Low molecular weight chitosans—Preparation with the aid of pronase,characterization and their bactericidal activity towards Bacillus cereus and Escherichia coli

The homogeneous low molecular weight chitosans (LMWC) of molecular weight 9.5–8.5 kDa, obtained by pronase catalyzed non-specific depolymerization (at pH 3.5, 37 °C) of chitosan showed lyses of Bacillus cereus and Escherichia coli more efficiently (100%) than native chitosan (< 50%). IR and ¹H-NMR data showed decrease in the degree of acetylation (14–19%) in LMWC compared to native chitosan (∼ 26%). Minimum inhibitory concentration of LMWC towards 10⁶ CFU ml^− 1 of B. cereus was 0.01% (w/v) compared to 0.03% for 10⁴ CFU ml^− 1 of E. coli. SEM revealed pore formation as well as permeabilization of the bacterial cells, as also evidenced by increased carbohydrate and protein contents as well as the cytoplasmic enzymes in the cell-free supernatants. N-terminal sequence analyses of the released proteins revealed them to be cytoplasmic/membrane proteins. Upon GLC, the supernatant showed characteristic fatty acid profiles in E. coli, thus subscribing to detachment of lipopolysaccharides into the medium, whereas that of B. cereus indicated release of surface lipids. The mechanism for the observed bactericidal activity of LMWC towards both Gram-positive and Gram-negative bacteria has been discussed.     

Conjugal transfer between Bacillus thuringiensis and Bacillus cereus strains is not directly correlated with growth of recipient strains

Bacillus thuringiensis and Bacillus cereus belong to the B. cereus species group. The two species share substantial chromosomal similarity and differ mostly in their plasmid content. The phylogenetic relationship between these species remains a matter of debate. There is genetic exchange both within and between these species, and current evidence indicates that insects are a particularly suitable environment for the growth of and genetic exchange between these species. We investigated the conjugation efficiency of B. thuringiensis var. kurstaki KT0 (pHT73-Em^R) as a donor and a B. thuringiensis and several B. cereus strains as recipients; we used one-recipient and two-recipient conjugal transfer systems in vitro (broth and filter) and in Bombyx mori larvae, and assessed multiplication following conjugation between Bacillus strains. The B. thuringiensis KT0 strain did not show preference for genetic exchange with the B. thuringiensis recipient strain over that with the B. cereus recipient strains. However, B. thuringiensis strains germinated and multiplied more efficiently than B. cereus strains in insect larvae and only B. thuringiensis maintained complete spore germination for at least 24 h in B. mori larvae. These findings show that there is no positive association between bacterial multiplication efficiency and conjugation ability in infected insects for the used strains.     

Optimization on Antimicrobial Effects of Natural Compound Preservative Against B. cereus and E. coli by RSM

In this paper, the sensitivity of food spoilage organisms (Bacillus cereus; Escherichia coli) to natural antimicrobial peptides (surfactin; polylysine; nisin) from microorganism was observed, and the optimization of antimicrobial effect in meat evaluated by a RSM was studied. Results showed that these strains were sensitive to them. MICs of surfactin and polylysine and nisin were 31.25 and 312.5 and 312.5???g/mL respectively against B. cereus, and MIC were 15.625 and 156.25 and 2,500???g/mL respectively against E. coli. The optimization result indicated that B. cereus and E. coli could be sterilized by six log cycles when the temperature was 14.05?°C, the action time was 10.95?h, and the concentration (surfactin/polylysine/nisin weight ratio 0.1:1:2) was 379.53???g/mL.     

Insulin action on Escherichia coli Regulation of the adenylate cyclase and phosphotransferase enzymes

Insulin action on Escherichia coli was studied using wild type E. coli B/r and K12 strains and a number of phosphoenolpyruvate phosphotranferase mutants. In vivo, the effects of insulin on the differential rate of tryptophanase synthesis, The rate of α-methyglucoside uptake and the rate of growth on glucose were determined in E. coli B/r. in vitro, the effect of insulin on the adenylate cyclase and the phosphotransferase activities was determined using toluenized cell preparations of E. coli B/r, E. coli K12 and phosphotransferase mutant strains. The specificity of insulin action on E. coli was determined using glucagon, vasopressin and somatropin as well as insulin antisera. Results show the specific action of insulin n E. coli, inhibiting tryptophanase induction and adenylate cyclase activity, while stimulating growth on glucose and uptake and phosphorylation of α-methylglucosode     

Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group,and pairwise genome comparisons of the species of the group by means of ANI calculations

Strain BCT-7112^T was isolated in 1966 in Japan from a survey designed to obtain naturally occurring microorganisms as pure cultures in the laboratory for use as probiotics in animal nutrition. This strain, which was primarily identified as Bacillus cereus var toyoi, has been in use for more than 30 years as the active ingredient of the preparation TOYOCERIN^®, an additive for use in animal nutrition (e.g. swine, poultry, cattle, rabbits and aquaculture). Despite the fact that the strain was initially classified as B. cereus, it showed significant genomic differences from the type strains of the B. cereus group that were large enough (ANI values below 92%) to allow it to be considered as a different species within the group. The polyphasic taxonomic study presented here provides sufficient discriminative parameters to classify BCT-7112^T as a new species for which the name Bacillus toyonensis sp. nov. is proposed, with BCT-7112^T (=CECT 876^T; =NCIMB 14858^T) being designated as the type strain. In addition, a pairwise comparison between the available genomes of the whole B. cereus group by means of average nucleotide identity (ANI) calculations indicated that besides the eight classified species (including B. toyonensis), additional genomospecies could be detected, and most of them also had ANI values below 94%. ANI values were on the borderline of a species definition only in the cases of representatives of B. cereus versus B. thuringiensis, and B. mycoides and B. weihenstephanensis.     

Evolution of pathogenicity in the Bacillus cereus group

The Bacillus cereus group of bacteria comprises soil-dwelling saprophytes but on occasion these bacteria can cause a wide range of diseases in humans, including food poisoning, systemic infections and highly lethal forms of anthrax. While anthrax is almost invariably caused by strains from a single evolutionary lineage, Bacillus anthracis, variation in the virulence properties of strains from other lineages has not been fully addressed. Using multi-locus sequence data from 667 strains, we reconstructed the evolutionary history of the B. cereus group in terms of both clonal inheritance and recombination. The strains included 155 clinical isolates representing B. anthracis, and isolates from emetic and diarrhoeal food poisoning, septicaemia and related infections, wound, and lung infections. We confirmed the existence of three major clades and found that clinical isolates of B. cereus (with the exception of emetic toxin-producing strains) are evenly distributed between and within clades 1 and 2. B. anthracis in particular and emetic toxin-producing B. cereus show more clonal structure and are restricted to clade 1. Our characterization of the patterns of genetic exchange showed that there exist partial barriers to gene flow between the three clades. The pathogenic strains do not exhibit atypically high or low rates of recombination, consistent with the opportunistic nature of most pathogenic infections. However, there have been a large number of recent imports in clade 1 of strains from external origins, which is indicative of an on-going shift in gene-flow boundaries for this clade.     

Genome Sequences of Escherichia coli B strains REL606 and BL21(DE3)

Escherichia coli K-12 and B have been the subjects of classical experiments from which much of our understanding of molecular genetics has emerged. We present here complete genome sequences of two E. coli B strains, REL606, used in a long-term evolution experiment, and BL21(DE3), widely used to express recombinant proteins. The two genomes differ in length by 72,304 bp and have 426 single base pair differences, a seemingly large difference for laboratory strains having a common ancestor within the last 67 years. Transpositions by IS1 and IS150 have occurred in both lineages. Integration of the DE3 prophage in BL21(DE3) apparently displaced a defective prophage in the λ attachment site of B. As might have been anticipated from the many genetic and biochemical experiments comparing B and K-12 over the years, the B genomes are similar in size and organization to the genome of E. coli K-12 MG1655 and have > 99% sequence identity over ∼ 92% of their genomes. E. coli B and K-12 differ considerably in distribution of IS elements and in location and composition of larger mobile elements. An unexpected difference is the absence of a large cluster of flagella genes in B, due to a 41 kbp IS1-mediated deletion. Gene clusters that specify the LPS core, O antigen, and restriction enzymes differ substantially, presumably because of horizontal transfer. Comparative analysis of 32 independently isolated E. coli and Shigella genomes, both commensals and pathogenic strains, identifies a minimal set of genes in common plus many strain-specific genes that constitute a large E. coli pan-genome.     

Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella

Infection of Galleria mellonella by feeding a mixture of Bacillus thuringiensis spores or vegetative bacteria in association with the toxin Cry1C results in high levels of larval mortality. Under these conditions the toxin or bacteria have minimal effects on the larva when inoculated separately. In order to evaluate whether G. mellonella can function as an oral infection model for human and entomo-bacterial pathogens, we tested strains of Bacillus cereus, Bacillus anthracis, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and a Drosophila targeting Pseudomonas entomophila strain. Six B. cereus strains (5 diarrheal, 1 environmental isolate) were first screened in 2nd instar G. mellonella larvae by free ingestion and four of them were analyzed by force-feeding 5th instar larvae. The virulence of these B. cereus strains did not differ from the B. thuringiensis virulent reference strain 407Cry⁻ with the exception of strain D19 (NVH391/98) that showed a lower virulence. Following force-feeding, 5th instar G. mellonella larvae survived infection with B. anthracis, L. monocytogenes, E. faecalis and P. aeruginosa strains in contrast to the P. entomophila strain which led to high mortality even without Cry1C toxin co-ingestion. Thus, specific virulence factors adapted to the insect intestine might exist in B. thuringiensis/B. cereus and P. entomophila. This suggests a co-evolution between host and pathogens and supports the close links between B. thuringiensis and B. cereus and more distant links to their relative B. anthracis.     

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.     

Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli

The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity.     

Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are closely related gram-positive, spore-forming bacteria of the B. cereus sensu lato group. While independently derived strains of B. anthracis reveal conspicuous sequence homogeneity, environmental isolates of B. cereus and B. thuringiensis exhibit extensive genetic diversity. Here we report the sequencing and comparative analysis of the genomes of two members of the B. cereus group, B. thuringiensis 97-27 subsp. konkukian serotype H34, isolated from a necrotic human wound, and B. cereus E33L, which was isolated from a swab of a zebra carcass in Namibia. These two strains, when analyzed by amplified fragment length polymorphism within a collection of over 300 of B. cereus, B. thuringiensis, and B. anthracis isolates, appear closely related to B. anthracis. The B. cereus E33L isolate appears to be the nearest relative to B. anthracis identified thus far. Whole-genome sequencing of B. thuringiensis 97-27and B. cereus E33L was undertaken to identify shared and unique genes among these isolates in comparison to the genomes of pathogenic strains B. anthracis Ames and B. cereus G9241 and nonpathogenic strains B. cereus ATCC 10987 and B. cereus ATCC 14579. Comparison of these genomes revealed differences in terms of virulence, metabolic competence, structural components, and regulatory mechanisms.     

Quantification methods for Bacillus cereus vegetative cells and spores in the gastrointestinal environment

There is an interest to understand the fate and behaviour of the food-borne pathogen Bacillus cereus in the gut, a challenging environment with a high bacterial background. We evaluated the current detection methods to select an appropriate strategy for B. cereus monitoring during gastrointestinal experiments. Application of quantitative real-time PCR (qPCR) in a gastrointestinal matrix required careful selection of the qPCR reaction and elaborate optimization of the DNA extraction protocol. Primer competition and depletion problems associated with qPCR reactions targeting general 16S rRNA gene can be avoided by the selection of a target sequence that is unique for and widespread among the target bacteria, such as the toxin gene nheB in the case of pathogenic B. cereus. Enumeration of B. cereus during the ileum phase was impossible by plating due to overgrowth by intestinal bacteria, while a carefully optimized qPCR enabled specific detection and quantification of B. cereus. On the other hand, plating allowed the distinction of viable, injured and dead bacteria and the germination of spores, which was not possible with qPCR. In conclusion, both plating and qPCR were necessary to yield the maximal information regarding the viability and physiology of the B. cereus population in various gastrointestinal compartments.     

Cloning and Expression of an Insecticidal k-73 Type Crystal Protein Gene from Bacillus thuringiensis var. kurstaki into Escherichia coli

A 75-kilobase plasmid from Bacillus thuringiensis var. kurstaki (HD-244) was associated with the k-73 type insecticidal crystal protein production by mating into B. cereus and subsequent curing of excess plasmids. This plasmid was partially digested with endonuclease R · Sau3A and the fragments were cloned into Escherichia coli (HB101) on vector pBR322. Candidate clones were screened for plasmid vectors which contained the expected insert size (at least 3 kilobases) and then with an enzyme-linked immunosorbent assay, using antisera prepared against electrophoretically purified, solubilized insecticidal crystal protein of 130,000 daltons. Several positive clones were isolated and were analyzed for expression, toxicity, and genetic content by restriction enzyme analysis. Electrophoretic transfer blots of proteins from a candidate E. coli clone, analyzed by enzyme-linked immunosorbent assay, demonstrated a predominant cross-reacting protein of about 140,000 daltons. Ouchterlony analysis also showed a single precipitin band. Extensive bioassays with Manduca sexta larvae revealed that the E. coli clones make toxin with a specific activity (50% lethal dose per microgram of cross-reacting protein) equivalent to that of the parental B. thuringiensis strain or a B. cereus trancipient carrying the toxin-encoding, 75-kilobase plasmid.     

Peptides panned from a phage-displayed random peptide library are useful for the detection of Bacillus anthracis surrogates B. cereus 4342 and B. anthracis Sterne

Recent use of Bacillus anthracis as a bioweapon has highlighted the need for a sensitive monitoring system. Current bacterial detection tests use antibodies as bio-molecular recognition elements which have limitations with regard to time, specificity and sensitivity, creating the need for new and improved cost-effective high-affinity detection probes. In this study, we screened a commercially available bacteriophage-displayed random peptide library using Bacillus cereus 4342 cells as bait to identify peptides that could be used for detection of Bacillus. The method enabled us to identify two 12-amino acid consensus peptide sequences that specifically bind to B. cereus 4342 and B. anthracis Sterne, the nonpathogenic surrogates of B. anthracis strain. The two Bacillus-binding peptides (named BBP-1 and BBP-2) were synthesized with biotin tag to confirm their binding by four independent detection assays. Dot-blot analysis revealed that the peptides bind specifically to B. cereus 4342 and B. anthracis Sterne. Quantitative analysis of this interaction by ELISA and fluorometry demonstrated a detection sensitivity of 10² colony forming U/ml (CFU/ml) by both assays. When the peptides were used in combination with Qdots, the sensitivity was enhanced further by enabling detection of even a single bacterium by fluorescence microscopy. Immunoblot analysis and protein sequencing showed that BBP-1 and BBP-2 bound to the S-layer protein of B. anthracis Sterne. Overall, our findings validate the usefulness of synthetic versions of phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity bioprobes for various microbial detection platforms.     

Cellular defense reactions of insect hemocytes in vivo: Nodule formation and development in Galleria mellonella and Pieris brassicae larvae

Galleria mellonella and Pieris brassicae larvae were injected with a standardized dose of killed Bacillus cereus and other bacteria and the reactions of hemocytes followed in the first 24 hr by dissection and histology. Nodules formed in all insects injected with nonpathogens, but a pathogen, Staphylococcus aureus, failed to provoke this reaction. Within 5 min, clumps consisting of granular hemocytes, plasmatocytes, and bacteria were found attached to the internal surfaces of the insects. In the following hours, the cells comprising the clumps broke down and merged with a melanizing acellular substance, and the necrosing masses became encapsulated by plasmatocytes to form mature nodules. The role of granular hemocytes in the formation of the initial cell/bacteria aggregates is discussed along with the possible importance of nodules to the cellular defense reactions of insects.     

DNA sequence of the araBAD-araC controlling region in Salmonella typhimurium LT2

The araB and araC genes of Salmonella typhimurium have been cloned onto the plasmid pBR322. Restriction analysis and subcloning of restriction fragments localized these genes to a 4.4 kb DNA fragment. Complementation analysis revealed that the cloned araB and araC genes from S. typhimurium complemented araB and araC mutant strains of Escherichia coli. Conversely, cloned araB and araC genes from E. coli complemented araB and araC mutant strains of S. typhimurium. The DNA sequences was determined for the S. typhimurium araB and araC controlling region and for the initially translated portions of these genes. The nucleotide sequence of the araB promoter was 87% homologous with the same region in E. coli and contained no deletions or insertions relative to the E. coli sequence. The presumed AUG codon corresponding to the amino terminus of the S. typhimurium araC protein was in the same location as in E. coli. There was, however, considerable divergence from the E. coli sequence preceding the translation start site. The nucleotide sequence of the initial 237 bp in the open reading frame of the S. typhimurium araC gene was 78% homologous with the same sequence in E. coli. By comparison, the amino acid sequence for this region was 91% conserved.     

The resistances of Micrococcus radiodurans to killing and mutation by agents which damage DNA

The resistance of Micrococcus radiodurans to the lethal and mutagenic action 3f ultraviolet (UV) light, ionising (γ) radiation, mitomycin C (MTC), nitrous acid (NA), hydroxylamine (HA), N-methyl-N′-nitro-N-nitrosoguanidine (NG), ethylmethanesulphonate (EMS) and β-propiolactone (βPL) has been compared with that of Escherichia coli B/r.M. radiodurans was much more resistant than E. coli B/r to the lethal effects of UV light (by a factor of 33), γ-radiation (55), NG (15) and NA (62), showed intermediate resistance to MTC (4) and HA(7), but was sensitive to EMS (1) and βPL (2). M. radiodurans was very resistant to mutagens producing damage which can be repaired by a recombination system, indicating that it possesses an extremely efficient recombination repair mechanism.Both species were equally sensitive to mutation to trimethoprim resistance by NG, but M. radiodurans was more resistant the E. coli B/r to the other multagens tested, being non-mutable by UV light, γ-radiation, MTC and HA, and only slightly sensitive to mutation by NA, EMS, and βPL. The resistance of M. radiodurans to mutation by UV-light, γ-radiation and MTC is consistent with an hypothesis that recombination repair in M. radiodurans is accurate since these mutagens may depend on an “error-prone” recombination system for their mutagenic effect in E. coli B/r. However, because M. radiodurans is also resistant to mutagens such as HA and EMS, which are mutagenic in E. coli in the absence of an “error-prone” system, we propose that all the mutagens tested may have a common mode of action in E. coli B/r, but that this mutagenic pathway is missing in M. radiodurans.     

Computational analysis of phenotypic space in heterologous polyketide biosynthesis—Applications to Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae

Polyketides represent a class of natural product small molecules with an impressive range of medicinal activities. In order to improve access to therapeutic polyketide compounds, heterologous metabolic engineering has been applied to transfer polyketide genetic pathways from often fastidious native hosts to more industrially-amenable heterologous hosts such as Escherichia coli, Saccharomyces cerevisiae, or Streptomyces coelicolor. Efforts thus far have resulted in titers either inferior to the native host and significantly below the theoretical yield, emphasizing the need to computationally investigate and engineer the interaction between native and heterologous metabolism for the improved production of heterologous polyketide compounds. In this work, we applied flux balance analysis on genome-scale models to simulate cellular metabolism and 6-deoxyerythronolide B (the cyclized polyketide precursor to erythromycin) production in three common heterologous hosts (E. coli, Bacillus subtilis, and S. cerevisiae) under a variety of carbon-source and medium compositions. We then undertook minimization of metabolic adjustment optimization to identify single and double gene-knockouts that resulted in increased polyketide production while maintaining cellular growth. For the production of 6-deoxyerythronolide B, the results suggest B. subtilis and E. coli are better heterologous hosts when compared to S. cerevisiae and that several single and multiple gene-knockout mutants are computationally predicted to improve specific production, in some cases, over 25-fold.     

Autoinducer-2 Could Affect Biofilm Formation by Food-Derived Bacillus cereus

Bacillus cereus is a foodborne pathogen and cause a frequent problem due to the biofilms forming in equipment of food production plants. Autoinducer-2 (AI-2) involved in interspecies communication, plays a role in the biofilm formation of B. cereus. In this study, biofilm formation by thirty-nine B. cereus strains isolated from foods produced in Korea was determined. To investigate the effect of AI-2 on biofilm formation by B. cereus SBC27, which had the highest biofilm-forming ability, biofilm densities formed after addition of the AI-2 from Staphylococcus aureus and Escherichia coli were analysed. As a result, it was found that the quorum sensing molecule AI-2 could induce biofilm formation by B. cereus within 24 h, but it may also inhibit biofilm formation when more AI-2 is added after 24 h. Thus, these results improve our understanding of biofilm formation by food-derived B. cereus and provide clues that could help to reduce the impact of biofilms, the biggest problem in food processing environments, which has an impact on public health as well as the economy.     

Molecular cloning and expression of a Bacillus subtilis β-glucanase gene in Escherichia coli

A Bacillus subtilis gene coding for an endo-β-1,3-1,4-glucanase has been transferred to Escherichia coli by molecular cloning using bacteriophage λ and plasmid vectors. The gene is contained within a 1.6-kb EcoRI-PvuI DNA fragment and directs the synthesis in E. coli of a β-glucanase which specifically degrades barley glucan and lichenan. A novel dye-staining method has been developed to detect β-glucanase activity in colonies on agar plates.