‐borne pathogenic bacteria

This study determines the inhibitory effect of Stevia rebaudiana leaf extracts and its purified bioactive compound ‘stevioside’ against food‐related pathogens. The S. rebaudiana solvent extracts (1000 μg/mL) displayed antibacterial activity to Serratia marcescens, Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, B. subtilis, Alcaligenes denitrificans and Salmonella typhimurium. Of the six solvents, ethanol and acetone extracts displayed the highest zone of inhibition. The bioactive compound from S. rebaudiana was purified by solvent extraction, thin‐layer chromatography followed by structural characterization by spectroscopy evidence. Purified stevioside prevented the growth of tested bacterial species, i.e. B. subtilis, K. pneumoniae and S. typhimurium. Significant zone of inhibition (12 mm) was observed against B. cereus which proposes potential application of stevioside in foods to increase their shelf life.     

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.     

Rapid Characterization of Spores of Bacillus cereus Group Bacteria by Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry

Matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry was used to characterize the spores of 14 microorganisms of the Bacillus cereus group. This group includes the four Bacillus species B. anthracis, B. cereus, B. mycoides, and B. thuringiensis. MALDI mass spectra obtained from whole bacterial spores showed many similarities between the species, except for B. mycoides. At the same time, unique mass spectra could be obtained for the different B. cereus and B. thuringiensis strains, allowing for differentiation at the strain level. To increase the number of detectable biomarkers in the usually peak-poor MALDI spectra of spores, the spores were treated by corona plasma discharge (CPD) or sonicated prior to MALDI analysis. Spectra of sonicated or CPD-treated spores displayed an ensemble of biomarkers common for B. cereus group bacteria. Based on the spectra available, these biomarkers differentiate B. cereus group spores from those of Bacillus subtilis and Bacillus globigii. The effect of growth medium on MALDI spectra of spores was also explored.     

Photoreactivation in the genus Bacillus

Photoreactivation of ultraviolet radiation-induced DNA damage was examined in exponential-phase cells of six mesophilic species of the genus Bacillus. Under the experimental conditions used, it was observed that the laboratory strains B. cereus strain T and B. thuringiensis var. thuringiensis strain NRRL-B4039 exhibited strong photoreactivation (86-fold and 70-fold respectively). Bacillus licheniformis strain ATCC 8480 exhibited moderate (15-fold) photoreactivation. Weak photoreactivation was observed in B. subtilis strain 168 (4-fold) and B. megaterium strain QM B1551 (3.4-fold). Bacillus amyloliquefaciens strain H demonstrated no detectable photoreactivation.     

Effect of glucose on the biosynthesis of the membranes of bacillus

In the presence of glucose in complex media, the following changes in the characters of the membranes of Bacillus subtilis and Bacillus cereus were observed. (1) The activity of succinate dehydrogenase and the amount of cytochromes of the membranes were greatly reduced. (2) The ratio of lipid to protein in the membranes was decreased and a membrane subfraction, which had a density of around 1.2 (B. subtilis) and 1.24 g/cm³ (B. cereus), was newly formed (B. subtilis) or increased (B. cereus). (3) The phospholipid and diglyceride contents in the membranes were reduced.Polyacrylamide gel electrophoresis of proteins of the two types (plus and minus glucose) of the membranes show that the patterns were very different between the two types of membranes, though the lost or newly formed membrane protein components were not observed.The cytochrome content was not increased when the cells were grown in glucose medium supplemented with haemin, therefore, glucose or its metabolite may not be involved in the inhibition of haem biosynthesis.     

Characterization of Bacillus Probiotics Available for Human Use

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.     

Effects of 5-fluoro-2′-deoxyuridine on bacterial growth its use for DNA labelling

Experiments on the action of 5-fluoro-2′-deoxyuridine on growth ofEscherichia coli B, CECT 101;Pseudomonas fluorescens, CECT 318;Pseudomonas savastanoi, CECT 93;Micrococcus luteus, ATCC 4698;Bacillus cereus, CIP 52.58;Bacillus macerans, ClP 52.58 andBacillus subtilis, ATCC 6633, are described. The inhibition of growth is reversed by thymine plus uracil in all cases except inPseudomonas strains in which uracil alone is active, and in which no exogenous thymine is taken up, not even in the presece of 2′-deoxyguanosine. Growth conditions for improved labelling of bacterial DNA are discussed in the light of the results.