Isolation, characterization, and identification of bacterial contaminants in semifinal gelatin extracts

Bacterial contamination of gelatin is of great concern. Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products. In a previous study (E. De Clerck and P. De Vos, Syst. Appl. Microbiol. 25:611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated. In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined. Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced. In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants. Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated. For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups. Representative strains were identified by means of 16S rRNA gene sequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity. The majority of isolates belonged to members of Bacillus or related endospore-forming genera. Representative strains were identified as Bacillus cereus, Bacillus coagulans, Bacillus fumarioli, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus sonorensis, Bacillus subtilis, Bacillus gelatini, Bacillus thermoamylovorans, Anoxybacillus contaminans, Anoxybacillus flavithermus, Brevibacillus agri, Brevibacillus borstelensis, and Geobacillus stearothermophilus. The majority of these species include strains exhibiting gelatinase activity. Moreover, some of these species have known pathogenic properties. These findings are of great concern with regard to the safety and quality of gelatin and its applications.     

Biology and taxonomy of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis

Three species of the Bacillus cereus group (Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis) have a marked impact on human activity. Bacillus cereus and B. anthracis are important pathogens of mammals, including humans, and B. thuringiensis is extensively used in the biological control of insects. The microbiological, biochemical, and genetic characteristics of these three species are reviewed, together with a discussion of several genomic studies conducted on strains of B. cereus group. Using bacterial systematic concepts, we speculate that to understand the taxonomic relationship within this group of bacteria, special attention should be devoted also to the ecology and the population genetics of these species.     

Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov.

Comparative 16S rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen. nov. An analysis of 16S rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organisms Bacillus stearothermophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species. The thermoacidophilic Bacillus species B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus also are unique in that they possess omega-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date. This phenotype, along with the 16S rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus.     

Structure, biosynthesis, and properties of kurstakins, nonribosomal lipopeptides from Bacillus spp.

A new family of lipopeptides produced by Bacillus thuringiensis, the kurstakins, was discovered in 2000 and considered as a biomarker of this species. Kurstakins are lipoheptapeptides displaying antifungal activities against Stachybotrys charatum. Recently, the biosynthesis mechanism, the regulation of this biosynthesis and the potential new properties of kurstakins were described in the literature. In addition, kurstakins were also detected in other species belonging to Bacillus genus such as Bacillus cereus. This mini-review gathers all the information about these promising bioactive molecules.     

Inactivation of spores of Bacillus anthracis Sterne, Bacillus cereus, and Bacillus thuringiensis subsp. israelensis by chlorination

Three species of Bacillus were evaluated as potential surrogates for Bacillus anthracis for determining the sporicidal activity of chlorination as commonly used in drinking water treatment. Spores of Bacillus thuringiensis subsp. israelensis were found to be an appropriate surrogate for spores of B. anthracis for use in chlorine inactivation studies.     

Isolation, Characterization, and Identification of Bacterial Contaminants in Semifinal Gelatin Extracts

Bacterial contamination of gelatin is of great concern. Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products. In a previous study (E. De Clerck and P. De Vos, Syst. Appl. Microbiol. 25:611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated. In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined. Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced. In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants. Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated. For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups. Representative strains were identified by means of 16S rRNA genesequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity. The majority of isolates belonged to members of Bacillus or related endospore-forming genera. Representative strains were identified as Bacillus cereus, Bacillus coagulans, Bacillus fumarioli, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus sonorensis, Bacillus subtilis, Bacillus gelatini, Bacillus thermoamylovorans, Anoxybacillus contaminans, Anoxybacillus flavithermus, Brevibacillus agri, Brevibacillus borstelensis, and Geobacillus stearothermophilus. The majority of these species include strains exhibiting gelatinase activity. Moreover, some of these species have known pathogenic properties. These findings are of great concern with regard to the safety and quality of gelatin and its applications.     

青海可可西里嗜碱芽胞杆菌资源调查

【目的】了解可可西里嗜碱芽胞杆菌资源多样性及产酶多样性,为芽胞杆菌功能资源挖掘和菌剂开发提供基础。【方法】采用Horikoshi I培养基,通过可培养法分离青海可可西里土壤中的嗜碱芽胞杆菌,利用16S r RNA基因序列初步鉴定分离获得的芽胞杆菌。采用透明圈法分析分离菌株的产蛋白酶、纤维素酶及木聚糖酶活性。【结果】从青海可可西里土壤中共分离获得66株嗜碱芽胞杆菌,根据16S r RNA基因序列相似性分析,发现它们隶属于6个属22个种,分别为芽胞杆菌属(Bacillus)、纤细芽胞杆菌属(Gracilibacillus)、喜盐芽胞杆菌属(Halobacillus)、咸海鲜芽胞杆菌属(Jeotgalibacillus)、类芽胞杆菌属(Paenibacillus)和嗜冷芽胞杆菌属(Psychrobacillus),其中以芽胞杆菌属(Bacillus)为优势属。2株嗜碱芽胞杆菌与它们最近匹配模式菌株的16S r RNA基因序列相似性为97.00%和98.65%,为潜在新种。三种酶活检测结果表明产酶菌株约占总分离菌株的95.00%,其中55株具有产蛋白酶活性,27株具有产纤维素酶活性,8株能够产木聚糖酶。【结论】青海可可西里蕴藏着较丰富的嗜碱芽胞杆菌资源及丰富的产酶资源,为后续嗜碱芽胞杆菌的挖掘提供理论基础。     

Biomineralization Potential of Bacillus subtilis,Rummeliibacillus Stabekisii and Staphylococcus Epidermidis Strains In Vitro Isolated from Speleothems,Khasi Hill Caves,Meghalaya, India

Microorganisms were isolated and identified from speleothems at Khasi hill caves, Meghalaya. The aim was to understand their biomineralization potential. Analyses of the speleothems from Krem Soitan, Krem Mawpun, and Krem Lawbah using scanning electron microscope (SEM) showed evidences for microbe–mineral interactions. SEM showed microbial reticulate and beaded filaments, cells, fiber calcites, and clusters of coccoid-like structures. A total of 113 bacterial strains were isolated and identified by a combination of conventional and molecular based tools. 105 strains that were sequenced belonged to the genus: Bacillus, Rummeliibacillus, Staphylococcus, and Brevibacterium. The BLASTn sequence search of 16S rRNA sequences with the National Centre for Biotechnology Information database to establish the identity of the strains yielded similarity scores of ≥99% with the respective organisms. The strains were identified as Bacillus simplex, Bacillus gaemokensis, Bacillus subtilis, Bacillus thuringiensis, Bacillus albus, Bacillus cereus, Bacillus anthracis, Bacillus weihenstephanensis, Rummeliibacillus stabekisii, Bacillus wiedmannii, Staphylococcus epidermidis, Rummeliibacillus pycnus, Kurthia zopfii, and Brevibacterium frigoritolerans. These strains were tested for biomineralization on B-4 medium. Five strains (B. subtilis, R. stabekisii, Staphylococcus epiderdimis, B. cereus, and B. wiedmannii) had the capability to precipitate biominerals in vitro. B. subtilis, R. stabekisii, and S. epidermidis precipitated 0.24, 0.36, and 0.35 g/L of biominerals at 22°C at the end of the four week experiment period. These strains increased the pH of the medium from 7 to 8.95. The precipitated biominerals were imaged using an ultra-high resolution field emission SEM. X-ray diffraction of the biomineral precipitated by R. stabekisii showed that it was composed of vaterite and jungite. Whereas S. epidermidis showed that it was composed of calcite, vaterite, and jungite. B. subtilis produced small, circular calcite crystals. This is the first comprehensive report on the possible evidences about the role of R. stabekisii and S. epidermidis in calcite precipitation isolated from speleothems in the Indian caves. These results allow us to postulate that the identified strains have biomineralization potential. Further evidences of the coexistence of exopolysaccharides, whisker fiber calcites, microbial filaments, and coccoid-like forms point to biogenic inputs in the cave mineral formations.     

Bioinformatics,functional genomics,and proteomics study of Bacillus sp

The ability of bioinformatics to characterize genomic and proteomic sequences from bacteria Bacillus sp. for prediction of genes and proteins has been evaluated. Genomics coupling with proteomics, which is relied on integration of the significant advances recently achieved in two-dimensional (2-D) electrophoretic separation of proteins and mass spectrometry (MS), are now important and high throughput techniques for qualifying and analyzing gene and protein expression, discovering new gene or protein products, and understanding of gene and protein functions including post-genomic study. In addition, the bioinformatics of Bacillus sp. is embraced into many databases that will facilitate to rapidly search the information of Bacillus sp. in both genomics and proteomics. It is also possible to highlight sites for post-translational modifications based on the specific protein sequence motifs that play important roles in the structure, activity and compartmentalization of proteins. Moreover, the secreted proteins from Bacillus sp. are interesting and widely used in many applications especially biomedical applications that are the highly advantages for their potential therapeutic values.     

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic evidence

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.     

Fatty acid composition and primer specificity of de novo fatty acid synthetase in Bacillus globispores, Bacillus insolitus, and Bacillus psychrophilus

The fatty acid compositions of three psychrophilic species of Bacillus were determined by gas--liquid chromatography. The proportions of straight-chain fatty acids, branched-chain fatty acids, and unsaturated fatty acids were found to be 13.3, 86.7, and 26.1% of the total cellular fatty acids for Bacillus globispores, 36.6, 63.4, and 25.1% for Bacillus insolitus, and 6.9, 93.1, and 18.4% for Bacillus psychrophilus, respectively. In all three organisms the de novo fatty acid synthetase specificity towards acyl-CoA primers was butyryl-CoA greater than propionyl-CoA much greater than acetyl-CoA. This shows that B. insolitus, which has an unusually large proportion of straight-chain fatty acids for Bacillus, does not possess a different de novo fatty acid synthetase than the other two organisms. Therefore, the greater proportion of straight-chain fatty acids in B. insolitus may be explained by a large supply of straight-chain primer.     

Comparative studies on extracellular penicillinases of the same structural gene, penP, expressed in Bacillus licheniformis and Bacillus subtilis

Extracellular penicillinases produced by Bacillus licheniformis ATCC 9945A and Bacillus subtilis from the same structural gene, penP, were compared. The two strains secreted the same exo-large penicillinase (mol. wt, 305000; isoelectric point, pI = 5.00-5.04; NH2-terminal amino acid, Ser). In contrast, the exo-small enzyme from Bacillus subtilis (mol. wt, 29500; pI = 5.00-5.04; NH2-terminal amino acid, Glu or Asn) was slightly different from that of Bacillus licheniformis (mol. wt, 29500; pI = 5.13; NH2-terminal amino acid, Lys). The difference in the NH2-terminal residue is most probably due to differences in degradation by host-specific proteolytic enzymes.     

作物根际联合固氮芽孢杆菌的分离鉴定及生态分布

本文报导一种富集和分离根际联合固氮芽孢杆菌的方法,该方法以果胶为唯一碳源的培养基进行富集,中性红为指示剂的培养基进行分离,根据固氮芽孢杆菌能产气以及在分离培养基平板上菌落为红色两个重要的鉴别性特征即可检出,准确性高,简便快速;应用此法从１５个地点６种作物共８８个根系样品中分离出５１株固氮芽孢杆菌,经鉴定均为多粘芽孢杆菌（Ｂａｃｉｌｕｓｐｏｌｙｍｙｘａ）;还初步讨论了多粘芽孢杆菌在几种作物根际的分布情况。     

Cloning, sequencing and expression of the gene encoding the extracellular neutral protease, vibriolysin, of Vibrio proteolyticus.

The gene (nprV), encoding the extracellular neutral protease, vibriolysin (NprV), of the Gram- marine microorganism, Vibrio proteolyticus, was isolated from a V. proteolyticus DNA library constructed in Escherichia coli. The recombinant E. coli produced a protease that co-migrated with purified neutral protease from V. proteolyticus on non-denaturing polyacrylamide gels, and that demonstrated enzymatic specificity towards the neutral protease substrate N-[3-(2-furyl)acryloyl]-L-alanylphenylalanine amide. The nucleotide (nt) sequence of the cloned nprV gene revealed an open reading frame encoding 609 amino acids (aa) including a putative signal peptide sequence followed by a long 'pro' sequence consisting of 172 aa. The N-terminal aa sequence of NprV purified from cultures of V. proteolyticus, identified the beginning of the mature protein within the aa sequence deduced from the nt sequence. Comparative analysis of mature NprV to the sequences of the neutral proteases from Bacillus thermoproteolyticus (thermolysin) and Bacillus stearothermophilus identified extensive regions of conserved aa homology, particularly with respect to active-site residues, zinc-binding residues, and calcium-binding sites. NprV was overproduced in Bacillus subtilis by placing the DNA encoding the 'pro' and mature enzyme downstream from a Bacillus promoter and signal sequence.     

Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene

Extracellular Bacillus proteases are used as additives in detergent powders. We identified a Bacillus strain that produces a protease with an extremely alkaline pH optimum; this protease is suitable for use in modern alkaline detergent powders. The alkalophilic strain Bacillus alcalophilus PB92 gene encoding this high-alkaline serine protease was cloned and characterized. Sequence analysis revealed an open reading frame of 380 amino acids composed of a signal peptide (27 amino acids), a prosequence (84 amino acids), and a mature protein of 269 amino acids. Amino acid comparison with other serine proteases shows good homology with protease YaB, which is also produced by an alkalophilic Bacillus strain. Both show moderate homology with subtilisins but show some remarkable differences from subtilisins produced by neutrophilic bacilli. The prosequence of PB92 protease has no significant homology with prosequences of subtilisins. The abundance of negatively charged residues in the prosequences of PB92 protease is especially remarkable. The cloned gene was used to increase the production level of the protease. For this purpose the strategy of gene amplification in the original alkalophilic Bacillus strain was chosen. When introduced on a multicopy plasmid, the recombinant strain was unstable; under production conditions, plasmid segregation occurred. More stable ways of gene amplification were obtained by chromosomal integration. This was achieved by (i) homologous recombination, resulting in a strain with two tandemly arranged genes, and (ii) illegitimate recombination, resulting in a strain with a second copy of the protease gene on a locus not adjacent to the originally present gene. Both strains showed increased production and were more stable than the plasmid-containing strain. Absolute stability was only found when nontandem duplication occurred. This method of gene amplification circumvents stability problems often encountered in gene amplification in Bacillus species when plasmids or tandemly arranged genes in the chromosome are used.     

大熊猫肠道芽孢杆菌的分离鉴定及部分生物学特性

【目的】分析大熊猫肠道中芽孢杆菌的种类、纤维素分解能力、抗微生物作用和常用抗生素药物敏感性。【方法】利用芽孢耐高温特性分离菌株,基于16S r RNA基因序列构建系统发育树,通过测量芽孢杆菌在刚果红纤维素培养基上的分解圈分析其纤维素分解能力,采用牛津杯法测定芽孢杆菌的抑菌能力,结合软件分析抑菌能力和进化树之间的关系,通过PCR调查芽孢杆菌的抗菌肽分布规律,最后通过药敏试验检测芽孢杆菌是否对常用抗生素敏感。【结果】共分离得到21株芽孢杆菌;进化树显示,这些芽孢杆菌分为6个类别(Category);羧甲基纤维素钠水解结果显示,所有菌株均能分解纤维素;大部分芽孢杆菌菌株对3种肠道病原菌有较强的抑制能力,聚类分析表明,菌株的抗菌能力与基于16S r RNA基因的分类有一定的关联性;66.67%(14/21)的菌株中可以检测到2个或3个抗菌肽基因;药敏试验结果显示,菌株整体药物耐受率低,仅为7.54%(19/264),但仍有少数菌株对抗生素耐受。【结论】分离菌株种类丰富,分布平均,且均具有纤维素分解能力。21株菌株都含有抗菌肽基因,代谢产物对3种肠道病原菌具有明显抑制作用。常用抗生素耐受性低,对规范临床用药具有指导性。     

Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene.

Extracellular Bacillus proteases are used as additives in detergent powders. We identified a Bacillus strain that produces a protease with an extremely alkaline pH optimum; this protease is suitable for use in modern alkaline detergent powders. The alkalophilic strain Bacillus alcalophilus PB92 gene encoding this high-alkaline serine protease was cloned and characterized. Sequence analysis revealed an open reading frame of 380 amino acids composed of a signal peptide (27 amino acids), a prosequence (84 amino acids), and a mature protein of 269 amino acids. Amino acid comparison with other serine proteases shows good homology with protease YaB, which is also produced by an alkalophilic Bacillus strain. Both show moderate homology with subtilisins but show some remarkable differences from subtilisins produced by neutrophilic bacilli. The prosequence of PB92 protease has no significant homology with prosequences of subtilisins. The abundance of negatively charged residues in the prosequences of PB92 protease is especially remarkable. The cloned gene was used to increase the production level of the protease. For this purpose the strategy of gene amplification in the original alkalophilic Bacillus strain was chosen. When introduced on a multicopy plasmid, the recombinant strain was unstable; under production conditions, plasmid segregation occurred. More stable ways of gene amplification were obtained by chromosomal integration. This was achieved by (i) homologous recombination, resulting in a strain with two tandemly arranged genes, and (ii) illegitimate recombination, resulting in a strain with a second copy of the protease gene on a locus not adjacent to the originally present gene. Both strains showed increased production and were more stable than the plasmid-containing strain. Absolute stability was only found when nontandem duplication occurred. This method of gene amplification circumvents stability problems often encountered in gene amplification in Bacillus species when plasmids or tandemly arranged genes in the chromosome are used.     

Differentiation of Bacillus anthracis,B. cereus,and B. thuringiensis on the Basis of the csaB Gene Reflects Host Source

csaB gene analysis clustered 198 strains of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis into two groups related to mammalian and insect hosts, respectively. Mammal-related group I strains also have more S-layer homology (SLH) protein genes than group II strains. This indicates that csaB-based differentiation reflects selective pressure from animal hosts.     

A cryptic plasmid, pAO1, from a compost bacterium, Bacillus sp

The complete nucleotide sequence of a new cryptic plasmid, pAO1 isolated from a compost bacterium Bacillus sp., has been analyzed. Analysis of the PCR-based 16S rRNA sequence showed the bacterium harboring pAO1 was closely related to Bacillus pallidus. The plasmid pAO1 was 3,325 bp in size. Two open reading frames, ORF1 and ORF2, encoding putative polypeptides of 248 and 290 amino acids, respectively, were identified within the sequence. The ORF1 has a limited sequence similarity to an integrase/recombinase, while the ORF2 has high similarity with the replication protein of pBC1 from Bacillus coagulans. A putative origin sequence for a plus-strand was located between ORFs. Southern blot analysis indicates this plasmid replicates via a rolling circle-type mechanism.