Characterization of emetic Bacillus weihenstephanensis, a new cereulide-producing bacterium

Cereulide production has until now been restricted to the species Bacillus cereus. Here we report on two psychrotolerant Bacillus weihenstephanensis strains, MC67 and MC118, that produce cereulide. The strains are atypical with regard to pheno- and genotypic characteristics normally used for identification of emetic B. cereus strains. MC67 and MC118 produced cereulide at temperatures of as low as 8 degrees C.     

Isolation and Characterization of a New Type of Collagenase Producing Bacterium,Bacillus alvei DC-1

A bacterium capable of using collagen as sole carbon and nitrogen sources was isolated and identified as Bacillus alvei DC-1. The specific activity of the ammonium sulfate fraction (80% saturation) was 3.81 x 10⁷ Mandle units/mg. Also, in the collagen substrate, the optimum pHs were 4.5, 6.0, and 7.0. This strain produced a new type of collagenase, which had optimal activity at an acidic pH.     

Pathogenic potential of fifty Bacillus weihenstephanensis strains

Sequence comparison of pseudomurein endoisopeptidases PeiW encoded by the defective prophage PsiM100 of Methanothermobacter wolfeii, and PeiP encoded by phage PsiM2 of Methanothermobacter marburgensis, revealed that the two enzymes share only limited similarity. Their amino acid sequences comprise an N-terminal domain characterized by the presence of direct repeats and a C-terminal domain with a catalytic triad C-H-D as in thiol proteases and animal transglutaminases. Both PeiW and PeiP catalyze the in vitro lysis of M. marburgensis cells under reducing conditions and exhibit characteristics of metal-activated peptidases. Optimal temperature and pH were determined to be 63 degrees C and 6.4 for His-tagged PeiP and 71 degrees C and 6.4 for His-tagged PeiW, respectively. Database search results suggest that PeiW and PeiP are the first two experimentally identified members of a novel family of proteases in a superfamily of archaeal, bacterial, and eukaryotic protein homologs of animal transglutaminases.     

Identification and Characterization of a Novel Staphylococcal Emetic Toxin

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have superantigenic and emetic activities, which cause toxic shock syndrome and staphylococcal food poisoning, respectively. Our previous study demonstrated that the sequence of SET has a low level of similarity to the sequences of other SEs and exhibits atypical bioactivities. Hence, we further explored whether there is an additional SET-related gene in S. aureus strains. One SET-like gene was found in the genome of S. aureus isolates that originated from a case of food poisoning, a human nasal swab, and a case of bovine mastitis. The deduced amino acid sequence of the SET-like gene showed 32% identity with the amino acid sequence of SET. The SET-like gene product was designated SElY. In the food poisoning and nasal swab isolates, mRNA encoding SElY was highly expressed in the early log phase of cultivation, whereas a high level of expression of this mRNA was found in the bovine mastitis isolate at the early stationary phase. To estimate whether SElY has both superantigenic and emetic activities, recombinant SElY was prepared. Cell proliferation and cytokine production were examined to assess the superantigenic activity of SElY. SElY exhibited superantigenic activity in human peripheral blood mononuclear cells but not in mouse splenocytes. In addition, SElY exhibited emetic activity in house musk shrews after intraperitoneal and oral administration. However, the stability of SElY against heating and pepsin and trypsin digestion was different from that of SET and SEA. From these results, we identified SElY to be a novel staphylococcal emetic toxin.     

Multilocus sequence analysis of Bacillus thuringiensis serovars navarrensis, bolivia and vazensis and Bacillus weihenstephanensis reveals a common phylogeny

The Bacillus cereus group sensu lato includes six closely-related bacterial species: Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides and Bacillus weihenstephanensis. B. thuringiensis is distinguished from the other species mainly by the appearance of an inclusion body upon sporulation. B. weihenstephanensis is distinguished based on its psychrotolerance and the presence of specific signature sequences in the 16S rRNA gene and cspA genes. A total of seven housekeeping genes (glpF, gmK, ilvD, pta, purH, pycA and tpi) from different B. thuringiensis serovars and B. weihenstephanensis strains were amplified and their nucleotide sequences determined. A maximum likelihood phylogenetic tree was inferred from comparisons of the concatenated sequences. B. thuringiensis serovars navarrensis, bolivia and vazensis clustered not with the other B. thuringiensis serovars but rather with the B. weihenstephanensis strains, indicative of a common phylogeny. In addition, specific signature sequences and single nucleotide polymorphisms common to B. thuringiensis serovars navarrensis, bolivia and vazensis and the B. weihenstephanensis strains, and absent in the other B. thuringiensis serovars, were identified.     

Psychrotolerant species from the Bacillus cereus group are not necessarily Bacillus weihenstephanensis

Twenty-six strains of Bacillus cereus from different sources were determined to be either mesophilic or psychrotrophic by growth at 6 and 42 degrees C. The strains were also screened by two polymerase chain reaction (PCR) methods designed to discriminate between mesophilic and psychrotrophic types. Seventeen of the 26 strains were able to grow at 6 degrees C, but only four conformed to the new psychrotolerant species Bacillus weihenstephanensis. Among the 26 strains were two which caused outbreaks of food poisoning in Norway, and three others that were isolated from food suspected of causing illness. The presence of the gene components encoding production of enterotoxins Nhe, Hbl, EntT and a recently described cytotoxin K was determined by PCR. All the strains possessed genes for at least one of these toxins, and 19 of the 26 strains were cytotoxic in a Vero cell assay. We conclude that there are psychrotrophic B. cereus strains which cannot be classified as B. weihenstephanensis, and that intermediate forms between the two species exist. No correlation between cytotoxicity and the growth temperature of the strains was found.     

Genome of Bacillus macauensis ZFHKF-1, a Long-Chain-Forming Bacterium

Here, we report the draft genome sequence of Bacillus macauensis ZFHKF-1, a novel long-chain bacterium previously isolated and identified by us (Zhang T, Fan XJ, Hanada S, Kamagata Y, Fang HHP, J. Syst. Evol. Microbiol. 56:349-353, 2006). The genome provides basic genetic information to understand this particular species and explore the potential mechanism of long-chain formation. The type strain is ZFHKF-1 (= JCM 13285 = DSM 17262).     

Natural isolates of Bacillus thuringiensis display genetic and psychrotrophic properties characteristic of Bacillus weihenstephanensis

Aim:  To determine the potential of Bacillus thuringiensis , known primarily for its entomopathogenicity, to be a psychrotolerant contaminant of stored products.
Methods and Results:  We determined the genetic properties and diversity of cold-adapted isolates of B. thuringiensis based on (i) the presence of cspA , a genetic determinant that confers psychrotolerance in Bacillus weihenstephanensis , (ii) 16S rRNA genes, and (iii) pulse-field gel electrophoretic (PFGE) genome profiles. We assessed the pathogenic potential of these isolates based on whether they harboured various combinations of known toxigenic-associated determinants ( nheA , hblA , cytK ). Of 36 nonclonal B. thuringiensis cultured from soil and milk, 21 harboured cspA , and of these, 16 (76%) were psychrotolerant and possessed genetic signatures typical of psychrotrophic Bacillus species. The majority of psychrotolerant isolates contained various combinations of nheA , hblA , and cytK .
Conclusion:  Our results show that natural isolates of psychrotolerant B. thuringiensis occur in soil and milk, and suggest that psychrotolerance is determined by cspA .
Significance and Impact of the study:  The presence of cspA in combination with nheA , hblA , and cytK could be of concern if commercial products are contaminated with strains that harbour these determinants.     

Bacillus pumilus XJU-13的分离鉴定及其碱性脂肪酶酶学特性分析

目的:对从新疆精河地区一处工业污水中分离得到的一株产碱性脂肪酶细菌进行研究.方法:通过生理生化检测,16SrDNA序列同源性分析和G+Cmol%含量的测定对命名为XJU-13的这株菌进行鉴定.结果:该菌株可在pH 3.0～12.5的广泛酸碱泛围的营养肉汤培养基中生长.最适生长温度为37℃.基于16S rDNA序列同源性构建系统进化树分析表明与Bacillus pumilus clone B257聚在同一亚分枝,序列相似性达100%.数据证明XJU-13属于Bacillus pumilus.由于在氧化酶反应及淀粉水解实验与伯杰氏鉴定手册有差异,具不可比拟的pH耐受性,且脂肪酸含量与参考菌株差异较大,认为这是Bacillus pumilus中的一个新品系.该菌株产生的脂肪酶最适pH为10,最适温度为35℃,且在广泛pH(pH4-10)范围具稳定性.酶活可被Mg2+、K+、Ba2+、Pb+盐强烈抑制,被Ca2+、Cu2+、Al+及Fe2+盐激活.Zn2+对酶活无影响.结论:实验表明,XJU-13应属于B.pumilus.B.pumilus XJu-13中分离到的碱性脂肪酶有很好的特性及潜能,以期为工业应用提供数据.     

Climatic influence on mesophilic Bacillus cereus and psychrotolerant Bacillus weihenstephanensis populations in tropical, temperate and alpine soil

Bacillus weihenstephanensis strains are psychrotolerant and grow from below 7°C to 38°C. Closely related mesophilic Bacillus cereus strains can grow from above 7°C to 46°C. We classified 1060 B. cereus group isolates from different soil samples with respect to their psychrotolerant and mesophilic genotypes by polymerase chain reaction (PCR) targeting of specific 16S rDNA and cold shock protein A gene signatures. In parallel, growth tests at 7°C were carried out to determine the thermal phenotype. The geographic distribution of psychrotolerant and mesophilic isolates was found to depend significantly on the prevalent annual average temperature. In one tropical, one temperate and two alpine habitats, the proportion of psychrotolerant cspA genotypes was found to be 0%, 45% and 86% and 98%, respectively, with the corresponding annual average temperatures being 28°C, 7°C, 4°C and 1°C. In the tropical habitat, only the mesophilic B. cereus was found, characterized by correspondence of thermal genotype and phenotype. In the alpine habitat, almost only the psychrotolerant B. weihenstephanensis was isolated. In the temperate habitat, mesophilic B. cereus and psychrotolerant B. weihenstephanensis as well as 'intermediate thermal types' occurred, the latter having opposite thermal genotypes and phenotypes or opposing sets of thermal DNA signatures, characterized by the coexistence of mesophilic and psychrotolerant 16S rDNA operon copies within a single isolate. Both sugar utilization and DNA fingerprinting patterns revealed a high, probably non-clonal microsite diversity within the population of the temperate habitat. We interpret our observations in terms of a temperature-dependent selection regime, acting on recombining B. cereus / B. weihenstephanensis populations in soil.     

Isolation, Identification, and Characterization of a Feather-Degrading Bacterium

A feather-degrading culture was enriched with isolates from a poultry waste digestor and adapted to grow with feathers as its primary source of carbon, sulfur, and energy. Subsequently, a feather-hydrolytic, endospore-forming, motile, rod-shaped bacterium was isolated from the feather-degrading culture. The organism was Gram stain variable and catalase positive and demonstrated facultative growth at thermophilic temperatures. The optimum rate of growth in nutrient broth occurred at 45 to 50°C and at pH 7.5. Electron microscopy of the isolate showed internal crystals. The microorganism was identified as Bacillus licheniformis PWD-1. Growth on hammer-milled-feather medium of various substrate concentrations was determined by plate colony count. Maximum growth (approximately 10⁹ cells per ml) at 50°C occurred 5 days postinoculation on 1% feather substrate. Feather hydrolysis was evidenced as free amino acids produced in the medium. The most efficient conditions for feather fermentation occurred during the incubation of 1 part feathers to 2 parts B. licheniformis PWD-1 culture (10⁷ cells per ml) for 6 days at 50°C. These data indicate a potential biotechnique for degradation and utilization of feather keratin.     

Production of an Emetic Toxin,Cereulide, Is Associated with a Specific Class of Bacillus cereus

The emetic toxin (cereulide) of Bacillus cereus was quantified in several isolates of B. cereus and in various food sources. When the emetic toxin was produced, vomiting-type food poisoning was observed in humans. We also found that the H-1 serovar phenotype was strongly associated with the production of cereulide and that none of the isolates that hydrolyzed starch or expressed diarrheal enterotoxin activity produced cereulide. Received: 4 December 1995 / Accepted: 30 January 1996     

Melanin-Like Pigment Synthesis by Soil Bacillus weihenstephanensis Isolates from Northeastern Poland

Although melanin is known for protecting living organisms from harmful physical and chemical factors, its synthesis is rarely observed among endospore-forming Bacillus cereus sensu lato. Here, for the first time, we reported that psychrotolerant Bacillus weihenstephanensis from Northeastern Poland can produce melanin-like pigment. We assessed physicochemical properties of the pigment and the mechanism of its synthesis in relation to B. weihenstephanensis genotypic and phenotypic characteristics. Electron paramagnetic resonance (EPR) spectroscopy displayed a stable free radical signal of the pigment from environmental isolates which are consistent with the commercial melanin. Fourier transform infrared spectroscopy (FT-IR) and physicochemical tests indicated the phenolic character of the pigment. Several biochemical tests showed that melanin-like pigment synthesis by B. weihenstephanensis was associated with laccase activity. The presence of the gene encoding laccase was confirmed by the next generation whole genome sequencing of one B. weihenstephanensis strain. Biochemical (API 20E and 50CHB tests) and genetic (Multi-locus Sequence Typing, 16S rRNA sequencing, and Pulsed-Field Gel Electrophoresis) characterization of the isolates revealed their close relation to the psychrotrophic B. weihenstephanensis DSMZ 11821 reference strain. The ability to synthesize melanin-like pigment by soil B. weihenstephanensis isolates and their psychrotrophic character seemed to be a local adaptation to a specific niche. Detailed genetic and biochemical analyses of melanin-positive environmental B. weihenstephanensis strains shed some light on the evolution and ecological adaptation of these bacteria. Moreover, our study raised new biotechnological possibilities for the use of water-soluble melanin-like pigment naturally produced by B. weihenstephanensis as an alternative to commercial non-soluble pigment.     

一株异养型亚硝酸盐氧化细菌的分离及其降解特性的研究

以亚硝酸盐和琥珀酸钠作为惟一氮、碳源从活性污泥中筛选分离一株能够高效氧化亚硝酸盐的硝化菌株,并对其形态学、生理生化及16S rDNA同源性进行分析,在此基础上研究pH、温度、转速、初始亚硝基氮的浓度以及盐浓度对其氧化亚硝酸盐的影响。结果显示,在好氧条件下,该菌株能在12 h内将356.004 mg/L亚硝酸盐降解99.53%。根据形态学特征、生理生化特性以及16S rRNA同源性分析,初步将该菌株鉴定为施氏假单胞菌(Pseudomonas stutzeri),并将其命名为LYS-86。该菌株氧化亚硝酸盐的最适pH8.0-10.0,温度30℃,转速180 r/min,盐浓度1 g/L。当培养基中初始亚硝酸盐浓度为0.5 g/L时,菌株LYS-86的硝化活性最高,随着培养基中初始亚硝基氮浓度的不断提高,菌株LYS-86的硝化活性会不断下降。本研究利用硝化细菌选择性培养基从活性污泥中筛选到了一株异养型亚硝酸氧化菌菌株,该菌株具有高效的硝化活性,为今后该菌株的实际应用及理论研究奠定了基础。     

Genetic Characterization of a New Thermotolerant Bacillus licheniformis Strain

A potentially new thermotolerant B. licheniformis strain (code name I89), producer of an antibiotic active against Gram-positive bacteria, was genetically characterized and compared with the type strain B. licheniformis ATCC 10716, producer of bacitracin. Studies on DNA base composition (G + C content) and DNA reassociation revealed that the two strains show around 76% homology. Nevertheless, results obtained by rRNA hybridization, with a heterologous probe coding for most of the 16S region of the rRNA operon of Bacillus subtilis, revealed differences in the number of copies for that gene and in the hybridization pattern. Additionally, a different restriction digestion pattern was obtained when DNA was digested with the enzymes NotI, SmaI and analyzed by PFGE. The I89 strain holds a 7.6-kb plasmid not present in the reference strain. The existence of various unique restriction sites and also the stability of this plasmid make it ideal for the future development of a cloning and expression vector. Received: 29 June 1999 / Accepted: 1 September 1999     

Semiautomated Metabolic Staining Assay for Bacillus cereus Emetic Toxin

This paper describes a specific, sensitive, semiautomated, and quantitative Hep-2 cell culture-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for Bacillus cereus emetic toxin. Of nine Bacillus, Brevibacillus, and Paenibacillus species assessed for emetic toxin production, only B. cereus was cytotoxic.     

Isolation and Characterization of a Chlorinated-Pyridinol-Degrading Bacterium

The isolation of a pure culture of bacteria able to use 3,5,6-trichloro-2-pyridinol (TCP) as a sole source of carbon and energy under aerobic conditions was achieved for the first time. The bacterium was identified as a Pseudomonas sp. and designated ATCC 700113. [2,6-(sup14)C]TCP degradation yielded (sup14)CO(inf2), chloride, and unidentified polar metabolites.