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.     

Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579

An electro-transformation procedure was established for Bacillus cereus ATCC 14579. Using early growth-stage culture and high electric field, the ectroporation efficiency was up to 2 x 10(9) cfu microg(-1) ml(-1) with pC194 plasmid DNA. The procedure was tested with three other plasmids, of various sizes, replication mechanisms and selection markers, and the transformation efficiencies ranged between 2 x 10(6) and 1 x 10(8) cfu microg(-1) ml(-)(1). The effects of two wall-weakening agents on electroporation rates were also evaluated. The transformation rate that was reached with our procedure is 10(3) times higher than that previously obtained with members of the Bacillus genus with similar plasmids, and 10(6) times superior than that achieved with available protocols for B. cereus. The proposed method is quick, simple, efficient with small rolling circle plasmids and large theta replicating plasmids with low copy number per cell, and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols.     

Characterization of type II and III restriction-modification systems from Bacillus cereus strains ATCC 10987 and ATCC 14579

The genomes of two Bacillus cereus strains (ATCC 10987 and ATCC 14579) have been sequenced. Here, we report the specificities of type II/III restriction (R) and modification (M) enzymes. Found in the ATCC 10987 strain, BceSI is a restriction endonuclease (REase) with the recognition and cut site CGAAG 24-25/27-28. BceSII is an isoschizomer of AvaII (G/GWCC). BceSIII cleaves at ACGGC 12/14. The BceSIII C terminus resembles the catalytic domains of AlwI, MlyI, and Nt.BstNBI. BceSIV is composed of two subunits and cleaves on both sides of GCWGC. BceSIV activity is strongly stimulated by the addition of cofactor ATP or GTP. The large subunit (R1) of BceSIV contains conserved motifs of NTPases and DNA helicases. The R1 subunit has no endonuclease activity by itself; it strongly stimulates REase activity when in complex with the R2 subunit. BceSIV was demonstrated to hydrolyze GTP and ATP in vitro. BceSIV is similar to CglI (GCSGC), and homologs of R1 are found in 11 sequenced bacterial genomes, where they are paired with specificity subunits. In addition, homologs of the BceSIV R1-R2 fusion are found in many sequenced microbial genomes. An orphan methylase, M.BceSV, was found to modify GCNGC, GGCC, CCGG, GGNNCC, and GCGC sites. A ParB-methylase fusion protein appears to nick DNA nonspecifically. The ATCC 14579 genome encodes an active enzyme Bce14579I (GCWGC). BceSIV and Bce14579I belong to the phospholipase D (PLD) family of endonucleases that are widely distributed among Bacteria and Archaea. A survey of type II and III restriction-modification (R-M) system genes is presented from sequenced B. cereus, Bacillus anthracis, and Bacillus thuringiensis strains.     

Isolation and characterization of flagellar filaments from Bacillus cereus ATCC 14579

Isolated flagellar filaments from the type strain of Bacillus cereus, ATCC 14579, were shown to consist of 34, 32 and 31 kDa proteins in similar proportions as judged by band intensities on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of these three proteins of strain ATCC 14579 were identical with the deduced sequences of three flagellin genes BC1657, BC1658 and BC1659 in the whole genome sequence. Strain ATCC 14579 was classified into serotype T2 by a flagellar serotyping scheme for B. cereus strains that are untypeable into known flagellar serotypes H1 to H23. Flagellar filaments from a reference strain of serotype T2 contained two protein bands at 34 and 32 kDa, but a single protein band at 39 kDa was detected in flagellar filaments of a reference strain of serotype H1. Two murine monoclonal antibodies, 1A5 and 2A5, which recognize both the 34 and 32 kDa flagellins and a single flagellin of 32 kDa, respectively, were specifically reactive with B. cereus strains ATCC 14579 and serotype T2 in whole-cell ELISA and bacterial motility inhibition tests. In immunoelectron microscopy with monoclonal antibodies 1A5 and 2A5, colloidal gold spheres were shown to localize almost evenly over the entire part of flagellar filaments. Since strain ATCC 14579, and presumably strain serotype T2, are unusual among B. cereus strains in possessing multiple genes that encode flagellin subunits, a possible unique mechanism may contribute to assembly of multiple flagellin subunits into the filament over its entire length.     

gerR, a novel ger operon involved in L-alanine- and inosine-initiated germination of Bacillus cereus ATCC 14579

Bacillus cereus endospores germinate in response to particular nutrients. Spores are able to sense these nutrients in the environment by receptors encoded by the gerA family of operons. Analysis of the Bacillus cereus ATCC 14579 genome revealed seven gerA family homologues. Using a transposon Tn917-based insertional mutagenesis approach followed by an enrichment procedure to select for l-alanine-induced germination mutants, we isolated a mutant with a defect in the l-alanine germination pathway. The transposon disrupted the last gene of a tricistronic gerA family operon, designated gerR, with the order gerRA, gerRC, gerRB. A second mutant was created by insertion of pMUTIN4 in gerRC. Both mutants showed the same phenotype for nutrient-induced germination. Spores of the gerR mutant strains were blocked in their l-alanine-initiated germination pathway and showed a delayed inosine-induced germination response. Apparently, germination mediated by l-alanine and inosine cannot be compensated for completely by the other germinant receptors, and this points towards an essential role of the gerR-encoded receptor in the receptor complex. In food products, spores of the mutant strains showed a reduced germination response compared to spores of the parental strain. High-pressure-initiated germination was not affected by the gerR mutations, as experiments with 100 and 550 MPa showed no difference with spores of the parental strain.     

Metabolic capacity of Bacillus cereus strains ATCC 14579 and ATCC 10987 interlinked with comparative genomics

Bacillus cereus is an important food-borne pathogen and spoilage organism. In this study, numerous phenotypes and the genomes of B. cereus strains ATCC 14579 and ATCC 10987 were analysed to compare their metabolic capacity and stress resistance potential. The growth performance of the two strains was assessed for nearly 2000 phenotypes, including use of nutrient sources, performance in acid and basic environments, osmo-tolerance and antibiotic resistance. Several food-relevant phenotypic differences were found between ATCC 14579 and ATCC 10987, such as differences in utilization of carbohydrates, peptides, amino acids and ammonia. Subsequently, the genomes of both strains were analysed with INPARANOID to search for strain-specific open reading frames (ORFs). B. cereus ATCC 14579 and ATCC 10987 were found to harbour 983 and 1360 strain-specific ORFs respectively. The strain-specific phenotypic features were interlinked with corresponding genetic features and for several phenotypic differences a related strain-specific genetic feature could be identified. In conclusion, the combination of phenotypic data with strain-specific genomic differences has led to detailed insight into the performance of the two B. cereus strains, and may supply indicators for the performance of these bacteria in different environments and ecological niches.     

蜡样芽孢杆菌ATCC14579毒力基因plcR缺失株的构建及其一般特性

蜡样芽孢杆菌是土壤中的优势菌,具有作为益生菌的潜在能力,同时它也是条件致病菌,能引起食物中毒等。蜡样芽孢杆菌的多种毒力因子受到多效性调控子(pleiotropic regulator,plcR)的调控,在其条件致病性作用中起着重要作用。真养产碱杆菌JMP34(Alcaligenes eutrophus)质粒上的2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid,2,4-D)单加氧酶(tfdA)基因可以降解2,4-D。本研究利用同源重组技术使tfdA基因置换掉蜡样芽孢杆菌的plcR基因,构建了蜡样芽孢杆菌ATCC14579突变株B.cereus△plcRΩtf-dA,并对其毒性、一般生理生化特性进行分析。研究结果表明,突变株B.cereus△plcRΩtfdA的毒性显著减弱;生理生化实验结果显示突变株与野生株并没有明显区别,且突变株并没有表现出tfdA酶活性。所有的结果表明plcR控制着蜡样芽孢杆菌ATCC14579的致病性,同时剔除plcR并不破坏其酶系统。本研究为今后构建蜡样芽孢杆菌工程菌提供了新的思路和依据。     

Identification of proteins involved in the heat stress response of Bacillus cereus ATCC 14579

To monitor the ability of the food-borne opportunistic pathogen Bacillus cereus to survive during minimal processing of food products, we determined its heat-adaptive response. During pre-exposure to 42 degrees C, B. cereus ATCC 14579 adapts to heat exposure at the lethal temperature of 50 degrees C (maximum protection occurs after 15 min to 1 h of pre-exposure to 42 degrees C). For this heat-adaptive response, de novo protein synthesis is required. By using two-dimensional gel electrophoresis, we observed 31 heat-induced proteins, and we determined the N-terminal sequences of a subset of these proteins. This revealed induction of stress proteins (CspB, CspE, and SodA), proteins involved in sporulation (SpoVG and AldA), metabolic enzymes (FolD and Dra), identified heat-induced proteins in related organisms (DnaK, GroEL, ClpP, RsbV, HSP16.4, YflT, PpiB, and TrxA), and other proteins (MreB, YloH, and YbbT). The upregulation of several stress proteins was confirmed by using antibodies specific for well-characterized heat shock proteins (HSPs) of B. subtilis. These observations indicate that heat adaptation of B. cereus involves proteins that function in a variety of cellular processes. Notably, a 30-min pre-exposure to 4% ethanol, pH 5, or 2.5% NaCl also results in increased thermotolerance. Also, for these adaptation processes, protein synthesis is required, and indeed, some HSPs are induced under these conditions. Collectively, these data show that during mild processing, cross-protection from heating occurs in pathogenic B. cereus, which may result in increased survival in foods.     

Identification of Proteins Involved in the Heat Stress Response of Bacillus cereus ATCC 14579

To monitor the ability of the food-borne opportunistic pathogen Bacillus cereus to survive during minimal processing of food products, we determined its heat-adaptive response. During pre-exposure to 42°C, B. cereus ATCC 14579 adapts to heat exposure at the lethal temperature of 50°C (maximum protection occurs after 15 min to 1 h of pre-exposure to 42°C). For this heat-adaptive response, de novo protein synthesis is required. By using two-dimensional gel electrophoresis, we observed 31 heat-induced proteins, and we determined the N-terminal sequences of a subset of these proteins. This revealed induction of stress proteins (CspB, CspE, and SodA), proteins involved in sporulation (SpoVG and AldA), metabolic enzymes (FolD and Dra), identified heat-induced proteins in related organisms (DnaK, GroEL, ClpP, RsbV, HSP16.4, YflT, PpiB, and TrxA), and other proteins (MreB, YloH, and YbbT). The upregulation of several stress proteins was confirmed by using antibodies specific for well-characterized heat shock proteins (HSPs) of B. subtilis. These observations indicate that heat adaptation of B. cereus involves proteins that function in a variety of cellular processes. Notably, a 30-min pre-exposure to 4% ethanol, pH 5, or 2.5% NaCl also results in increased thermotolerance. Also, for these adaptation processes, protein synthesis is required, and indeed, some HSPs are induced under these conditions. Collectively, these data show that during mild processing, cross-protection from heating occurs in pathogenic B. cereus, which may result in increased survival in foods.     

Exploring the Biosynthesis of Unsaturated Fatty Acids in Bacillus cereus ATCC 14579 and Functional Characterization of Novel Acyl-Lipid Desaturases

At low temperatures, Bacillus cereus synthesizes large amounts of unsaturated fatty acids (UFAs) with double bonds in positions Δ5 and Δ10, as well as Δ5,10 diunsaturated fatty acids. Through sequence homology searches, we identified two open reading frames (ORFs) encoding a putative Δ5 desaturase and a fatty acid acyl-lipid desaturase in the B. cereus ATCC 14579 genome, and these were named BC2983 and BC0400, respectively. Functional characterization of ORFs BC2983 and BC0400 by means of heterologous expression in Bacillus subtilis confirmed that they both encode acyl-lipid desaturases that use phospholipids as the substrates and have Δ5 and Δ10 desaturase activities. Thus, these ORFs were correspondingly named desA (Δ5 desaturase) and desB (Δ10 desaturase). We established that DesA utilizes ferredoxin and flavodoxins (Flds) as electron donors for the desaturation reaction, while DesB preferably employs Flds. In addition, increased amounts of UFAs were found when B. subtilis expressing B. cereus desaturases was subjected to a cold shock treatment, indicating that the activity or the expression of these enzymes is upregulated in response to a decrease in growth temperature. This represents the first work reporting the functional characterization of fatty acid desaturases from B. cereus.     

Direct-Imaging-Based Quantification of Bacillus cereus ATCC 14579 Population Heterogeneity at a Low Incubation Temperature

Bacillus cereus ATCC 14579 was cultured in microcolonies on Anopore strips near its minimum growth temperature to directly image and quantify its population heterogeneity at an abusive refrigeration temperature. Eleven percent of the microcolonies failed to grow during low-temperature incubation, and this cold-induced population heterogeneity could be partly attributed to the loss of membrane integrity of individual cells.Bacillus cereus is a food poisoning- and food spoilage-causing organism that can be found in a large variety of foods (4, 23). There are two illnesses associated with B. cereus, namely, emetic and diarrheal intoxication (17, 24). Most of the strains related to cases or outbreaks of B. cereus food-borne poisoning were shown to be unable to grow at 7°C (1, 12). The average temperatures of domestic refrigerators have been investigated in various surveys around the world and often ranged from 5°C to 7°C, but extreme values exceeded 10°C to 12°C (5, 16). Inadequate chilling was indeed reported in various incidents of B. cereus food-borne illness (7, 8, 18, 19), pointing to the importance of appropriate refrigeration of foods contaminated with B. cereus to control its growth and toxin production in foods (9).Several studies have demonstrated that microorganisms can show diversity in their population stress response, even in an apparently homogeneous stress environment (6, 11, 21, 22). However, only very limited data describing the heterogeneity in growth performance of individual cells from food-borne pathogens cultured at low temperatures are available (10). Because inadequate chilling of food is one of the factors that contribute to the number of incidents of B. cereus food-borne illness, there is a need for better understanding of its growth performance at lowered incubation temperatures. In this study, we used the direct-imaging-based Anopore technology (6, 13-15) to quantitatively describe the population heterogeneity of B. cereus ATCC 14579 cells at 12°C. The minimum temperature for the growth of B. cereus ATCC 14579 in brain heart infusion (BHI) broth is 7.5°C (personal communication from F. Carlin), but various food-borne-associated B. cereus isolates were shown to be unable to grow at 10°C (1). Therefore, in this study, a culturing temperature of 12°C was chosen, to mimic temperature abuse of refrigerated foods. In addition, the membrane integrity of individual cells was assessed using both membrane permeant and impermeant nucleic acid dyes in order to get more insight into cellular characteristics that may contribute to heterogeneity in growth response.     

Deletion of the sigB Gene in Bacillus cereus ATCC 14579 Leads to Hydrogen Peroxide Hyperresistance

The sigB gene of Bacillus cereus ATCC 14579 encodes the alternative sigma factor σ^B. Deletion of sigB in B. cereus leads to hyperresistance to hydrogen peroxide. The expression of katA, which encodes one of the catalases of B. cereus, is upregulated in the sigB deletion mutant, and this may contribute to the hydrogen peroxide-resistant phenotype.     

Deletion of the sigB gene in Bacillus cereus ATCC 14579 leads to hydrogen peroxide hyperresistance

The sigB gene of Bacillus cereus ATCC 14579 encodes the alternative sigma factor sigma(B). Deletion of sigB in B. cereus leads to hyperresistance to hydrogen peroxide. The expression of katA, which encodes one of the catalases of B. cereus, is upregulated in the sigB deletion mutant, and this may contribute to the hydrogen peroxide-resistant phenotype.     

Impact of Sorbic Acid on Germination and Outgrowth Heterogeneity of Bacillus cereus ATCC 14579 Spores

Population heterogeneity complicates the predictability of the outgrowth kinetics of individual spores. Flow cytometry sorting and monitoring of the germination and outgrowth of single dormant spores allowed the quantification of acid-induced spore population heterogeneity at pH 5.5 and in the presence of sorbic acid. This showed that germination efficiency was not a good predictor for heterogeneity in final outgrowth.     

Role of Germinant Receptors in Caco-2 Cell-Initiated Germination of Bacillus cereus ATCC 14579 Endospores

Spores obtained from Bacillus cereus ATCC 14579 and mutant strains lacking each of seven germinant receptor operons were exposed to differentiated Caco-2 cells and monitored for germination. Spores of the gerI and gerL mutants showed a reduced germination response, pointing to a role for these receptors in Caco-2-induced germination.