Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies

Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".     

Lipase-producing microorganisms from a Kenyan alkaline soda lake

Lipolytic enzyme production of 150 isolated strains from samples of Lake Bogoria (Kenya) was examined. Among these, fifteen isolates were selected on the basis of their lipolytic activities and subjected to morphological and 16S rRNA gene sequencing analyses for their identification. All the microorganisms have been selected under culture conditions with pH ranges between 7-10 and temperatures of 37-55 degrees C. Most of them showed optimal growth at 37 degrees C and tolerated salinity up to 10% (w/v). Ten of the isolates were Gram-negative, nine of which were closely related to the Pseudomonas cluster and one to the Halomonas cluster sharing high similarity profile with Halomonas desiderata. The remaining Gram-positive isolates were closely related to the Bacillus cluster, and were grouped with Bacillus halodurans, Bacillus alcalophilus and Bacillus licheniformis. Four members of the Bacillus cluster and the Halomonas sp. produced lipolytic activity under alkaline conditions, while others did so at neutral pH values.     

Identification of two genes encoding putative new members of the ECF subfamily of eubacterial RNA polymerase sigma factors in Clostridium acetobutylicum

Two genes from Clostridium acetobutylicum DSM 792 were identified which are predicted to encode new members of the ECF subfamily of eubacterial RNA polymerase sigma factors. The sigX gene has the potential to encode a 184-amino acid protein with a molecular mass of 21,870 Da and with the highest overall similarity to Fecl of Escherichia coli (27 % identical residues). The second gene, which is predicted to encode an alternative sigma factor of the ECF subfamily, is the previously described orf2 gene (Gerischer and Dürre, 1990) located in the adc gene region of C. acetobutylicum. The deduced protein of orf2 has significant similarity to SigX of C. acetobutylicum (22 % identical residues) and shares structural features with other alternative sigma factors. Therefore, it is proposed to rename orf2 as sigY. Analysis of the phylogenetic relationship revealed that SigX from C. acetobutylicum, together with sigmaE from Streptomyces coelicolor and SigX from Bacillus subtilis, form a gram-positive cluster within the ECF subfamily and that SigY from C. acetobutylicum together with UviA from Clostridium perfringens, form a separate cluster located between the gram-positive cluster and the sporulation sigma factor sigmaH from B. subtilis.     

Numerical taxonomy of Bacillus isolated from orally administered drugs

Numerical taxonomy procedures were used to study 118 strains of Bacillus isolated from non-sterile drugs prepared for oral administration. Similarities between pairs of strains were calculated by the simple matching coefficient of Sokal and Michener (S_SM). Each strain was tested for 60 unit characters and three clusters were defined. The strains in each cluster presented a similarity level of at least 60%. Cluster A comprised the strains identified as Bacillus cereus (S_SM= 93·13%), cluster B contained three subgroups corresponding to the species B. pumilus, B. subtilis and B. licheniformis (S_SM= 84·35%) and cluster C also included three subgroups that belonged to the species B. firmus, B. lentus and B. badius (S_SM= 80·14%). The most discriminating tests were selected to differentiate the clusters from the subgroups. The feature with the highest discriminating power between clusters A and B was the lack of acid production from arabinose and mannitol. The Voges-Proskauer, methyl red tests and sensitivity to polymyxin B clearly distinguished cluster A from C. The Voges-Proskauer test and acid production from arabinose were the best to differentiate between B and C. Bacillus pumilus and B. subtilis differed in starch hydrolysis and B. licheniformis in growing anaerobically. To discriminate B. firmus from B. lentus the most important tests were the acid production from glucose and sucrose; intermediate strains were found. Bacillus badius was differentiated from B. firmus by 10 tests, and from B. lentus by the production of urease.     

Numerical taxonomy of Bacillus isolated from orally administered drugs

Numerical taxonomy procedures were used to study 118 strains of Bacillus isolated from non-sterile drugs prepared for oral administration. Similarities between pairs of strains were calculated by the simple matching coefficient of Sokal and Michener (SSM). Each strain was tested for 60 unit characters and three clusters were defined. The strains in each cluster presented a similarity level of at least 60%. Cluster A comprised the strains identified as Bacillus cereus (SSM = 93.13%), cluster B contained three subgroups corresponding to the species B. pumilus, B. subtilis and B. licheniformis (SSM = 84.35%) and cluster C also included three subgroups that belonged to the species B. firmus, B. lentus and B. badius (SSM = 80.14%). The most discriminating tests were selected to differentiate the clusters from the subgroups. The feature with the highest discriminating power between clusters A and B was the lack of acid production from arabinose and mannitol. The Voges-Proskauer, methyl red tests and sensitivity to polymyxin B clearly distinguished cluster A from C. The Voges-Proskauer test and acid production from arabinose were the best to differentiate between B and C. Bacillus pumilus and B. subtilis differed in starch hydrolysis and B. licheniformis in growing anaerobically. To discriminate B. firmus from B. lentus the most important tests were the acid production from glucose and sucrose; intermediate strains were found. Bacillus badius was differentiated from B. firmus by 10 tests, and from B. lentus by the production of urease.     

Multiple-locus sequence typing analysis of Bacillus cereus and Bacillus thuringiensis reveals separate clustering and a distinct population structure of psychrotrophic strains

We used multilocus sequence typing (MLST) to characterize phylogenetic relationships for a collection of Bacillus cereus group strains isolated from forest soil in the Paris area during a mild winter. This collection contains multiple strains isolated from the same soil sample and strains isolated from samples from different sites. We characterized 115 strains of this collection and 19 other strains based on the sequences of the clpC, dinB, gdpD, panC, purF, and yhfL loci. The number of alleles ranged from 36 to 53, and a total of 93 allelic profiles or sequence types were distinguished. We identified three major strain clusters-C, T, and W-based on the comparison of individual gene sequences or concatenated sequences. Some less representative clusters and subclusters were also distinguished. Analysis of the MLST data using the concept of clonal complexes led to the identification of two, five, and three such groups in clusters C, T, and W, respectively. Some of the forest isolates were closely related to independently isolated psychrotrophic strains. Systematic testing of the strains of this collection showed that almost all the strains that were able to grow at a low temperature (6 degrees C) belonged to cluster W. Most of these strains, including three independently isolated strains, belong to two clonal complexes and are therefore very closely related genetically. These clonal complexes represent strains corresponding to the previously identified species Bacillus weihenstephanensis. Most of the other strains of our collection, including some from the W cluster, are not psychrotrophic. B. weihenstephanensis (cluster W) strains appear to comprise an effectively sexual population, whereas Bacillus thuringiensis (cluster T) and B. cereus (cluster C) have clonal population structures.     

Potential contact sites between the protein and RNA subunit in the Bacillus subtilis RNase P holoenzyme.

We have detected by nucleotide analog interference mapping (NAIM) AMPalphaS and IMPalphaS modifications in Bacillus subtilis RNase P RNA that interfere with binding of the homologous protein subunit. Interference as well as some enhancement effects were clustered in two main areas, in P10.1a/L10.1 and P12 of the specificity domain (cluster 1, domain I) and in P2, P3, P15.1, J18/2 and J19/4 of the catalytic domain (cluster 2a, domain II). Minor interferences in P1 and P19 and a strong and weak enhancement effect in P19 represent a third area located in domain II (cluster 2b). Our results suggest that P3, P2-J18/2 and J19/4 are key elements for anchoring of the protein to the catalytic domain close to the scissile phosphodiester in enzyme-substrate complexes. Sites of interference or enhancement in clusters 1 and 2a are located at distances between 65 and 130 A from each other in the current 3D model of a full-length RNase P RNA-substrate complex. Taking into account that the RNase P protein monomer can bridge a maximum distance of about 40 A, simultaneous direct contacts to the two aforementioned potential RNA-binding areas would be incompatible with our current understanding of bacterial RNase P RNA architecture. Our findings suggest that the current 3D model has to be rearranged in order to reduce the distance between clusters 1 and 2a. Alternatively, based on the recent finding that B. subtilis RNase P forms a tetramer consisting of two protein and two RNA subunits, cluster 1 may reflect one protein contact site in domain I, and cluster 2a a separate one in domain II.     

金矿床区蜡状芽孢杆菌孢壁蛋白SDS-PAGE图谱及聚类分析

用SDS PAGE方法对 5株从我国金矿床区采集到的典型的蜡状芽孢杆菌 (Bacilluscereus)C6,C14 ,B2 ,JY I T1,JY X T9的孢壁蛋白同标准株AS1.12 6的孢壁蛋白共同进行比较分析 ,计算出了各蛋白电泳带的近似分子量 ,又以它们的迁移距离为标准同苏云金芽孢杆菌 (Bacillusthuringiensis)的孢壁蛋白相比较 ,得到聚类分析树状图谱 ,表明具有聚金作用的蜡状芽孢杆菌在分类学上具有相似性 ,而与具有杀虫作用的Bt菌株在分类学上则相差较远。     

EPR characterization of soluble fragments of succinate dehydrogenase from mutant strains of Bacillus subtilis

Succinate dehydrogenase is a membrane-bound metallo-flavo-enzyme containing a bi- (S-1), a tri- (S-3) and a tetranuclear (S-2) iron-sulfur cluster. The catalytic portion of the enzyme contains two distinct subunits designated Fp and Ip. Using concentrated extracts from mutant strains of Bacillus subtilis it was demonstrated, by using low temperature EPR, that cluster S-2 can be assembled in a soluble succinate dehydrogenase. In a mutant with a truncated Ip subunit which lacks 7 of the 11 conserved cysteine residues, cluster S-1 lacked the spin relaxation properties attributable to an adjacent cluster S-2. These data are consistent with a model where one or more cysteine residues from the middle set of 4 conserved cysteines in the Ip subunit are ligands to the tetranuclear cluster.     

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.     

Staphylococcus aureus has clustered tRNA genes.

The polymerase chain reaction (PCR) was used to detect large tRNA gene clusters in Bacillus subtilis, Bacillus badius, Bacillus megaterium, Lactobacillus brevis, Lactobacillus casei, and Staphylococcus aureus. The primers were based on conserved sequences of known gram-positive bacterial tRNA(Arg) and tRNA(Phe) genes. This PCR procedure detected an unusually large tRNA gene cluster in S. aureus. PCR-generated probes were used to identify a 4.5-kb EcoRI fragment that contained 27 tRNA genes immediately 3' to an rRNA operon. Some of these 27 tRNA genes are very similar, but only 1 is exactly repeated in the cluster. The 5' end of this cluster has a gene order similar to that found in the 9- and 21-tRNA gene clusters of B. subtilis. The 3' end of this S. aureus cluster exhibits more similarity to the 16-tRNA gene cluster of B. subtilis. The 24th, 25th, and 26th tRNA genes of this S. aureus tRNA gene cluster code for three similar, unusual Gly-tRNAs that may be used in the synthesis of the peptidoglycan in the cell wall but not in protein synthesis. Southern analysis of restriction digests of S. aureus DNA indicate that there are five to six rRNA operons in this bacterium's genome and that most or all may have large tRNA gene clusters at the 3' end.     

Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from a fermented locust bean product manufactured in West Africa

Twenty-five Bacillus strains capable of producing gamma-polyglutamic acid (PGA) were isolated from fermented locust bean products manufactured in the savanna area of Ghana. To clarify the phylogeny of these PGA-producing strains, phylogenetic analyses based on sequences of 16S rDNA, rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) genes were performed. A phylogenetic tree based on 16S rDNA indicated that ten isolates were clustered in the same group of Bacillus subtilis. Another ten isolates were located in the cluster of B. amyloliquefaciens, and the remaining isolates were identified as B. pumilus (three isolates) and B. licheniformis (two isolates), respectively. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. Thirty-four strains in 27 species belonging to the genus Bacillus and its neighbors were also investigated for PGA production. It was found that PGA was produced by B. amyloliquefaciens NBRC 14141 and NBRC 15535(T), B. atrophaeus NBRC 15539(T), B. licheniformis NBRC 12107, B. mojavensis NBRC 15718(T), B. pumilus NBRC 12094, B. subtilis NBRC 16449, and Lysinibacillus sphaericus NBRC 3525. Except for L. sphaericus, the above Bacillus species are very closely related in phylogeny, indicating that PGA-producing Bacillus strains constitute a cluster.     

Multiple-Locus Sequence Typing Analysis of Bacillus cereus and Bacillus thuringiensis Reveals Separate Clustering and a Distinct Population Structure of Psychrotrophic Strains

We used multilocus sequence typing (MLST) to characterize phylogenetic relationships for a collection of Bacillus cereus group strains isolated from forest soil in the Paris area during a mild winter. This collection contains multiple strains isolated from the same soil sample and strains isolated from samples from different sites. We characterized 115 strains of this collection and 19 other strains based on the sequences of the clpC, dinB, gdpD, panC, purF, and yhfL loci. The number of alleles ranged from 36 to 53, and a total of 93 allelic profiles or sequence types were distinguished. We identified three major strain clusters—C, T, and W—based on the comparison of individual gene sequences or concatenated sequences. Some less representative clusters and subclusters were also distinguished. Analysis of the MLST data using the concept of clonal complexes led to the identification of two, five, and three such groups in clusters C, T, and W, respectively. Some of the forest isolates were closely related to independently isolated psychrotrophic strains. Systematic testing of the strains of this collection showed that almost all the strains that were able to grow at a low temperature (6°C) belonged to cluster W. Most of these strains, including three independently isolated strains, belong to two clonal complexes and are therefore very closely related genetically. These clonal complexes represent strains corresponding to the previously identified species Bacillus weihenstephanensis. Most of the other strains of our collection, including some from the W cluster, are not psychrotrophic. B. weihenstephanensis (cluster W) strains appear to comprise an effectively sexual population, whereas Bacillus thuringiensis (cluster T) and B. cereus (cluster C) have clonal population structures.     

Characterisation of oxidised 7Fe dicluster ferredoxins with NMR spectroscopy

Dicluster ferredoxins (Fds) from Sulfolobus acidocaldarius and Desulfovibrio africanus (FdIII) have been studied using 1H NMR. Both wild-type proteins contain a [3Fe-4S]+/0 and a [4Fe-4S]2+/+ cluster as isolated. The [4Fe-4S]2+/+ cluster (cluster II) is bound by cysteine residues arranged in a classic ferredoxin motif: CysI-(Xaa)2-CysII-(Xaa)2-CysIII-(Xaa)n-CysIV-Pro , whilst the binding motif of the [3Fe-4S]+/0 cluster (cluster I) has a non-ligating aspartic acid (Asp14) at position II, i.e. CysI-(Xaa)2-Asp-(Xaa)2-CysIII. D. africanus FdIII undergoes facile cluster transformation from the 7Fe form to the 8Fe form, but S. acidocaldarius Fd does not. Many factors determine the propensity of a cluster to undergo interconversion, including the presence, and correct orientation, of a suitable ligand. We have investigated this using 1H NMR by introducing a potential fourth ligand into the binding motif of cluster I of D. africanus FdIII. Asp14 has been mutated to cysteine (D14C), glutamic acid (D14E) and histidine (D14H). Cluster incorporation was performed in vitro. The cluster types present were identified from the chemical shift patterns and temperature-dependent behaviour of the hyperfine-shifted resonances. Factors influencing cluster ligation and cluster interconversion, in vitro, are discussed. Furthermore, the data have established that the residue at position II in the cluster binding motif of cluster I is influential in determining the chemical shift pattern observed for a [3Fe-4S]+ cluster when a short/symmetric binding motif is present. Based on this, a series of rules for characterising the 1H NMR chemical shifts of mono- and di-cluster [3Fe-4S]+ cluster-containing ferredoxins is given.     

New evidence for the role of calcium in the glycosidase reaction of GH43 arabinanases

Endo-1,5-α-L-arabinanases are glycosyl hydrolases that are able to cleave the glycosidic bonds of α-1,5-L-arabinan, releasing arabino-oligosaccharides and L-arabinose. Two extracellular endo-1,5-α-L-arabinanases have been isolated from Bacillus subtilis, BsArb43A and BsArb43B (formally named AbnA and Abn2, respectively). BsArb43B shows low sequence identity with previously characterized 1,5-α-L-arabinanases and is a much larger enzyme. Here we describe the 3D structure of native BsArb43B, biochemical and structure characterization of two BsArb43B mutant proteins (H318A and D171A), and the 3D structure of the BsArb43B D171A mutant enzyme in complex with arabinohexose. The 3D structure of BsArb43B is different from that of other structurally characterized endo-1,5-α-L-arabinanases, as it comprises two domains, an N-terminal catalytic domain, with a 3D fold similar to that observed for other endo-1,5-α-L-arabinanases, and an additional C-terminal domain. Moreover, this work also provides experimental evidence for the presence of a cluster containing a calcium ion in the catalytic domain, and the importance of this calcium ion in the enzymatic mechanism of BsArb43B.     

Genetic relationship among Bacillus licheniformis rolling-circle-replicating plasmids and complete nucleotide sequence of pBL63.1, an atypical replicon

The degree of biodiversity among Bacillus licheniformis plasmids and their relation to other Bacillus subtilis group plasmids has been evaluated. To attain this goal we surveyed the diversity and linkage of replication modules in a collection of 21 naturally occurring plasmids of B. licheniformis strains, isolated from different geographical areas. On the basis of rep gene sequence analysis it was possible to group the B. licheniformis plasmids rep genes in two main cluster. Comparison with known rep genes from Bacillus rolling-circle-replicating (RCR) plasmids revealed the presence in B. licheniformis plasmids of replication genes with a DNA sequence peculiar to B. licheniformis species together with rep genes with a very high sequence similarity to B. subtilis plasmids. Furthermore, the molecular organization of an atypical replicon, pBL63.1, was shown. This plasmid did not display any significant similarity with known Bacillus RCR plasmids. The complete nucleotide sequence evidenced a replication module with an unexpected similarity with Rep proteins from RCR plasmids of bacterial species phylogenetically distantly related to Bacillus. pBL63.1 represents an exception to the low-level diversity hypothesis among Bacillus RC replicons.     

Numerical survey of some bacterial taxa

Focht, D. D. (Iowa State University, Ames), and W. R. Lockhart. Numerical survey of some bacterial taxa. J. Bacteriol. 90:1314-1319. 1965.-A numerical analysis was made of 77 properties of each of 43 bacterial strains, representing 25 genera from 8 families in the orders Eubacteriales and Pseudomonadales. Four major groups were found, related to one another at approximately the same level of similarity: (1) a large cluster containing the subgroups (1a) Athiorhodaceae-Spirillaceae, (1b) Xanthomonas, and (1c) "inactive" Micrococcaceae-Achromobacteraceae; (2) a cluster containing the "active" Micrococcaceae and Lactobacillaceae; (3) the enterobacteria; and (4) Aeromonas. There was a sharp distinction between the branches of groups 1a, 1c, and 2. The composition of groups was essentially the same whether or not fermentation of carbohydrates (28 characters) was included in the analysis. Several individual strains, notably, Bacillus subtilis, Leuconostoc mesenteroides, Pseudomonas aeruginosa, and Erwinia amylovora, were related to none of the groups, and others (two species of Proteus, Flavobacterium devorans, and Lactobacillus casei) showed only minimal quantitative relationships with their groups. These results suggest that there may be significant variation in levels of similarity within microbial groups presently accorded equivalent taxonomic rank, and that some present distinctions among taxa, particularly at the generic level, cannot be confirmed on the basis of overall similarity.     

Generation of auxotrophic mutants of Enterococcus faecalis.

A 22-kb segment of chromosomal DNA from Enterococcus faecalis OG1RF containing the pyrimidine biosynthesis genes pyrC and pyrD was previously detected as complementing Escherichia coli pyrC and pyrD mutations. In the present study, it was found that the E. faecalis pyrimidine biosynthetic genes in this clone (designated pKV48) are part of a larger cluster resembling that seen in Bacillus spp. Transposon insertions were isolated at a number of sites throughout the cluster and resulted in loss of the ability to complement E. coli auxotrophs. The DNA sequences of the entire pyrD gene of E. faecalis and selected parts of the rest of the cluster were determined, and computer analyses found these to be similar to genes from Bacillus subtilis and Bacillus caldolyticus pyrimidine biosynthesis operons. Five of the transposon insertions were introduced back into the E. faecalis chromosome, and all except insertions in pyrD resulted in pyrimidine auxotrophy. The prototrophy of pyrD knockouts was observed for two different insertions and suggests that E. faecalis is similar to Lactococcus lactis, which has been shown to possess two pyrD genes. A similar analysis was performed with the purL gene from E. faecalis, contained in another cosmid clone, and purine auxotrophs were isolated. In addition, a pool of random transposon insertions in pKV48, isolated in E. coli, was introduced into the E. faecalis chromosome en masse, and an auxotroph was obtained. These results demonstrate a new methodology for constructing defined knockout mutations in E. faecalis.     

Detection of molecular diversity in Bacillus atrophaeus by amplified fragment length polymorphism analysis

Phenotypically, Bacillus atrophaeus is indistinguishable from the type strain of Bacillus subtilis except by virtue of pigment production on certain media. Several pigmented variants of B. subtilis have been reclassified as B. atrophaeus, but several remain ambiguous in regard to their taxonomic placement. In this study, we examined strains within the American Type Culture Collection originally deposited as Bacillus globigii, B. subtilis var. niger, or Bacillus niger using 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis to determine the level of molecular diversity among these strains and their relationship with closely related taxa. The 16S rRNA gene sequences revealed little variation with one base substitution between the B. atrophaeus type strain ATCC 49337 and the other pigmented bacilli. AFLP analysis produced high-quality DNA fingerprints with sufficient polymorphism to reveal strain-level variation. Cluster analysis of Dice similarity coefficients revealed that three strains, ATCC 31028, ATCC 49760, and ATCC 49822, are much more closely related to B. atrophaeus than to B. subtilis and should be reclassified as B. atrophaeus. A very closely related cluster of B. atrophaeus strains was also observed; this cluster was genetically distinct from the type strain. The level of variation between the two groups was approximately the same as the level of variation observed between members of the two B. subtilis subspecies, subtilis and spizizenii. It is proposed that the cluster of strains typified by ATCC 9372 be designated a new subspecies, B. atrophaeus subsp. globigii.