Purification and properties of rabbit reticulocyte elongation factor 1.

Elongation factor 1 has been purified about 100-fold from the lysate of rabbit reticulocytes. The native enzyme is highly asymmetric ($\text{f}\text{f}{}_0\text{= 1.53}$) and has a molecular weight of 450,000. Polyacrylamide-gel electrophoresis in sodium dodecyl sulfate shows two major bands with molecular weights of about 53,000 and 50,000. Partially purified phospholipase C and AB preparations and elastase cause dissociation of the aggregate form of the enzyme to an active form which has a molecular weight of about 50,000. The effect of these phospholipase preparations is unexplained since rabbit reticulocyte elongation factor 1 contains little or no phospholipid. A protease contamination has been considered but no evidence of protease activity has been detected in the phospholipase preparations. In aminoacyl-tRNA binding, elongation factor 1 appears to show very little, if any, turnover. However, in the presence of elongation factor 2, under conditions where polymerization occurs, elongation factor 1 functions catalytically.     

Proteomic Profiling and Identification of Immunodominant Spore Antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Δ-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.     

A Novel Sensitive Bioassay for Detection of Bacillus cereus Emetic Toxin and Related Depsipeptide Ionophores

Of the toxins produced by Bacillus cereus, the emetic toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known. In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of B. cereus is described. The assay is based on the loss of motility of boar spermatozoa upon 24 h of exposure to extracts of emetic B. cereus strains or contaminated food. The paralyzed spermatozoa exhibited swollen mitochondria, but no depletion of cellular ATP or damage to plasma membrane integrity was observed. Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide. The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of purified toxin ml of extended boar semen⁻¹. This amount corresponds to 10⁴ to 10⁵ CFU of B. cereus cells. No toxicity was detected for 27 other B. cereus strains up to 10⁸ CFU ml⁻¹. The detection limit for food was 3 g of rice containing 10⁶ to 10⁷ CFU of emetic B. cereus per gram. Effects similar to those provoked by emetic B. cereus toxin were also induced in boar spermatozoa by valinomycin and gramicidin at 2 and 3 ng ml of extended boar semen⁻¹, respectively. The symptoms provoked by the toxin in spermatozoa indicated that B. cereus emetic toxin was acting as a membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa.     

Production and Characterization of Antibodies against Each of the Three Subunits of the Bacillus cereus Nonhemolytic Enterotoxin Complex

The nonhemolytic enterotoxin (Nhe) is one of the two three-component enterotoxins which are responsible for diarrheal food poisoning syndrome caused by Bacillus cereus. To facilitate the detection of this toxin, consisting of the subunits NheA, NheB, and NheC, a complete set of high-affinity antibodies against each of the three components was established and characterized. A rabbit antiserum specific for the C-terminal part (15 amino acids) of NheC was produced using a respective synthetic peptide coupled to a protein carrier for immunization. Using purified B. cereus exoprotein preparations as immunogens, one monoclonal antibody against NheA and several antibodies against NheB were obtained. No cross-reactivity with other proteins produced by different strains of B. cereus was observed. Antibodies against the NheB component were able to neutralize the cytotoxic activity (up to 98%) of Nhe. Based on indirect enzyme immunoassays, the antibodies developed in this study were successfully used in the characterization of the enterotoxic activity of several B. cereus strains. For the first time, it could be shown that strains carrying the nhe genes usually express the complete set of the three components, including NheC. However, the amount of toxin produced varies considerably between the different strains.     

Complementary screening, identification and application of a novel class II 5-enopyruvylshikimate-3-phosphate synthase from Bacillus cereus

A novel aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase from Bacillus cereus was identified and overexpressed by genomic library construction and complementary screening. The enzyme was then purified to homogeneity. We also transformed the aroA _{B. cereus} gene into Arabidopsis thaliana by a floral dip method, and demonstrated that transgenic A. thaliana plants exhibited significant glyphosate resistance compared with the wild type. These results strongly suggested that the strategy was highly efficient and advantageous for rapidly cloning aroA genes from microorganisms in natural environments.     

Development of a Fluorogenic Probe-Based PCR Assay for Detection of Bacillus cereus in Nonfat Dry Milk

A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5′-to-3′ exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72 B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereus strains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negative B. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated with B. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.     

Translation Elongation Factor 1A Mutants with Altered Actin Bundling Activity Show Reduced Aminoacyl-tRNA Binding and Alter Initiation via eIF2α Phosphorylation

Apart from its canonical function in translation elongation, eukaryotic translation elongation factor 1A (eEF1A) has been shown to interact with the actin cytoskeleton. Amino acid substitutions in eEF1A that reduce its ability to bind and bundle actin in vitro cause improper actin organization in vivo and reduce total translation. Initial in vivo analysis indicated the reduced translation was through initiation. The mutant strains exhibit increased levels of phosphorylated initiation factor 2α (eIF2α) dependent on the presence of the general control nonderepressible 2 (Gcn2p) protein kinase. Gcn2p causes down-regulation of total protein synthesis at initiation in response to increases in deacylated tRNA levels in the cell. Increased levels of eIF2α phosphorylation are not due to a general reduction in translation elongation as eEF2 and eEF3 mutants do not exhibit this effect. Deletion of GCN2 from the eEF1A actin bundling mutant strains revealed a second defect in translation. The eEF1A actin-bundling proteins exhibit changes in their elongation activity at the level of aminoacyl-tRNA binding in vitro. These findings implicate eEF1A in a feedback mechanism for regulating translation at initiation.     

Purification of a thermostable chitinase from Bacillus cereus by chitin affinity and its application in microbial community changes in soil

A thermostable chitinase was purified by chitin affinity from the culture supernatant of Bacillus cereus TKU028 with shrimp head powder (SHP) as the sole carbon/nitrogen source. TKU028 chitinase was purified using a one-step affinity adsorbent system, and the molecular mass of TKU028 chitinase (approximately 40 kDa) was then determined using SDS-PAGE. The enzyme was stable for 60 min at temperatures below 60 °C and stable over a broad pH range of 4–9 for 60 min. In addition, the temporal changes of a bacterial community in mangrove river sediment of the Tamsui River with added SHP were also analysed by PCR–denaturing gradient gel electrophoresis to investigate the effects of B. cereus TKU028 on the degradation of SHP. The 6-week incubation sample of SHP and B. cereus TKU028-amended mangrove river sediment displayed the highest amount of biomass, reducing sugar and total sugar, and some variance of bacterial community composition existed in the soils.     

Effects of juvenile hormone on polysome integrity

Juvenile hormone inhibits protein and RNA synthesis in cell cultures from Trichoplusia ni and in the testicular germinal cysts of Hyalophora cecropia pupae in vitro. Sucrose gradient analyses revealed that the polysomes of both the T. ni cells and the germinal cysts were disaggregated almost immediately after the addition of juvenile hormone in vitro with a corresponding dose-dependent increase in monosomes. It is suggested that previous reports revealing juvenile hormone inhibition of ecdysone stimulated RNA and protein synthesis may be due to polysome disaggregation. Further studies demonstrated that the effect is not restricted to insect cells and can be elicited by several other lipids devoid of juvenile hormone morphogenetic activity. Experiments with broken cell preparations and isolated polysomes suggest the necessity of cell membrane integrity for the effect on the polysomes. Several probing studies utilizing cycloheximide, ribonuclease, and high K⁺ concentrations were conducted on the means by which juvenile hormone and other lipids may elicit polysome disaggregation.     

Behavior of Pythium torulosum Zoospores During Their Interaction with Tobacco Roots and Bacillus cereus

Bacillus cereus UW85 suppresses seedling damping-off diseases caused by Oomycetes and produces antibiotics that inhibit development of Oomycetes in culture. The goal of this study was to determine how UW85 and its antibiotics affected the behavior of an Oomycete, Pythium torulosum, in its interaction with plant roots. We studied tobacco seedlings inoculated with zoospores of P. torulosum and UW85 culture, culture filtrate, washed cells, antibiotics (zwittermicin A or kanosamine), purified from cultures of UW85, and UW030, a mutant of UW85 that does not suppress disease and does not produce the antibiotics. Microscopic observation revealed that all of the treatments inhibited zoospore activity around roots and encystment on roots. Treatment with UW85 culture, culture filtrate, zwittermicin A, or kanosamine delayed cyst germination and the elongation rate of germ tubes, whereas treatment with UW030 or washed UW85 cells did not. In an in vitro seedling bioassay of disease suppression, the antibiotics, zwittermicin A and kanosamine, suppressed disease singly or together, although UW85 culture suppressed disease more effectively than did the antibiotics. The results show that B. cereus cultures affect zoospore behavior in the presence of roots, and B. cereus-produced antibiotics, zwittermicin A and kanosamine, contribute to disease suppression and inhibition of germ tube elongation in the presence of the plant root. Received: 9 September 1998 / Accepted: 13 October 1998     

A COMPARATIVE ELECTRON MICROSCOPICAL STUDY OF RNA FROM DIFFERENT SOURCES

Electron micrographs of ribosomal RNA from Escherichia coli, microsomal RNA from calf, rat, and chick liver, Bacillus cereus RNA and E. coli soluble RNA are presented. Filaments of about 10 A in diameter could be observed in preparations obtained from aqueous solutions of high molecular weight RNA. When ammonium acetate solutions were used a tendency for coiling and aggregation was observed. E. coli soluble RNA appears as small, sometimes elongated particles the smallest diameter being of about 10 A.     

Heterologous expression of CYP102A5 variant from Bacillus cereus CYPPB-1: Validation of model for predicting drug metabolism of human P450 probe substrates

CYP102A5 variant (ADL27534) from isolated Bacillus cereus CYPPB-1 was heterologously expressed in Escherichia coli Top 10 cells. Comparative sequence analysis of purified CYP102A5 variant with respect to reported CYP102A5 (AAP10153) from Bacillus cereus ATCC 14579 revealed amino acid sequence changes at positions P245S and M318I of heme domain. The binding affinities of 15 selected human P450 probe substrates towards isolated CYP102A5 were analyzed in silico using a homology model together with molecular docking techniques to predict the human drug metabolism. In vitro analysis suggested that the purified CYP102A5 metabolizes typical substrates of human CYP2C9, CYP2D6, CYP2E1, and CYP3A4, such as coumarin, propranolol, aniline, chlorzoxazone, p-nitrophenol, and nifedipine. The calculated K _M values for propranolol, chloroxazone, coumarin, aniline, and 4-nitrophenol were calculated to be 0.962?±?0.041, 1.254?±?0.057, 2.859?±?0.083, 2.732?±?0.106, and 2.528?±?0.11 mM, respectively. Importantly, taking a ChemScore cutoff value of ?31 kJ/mol, substrate binding at active site and in vitro activity as the distinguishing lines between “substrates” and “nonsubstrates” revealed one false-positive and one false-negative results out of the 15 compounds examined. This is the first report on validation of CYP102A family homology model for in silico prediction of human drug metabolism.     

Bacillus cereus NVH 0500/00 Can Adhere to Mucin but Cannot Produce Enterotoxins during Gastrointestinal Simulation

Adhesion to the intestinal epithelium could constitute an essential mechanism of Bacillus cereus pathogenesis. However, the enterocytes are protected by mucus, a secretion composed mainly of mucin glycoproteins. These may serve as nutrients and sites of adhesion for intestinal bacteria. In this study, the food poisoning bacterium B. cereus NVH 0500/00 was exposed in vitro to gastrointestinal hurdles prior to evaluation of its attachment to mucin microcosms and its ability to produce nonhemolytic enterotoxin (Nhe). The persistence of mucin-adherent B. cereus after simulated gut emptying was determined using a mucin adhesion assay. The stability of Nhe toward bile and pancreatin (intestinal components) in the presence of mucin agar was also investigated. B. cereus could grow and simultaneously adhere to mucin during in vitro ileal incubation, despite the adverse effect of prior exposure to a low pH or intestinal components. The final concentration of B. cereus in the simulated lumen at 8 h of incubation was 6.62 ± 0.87 log CFU ml⁻¹. At that point, the percentage of adhesion was approximately 6%. No enterotoxin was detected in the ileum, due to either insufficient bacterial concentrations or Nhe degradation. Nevertheless, mucin appears to retain B. cereus and to supply it to the small intestine after simulated gut emptying. Additionally, mucin may play a role in the protection of enterotoxins from degradation by intestinal components.     

Concerted Action of Sphingomyelinase and Non-Hemolytic Enterotoxin in Pathogenic Bacillus cereus

Bacillus cereus causes food poisoning and serious non-gastrointestinal-tract infections. Non-hemolytic enterotoxin (Nhe), which is present in most B. cereus strains, is considered to be one of the main virulence factors. However, a B. cereus ΔnheBC mutant strain lacking Nhe is still cytotoxic to intestinal epithelial cells. In a screen for additional cytotoxic factors using an in vitro model for polarized colon epithelial cells we identified B. cereus sphingomyelinase (SMase) as a strong inducer of epithelial cell death. Using single and double deletion mutants of sph, the gene encoding for SMase, and nheBC in B. cereus we demonstrated that SMase is an important factor for B. cereus cytotoxicity in vitro and pathogenicity in vivo. SMase substantially complemented Nhe induced cytotoxicity in vitro. In addition, SMase but not Nhe contributed significantly to the mortality rate of larvae in vivo in the insect model Galleria mellonella. Our study suggests that the role of B. cereus SMase as a secreted virulence factor for in vivo pathogenesis has been underestimated and that Nhe and SMase complement each other significantly to cause full B. cereus virulence hence disease formation.     

Inflammation in insects: The release of a plasmatocyte depletion factor following interaction between bacteria and haemocytes

Injection of a suspension of the Gram-positive bacterium Bacillus cereus into the haemocoel of Galleria mellonella larvae caused a rapid decrease in the number of circulating plasmatocytes. A similar phenomenon was observed following the injection of cell-free haemolymph collected from larvae previously challenged with bacteria, indicating that plasmatocyte depletion was mediated by a humoral factor released into the haemolymph. The release of plasmatocyte depletion factor could be effected in vitro by incubating adherent haemocytes with a suspension of B. cereus. The in vitro production of the factor did not require the presence of exogenous humoral components, was dose-dependent, was inhibited at low temperatures, and showed the same specificity with respect to bacterial species as observed in vivo. Activity of the plasmatocyte depletion factor destroyed by a 15-min incubation at 56°C. The release of the factor appears to be an early biochemical signal in the complex cellular response of this insect to bacterial infection.     

动物用芽孢杆菌微生态制剂中蜡样芽孢杆菌的分离及安全性

【背景】芽孢杆菌是仅次于乳酸菌常用于微生态制剂中的菌种,然而部分芽孢杆菌微生态制剂规范不严,应用存在安全隐患。【目的】调查我国在售动物用芽孢杆菌微生态制剂中蜡样芽孢杆菌携带情况,揭示蜡样芽孢杆菌应用的潜在风险。【方法】对微生态制剂预处理,选择性筛选分离蜡样芽孢杆菌,通过全基因组测序测绘细菌毒素基因谱与耐药基因谱,细胞计数试剂盒-8法测定菌株对细胞的毒性,利用微量肉汤稀释法确定菌株耐药值。【结果】从50份微生态制剂产品中筛选分离得到23株蜡样芽孢杆菌群细菌,它们对氨苄西林、林可霉素和泰妙菌素3种抗生素均耐药,主要毒力基因nhe、hbl、cytK、ces的检出率分别为100%、30%、39%和4%,分离株均有溶血性且39%菌株产生热稳定毒素,不同菌株对非洲绿猴肾细胞呈现出不同程度的毒性。【结论】微生态制剂来源的蜡样芽孢杆菌毒性与耐药性严重,携带毒素基因与耐药基因广泛,多株菌株呈高细胞毒性且产生热稳定毒素。芽孢杆菌微生态制剂存在安全性问题,应加强对蜡样芽孢杆菌的质量安全监管力度,规范微生态制剂的市场秩序,杜绝安全隐患。     

Microtubules and tubulin sheet polymers elongate from isolated axonemes in vitro as observed by negative-stain electron microscopy

Microtubules are an important cytoskeletal component involved in cell motility and morphogenesis. They are unique polymers because they are highly dynamic in vivo and in vitro, displaying spontaneous transitions between phases of elongation and rapid shortening. This property has been termed microtubule dynamic instability. Here we describe the application of negative-stain electron microscopy to examine the morphology of microtubules. The purpose was to provide insight into the structural basis of dynamic instability. Highly purified porcine brain tubulin was seeded from isolated axoneme seeds and the morphologies of the tubulin polymers were examined. As previously reported, tubulin polymer sheets in addition to intact MTs were observed during elongation. Cross-sections of identical preparations displayed intact circles (MTs) and c-shaped polymers. The fraction of sheets (28%) observed by negative staining was identical to the fraction of c-shaped polymers in cross-sections. These results suggest that tubulin polymer elongation is not strictly helical due to the lack of helical symmetry of tubulin sheets. Finally, we document the novel effect of glutaraldehyde fixation on MTs. Fixation of MTs in 1% glutaraldehyde for longer than 2 min severely disrupted MT protofilament structure and induced MT curvature at a gross level. Even at 1 min, thin, thread-like structures were observed that were only present in glutaraldehyde-fixed samples. It is therefore an advantage to minimize the extent of glutaraldehyde fixation.     

Bacteriophage PBC1 and Its Endolysin as an Antimicrobial Agent against Bacillus cereus

Bacillus cereus is an opportunistic human pathogen responsible for food poisoning and other, nongastrointestinal infections. Due to the emergence of multidrug-resistant B. cereus strains, the demand for alternative therapeutic options is increasing. To address these problems, we isolated and characterized a Siphoviridae virulent phage, PBC1, and its lytic enzymes. PBC1 showed a very narrow host range, infecting only 1 of 22 B. cereus strains. Phylogenetic analysis based on the major capsid protein revealed that PBC1 is more closely related to the Bacillus clarkii phage BCJA1c and phages of lactic acid bacteria than to the phages infecting B. cereus. Whole-genome comparison showed that the late-gene region, including the terminase gene, structural genes, and holin gene of PBC1, is similar to that from B. cereus temperate phage 250, whereas their endolysins are different. Compared to the extreme host specificity of PBC1, its endolysin, LysPBC1, showed a much broader lytic spectrum, albeit limited to the genus Bacillus. The catalytic domain of LysPBC1 when expressed alone also showed Bacillus-specific lytic activity, which was lower against the B. cereus group but higher against the Bacillus subtilis group than the full-length protein. Taken together, these results suggest that the virulent phage PBC1 is a useful component of a phage cocktail to control B. cereus, even with its exceptionally narrow host range, as it can kill a strain of B. cereus that is not killed by other phages, and that LysPBC1 is an alternative biocontrol agent against B. cereus.