Gram-negative bacteria associated with brewery yeasts: reclassification of Obesumbacterium proteus biogroup 2 as Shimwellia pseudoproteus gen. nov., sp. nov., and transfer of Escherichia blattae to Shimwellia blattae comb. nov.

Phylogenetic analyses of type and reference strains of Obesumbacterium proteus biogroups 1 and 2 plus a novel isolate of biogroup 2 were carried out based on 16S rRNA gene sequences and partial sequences of four protein-coding genes (fusA, leuS, pyrG and rpoB). Both approaches revealed that O. proteus biogroup 1 strains were closely related to Hafnia alvei. Biogroup 2 strains, however, formed a distinct monophyletic clade of generic status that included Escherichia blattae. Phenotypic tests were consistent with the molecular classification and provided diagnostic features. It is proposed that biogroup 2 strains be placed in a new genus, Shimwellia gen. nov., as Shimwellia pseudoproteus sp. nov., with strain 521(T) (=DSM 3038(T)=LMG 24835(T)=NCIMB 14534(T)) as the type strain, and that Escherichia blattae be transferred to the genus Shimwellia as Shimwellia blattae comb. nov., with strain ATCC 29907( T) (=DSM 4481(T)) as the type strain.

Evolution of pathogenicity in the Bacillus cereus group.

The Bacillus cereus group of bacteria comprises soil-dwelling saprophytes but on occasion these bacteria can cause a wide range of diseases in humans, including food poisoning, systemic infections and highly lethal forms of anthrax. While anthrax is almost invariably caused by strains from a single evolutionary lineage, Bacillus anthracis, variation in the virulence properties of strains from other lineages has not been fully addressed. Using multi-locus sequence data from 667 strains, we reconstructed the evolutionary history of the B. cereus group in terms of both clonal inheritance and recombination. The strains included 155 clinical isolates representing B. anthracis, and isolates from emetic and diarrhoeal food poisoning, septicaemia and related infections, wound, and lung infections. We confirmed the existence of three major clades and found that clinical isolates of B. cereus (with the exception of emetic toxin-producing strains) are evenly distributed between and within clades 1 and 2. B. anthracis in particular and emetic toxin-producing B. cereus show more clonal structure and are restricted to clade 1. Our characterization of the patterns of genetic exchange showed that there exist partial barriers to gene flow between the three clades. The pathogenic strains do not exhibit atypically high or low rates of recombination, consistent with the opportunistic nature of most pathogenic infections. However, there have been a large number of recent imports in clade 1 of strains from external origins, which is indicative of an on-going shift in gene-flow boundaries for this clade.

Composition of peats used in the preparation of malt for scotch whisky productioninfluence of geographical source and extraction depth.

Peat is burned during malt kilning to provide flavor compounds in Scotch malt whisky. The aim of this work was to establish whether peats from different locations in Scotland are chemically distinct and could impart different flavors. Peat samples from four locations (Islay, Orkney, St. Fergus, and Tomintoul) were analyzed using Curie point pyrolysis in combination with gas chromatography-mass spectrometry (Py-GC-MS). Peat pyrolysates from Islay and St. Fergus were rich in lignin derivatives, while those from Orkney and Tomintoul had higher levels of carbohydrate derivatives. Also, Islay and Orkney peat pyrolysates were rich in nitrogen-containing compounds and aromatic hydrocarbons, respectively. The depth of peat extraction was found to have an additional effect on peat composition as the levels of carbohydrate derivatives reduced with increasing depth. Where peat is used in whisky production, the observed differences in peat composition could potentially impact flavor, an important consideration if the peat used for malt production is changed by either choice or necessity.

A Bacillus thuringiensis strain producing a polyglutamate capsule resembling that of Bacillus anthracis.

Bacillus thuringiensis serovar Monterrey strain BGSC 4AJ1 produced a microscopically visible capsule that reacted with a fluorescent antibody specific for the poly-gamma-d-glutamic acid (PGA) capsule of Bacillus anthracis. PGA capsule biosynthesis genes with 75%, 81%, 72%, 65% and 63% similarity, respectively, to those of the B. anthracis capBCADE cluster were present on a plasmid (pAJ1-1). Strain BGSC 4AJ1, together with five strains of Bacillus cereus that hybridized to a PGA cap gene probe, were analyzed phylogenetically using six housekeeping genes of a B. cereus multilocus sequence typing scheme. Bacillus thuringiensis BGSC 4AJ1 shared four identical alleles with B. anthracis and was the second most closely related to this bacterium of the 674 isolates in the multilocus sequence typing database. The other cap+ strains were distributed among various lineages of Clade 1 of the B. cereus group.

Sneathiella chinensis gen. nov., sp. nov., a novel marine alphaproteobacterium isolated from coastal sediment in Qingdao, China.

The taxonomic position of strain LMG 23452(T), which was isolated from coastal sediment from an aquaculture site near Qingdao, China, in 2000, was determined. Strain LMG 23452(T) comprised Gram-negative, non-spore-forming, motile rods and was found to be a halotolerant, aerobic, chemoheterotroph that produces catalase and oxidase. Comparative 16S rRNA gene sequence analysis revealed that strain LMG 23452(T) shared approximately 89 % sequence similarity with members of the genera Devosia, Hyphomonas, Ensifer and Chelatococcus, which belong to two different orders within the Alphaproteobacteria. Further phylogenetic analysis of the 16S rRNA gene sequence showed that strain LMG 23452(T) formed a separate branch within the order Rhizobiales, falling between the genera Devosia and Ensifer of the families Hyphomicrobiaceae and Rhizobiaceae, respectively. Strain LMG 23452(T) could be differentiated from its closest phylogenetic neighbours on the basis of several phenotypic features, including hydrolysis of the substrates starch and casein and assimilation of the carbohydrates d-glucose, d-mannose, mannitol, maltose and l-arabinose, and chemotaxonomically by the presence of the fatty acids C(14 : 0) 3-OH, C(16 : 1)omega11c, C(16 : 1)omega5c and C(18 : 1)omega5c. The major fatty acids detected in strain LMG 23452(T) were C(18 : 1)omega7c, C(16 : 0), C(19 : 0) cyclo omega8c, C(16 : 1)omega7c and C(17 : 1)omega6c and the G+C content of the genomic DNA was 57.1 mol%. Therefore, the polyphasic data support the placement of strain LMG 23452(T) within a novel genus and species, for which the name Sneathiella chinensis gen. nov., sp. nov. is proposed. The type strain is LMG 23452(T) (=CBMAI 737(T)).

Crystal protein synthesis is dependent on early sporulation gene expression in Bacillus sphaericus.

Insertional mutations in the spo0A and spoIIAC genes of Bacillus sphaericus 2362 were prepared by conjugation with Escherichia coli using a suicide plasmid containing cloned portions of the target genes. The mutants resembled their Bacillus subtilis counterparts phenotypically and were devoid of crystal proteins as determined by electron microscopy, SDS-PAGE and Western blots. The mutants had greatly reduced toxicity to anopheline mosquito larvae compared to the parental strain. We conclude that crystal protein synthesis in this bacterium is dependent on expression of early sporulation genes.

Multilocus sequence typing reveals that Bacillus cereus strains isolated from clinical infections have distinct phylogenetic origins.

Eight strains of Bacillus cereus isolated from bacteremia and soft tissue infections were assigned to seven sequence types (STs) by multilocus sequence typing (MLST). Two strains from different locations had identical STs. The concatenated sequences of the seven STs were aligned with 65 concatenated sequences from reference STs and a neighbor-joining tree was constructed. Two strains were distantly related to all reference STs. Three strains were recovered in a clade that included Bacillus anthracis, B. cereus and rare Bacillus thuringiensis strains while the other three strains were assigned to two STs that were more closely affiliated to most of the B. thuringiensis STs. We conclude that invasive B. cereus strains do not form a single clone or clonal complex of highly virulent strains.

Lactobacillus suntoryeus sp. nov., isolated from malt whisky distilleries.

Eight strains of Lactobacillus with identical partial 16S rRNA gene sequences and similar randomly amplified polymorphic DNA patterns were isolated from fermentation samples from Japanese and Scottish malt whisky distilleries. Phylogenetic analysis of almost complete 16S rRNA gene sequences from three representative strains (two from Japan, one from Scotland) placed them in the genus Lactobacillus as members of the Lactobacillus acidophilus group. Lactobacillus helveticus and Lactobacillus gallinarum were the most closely related species, with 16S rRNA gene similarities of 99.3 and 98.1 %, respectively. A similar phylogeny was derived from partial sequences of elongation factor Tu (tuf) genes in which the alleles from the three distillery isolates were identical and shared 99.0 % similarity with L. helveticus and L. gallinarum tuf genes. S-layer (slp) gene sequences suggested different relationships among the strains and the distillery isolates no longer formed a monophyletic group. The alleles from the Japanese and Scottish strains shared only 54 % similarity. Chromosomal DNA from the distillery strains gave DNA-DNA hybridization values between 79 and 100 % but showed less than 43 and 22 % reassociation with L. helveticus and L. gallinarum DNA, respectively. The name Lactobacillus suntoryeus sp. nov. is proposed for this novel taxon; the type strain is strain SAT (=LMG 22464T=NCIMB 14005T).

Population structure and evolution of the Bacillus cereus group.

Representative strains of the Bacillus cereus group of bacteria, including Bacillus anthracis (11 isolates), B. cereus (38 isolates), Bacillus mycoides (1 isolate), Bacillus thuringiensis (53 isolates from 17 serovars), and Bacillus weihenstephanensis (2 isolates) were assigned to 59 sequence types (STs) derived from the nucleotide sequences of seven alleles, glpF, gmk, ilvD, pta, pur, pycA, and tpi. Comparisons of the maximum likelihood (ML) tree of the concatenated sequences with individual gene trees showed more congruence than expected by chance, indicating a generally clonal structure to the population. The STs followed two major lines of descent. Clade 1 comprised B. anthracis strains, numerous B. cereus strains, and rare B. thuringiensis strains, while clade 2 included the majority of the B. thuringiensis strains together with some B. cereus strains. Other species were allocated to a third, heterogeneous clade. The ML trees and split decomposition analysis were used to assign STs to eight lineages within clades 1 and 2. These lineages were defined by bootstrap analysis and by a preponderance of fixed differences over shared polymorphisms among the STs. Lineages were named with reference to existing designations: Anthracis, Cereus I, Cereus II, Cereus III, Kurstaki, Sotto, Thuringiensis, and Tolworthi. Strains from some B. thuringiensis serovars were wholly or largely assigned to a single ST, for example, serovar aizawai isolates were assigned to ST-15, serovar kenyae isolates were assigned to ST-13, and serovar tolworthi isolates were assigned to ST-23, while other serovars, such as serovar canadensis, were genetically heterogeneous. We suggest a revision of the nomenclature in which the lineage and clone are recognized through name and ST designations in accordance with the clonal structure of the population.

Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax.

Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-gamma-d-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.

Taxonomic characterization and plant colonizing abilities of some bacteria related to Bacillus amyloliquefaciens and Bacillus subtilis.

The phylogenetic relationships of 17 Bacillus strains isolated from plants and soil were determined from partial sequences of genes encoding 16S rRNA, gyraseA (gyrA) and the cheA histidine kinase. Five strains were closely related to Bacillus subtilis subsp. subtilis, three strains were more closely related to B. subtilis subsp. spizizeni and two strains were identified as B. mojavensis. The remaining seven strains formed a cluster closely related to, but distinct from, Bacillus amyloliquefaciens. Some of these strains formed red-pigmented colonies. The abilities of selected strains to survive in the rhizosphere and to colonize plants were studied using oilseed rape (Brassica napus), barley (Hordeum vulgare) and thale cress (Arabidopsis thaliana) as model plants. It was shown by following the titre of bacteria in seedlings and by scanning electron microscopy that survival of Bacillus cells on the roots of seedlings during the first week after treatment of seeds with spore suspensions was crucial for colonization of the rhizosphere and for biocontrol activity. The group of strains related to B. amyloliquefaciens were generally better adapted to colonization of the rhizosphere of plants than other members of the B. subtilis group and could be considered a distinct ecotype of B. amyloliquefaciens. Bacteria in this taxon could be recognized on the basis of amplification of a PCR product with primers directed to the tetB(L) locus but no product with primers directed to the alpha-amylase gene of B.amyloliquefaciens sensu stricto.

Molecular typing of Bacillus thuringiensis serovars by RAPD-PCR.

One hundred and twenty-six strains of Bacillus thuringiensis representing 57 serovars were allocated to 58 genomic types using random amplified polymorphic DNA (RAPD)-PCR patterns. Serovars darmstadiensis, israelensis, kenyae, kumamotoensis, kurstaki, morrisoni, pakistani, sotto, thuringiensis and tolworthi each encompassed identical or closely related strains. Despite this genomic homogeneity, most of these serovars also included at least one variant strain. Serovars aizawai, canadensis, entomocidus and sotto biotype dendrolimus, on the other hand, were genomically heterogeneous. Of the 57 serovars examined, 31 contained at least one strain with a closely related or identical RAPD pattern to a strain from a different serovar. We conclude that while the species is genomically diverse, the homogeneous serovars represent clonal lineages of successful insect pathogens.

Pulsed field gel electrophoresis of chromosomal DNA reveals a clonal population structure to Bacillus thuringiensis that relates in general to crystal protein gene content.

Seventy strains of Bacillus thuringiensis representing 21 serovars were allocated to 38 genomic groups using pulsed field gel electrophoresis (PFGE) of restriction enzyme-digested DNA. There was a broad correlation between PFGE type and serotype for serovars darmstadiensis, israelensis, kenyae, kumamotoensis, kurstaki, sotto, thuringiensis, and tolworthi, although some serovars included atypical strains. Serovars canadensis and entomocidus were heterogeneous. Detection of crystal protein genes by polymerase chain reaction indicated an approximate correlation between PFGE type and cry gene complement. For example, cry1 products were amplified from DNA from PFGE type 17 strains of serovar aizawai and from PFGE type 23 strains of serovar tolworthi but not from a PFGE 18 strain of aizawai nor from a PFGE type 24 strain of tolworthi. These data suggest a clonal population structure to B. thuringiensis with some consistency of Cry-plasmid composition within PFGE types.

Bacillus endophyticus sp. nov., isolated from the inner tissues of cotton plants (Gossypium sp.).

Four strains of aerobic, endospore-forming bacteria were isolated from the inner tissues of healthy cotton plants (Gossypium sp., Dushanbe, Tajikistan). The organisms had identical randomly amplified polymorphic DNA patterns that distinguished them from other bacilli that are commonly isolated from plant tissues, e.g. Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus and Bacillus subtilis. PCR amplification of 16S-23S rRNA spacer regions suggested that the four strains could be assigned to two highly related taxa, which correlated with differences in cell morphology. However, the cloned spacer region provided a simple and specific hybridization probe for all four strains. The virtually complete 16S rDNA sequences were prepared for representatives of the two groups (strains 2DT(T) and 12DX) and differed by only two bases, thus supporting classification of the four strains in a single taxon at the species level. Phylogenetic analyses indicated that strain 2DT(T) belonged to the genus Bacillus and was most closely related to Bacillus sporothermodurans DSM 10599T with a sequence similarity of 94.8%. It is concluded that the four strains belong to a novel species of Bacillus for which the name Bacillus endophyticus sp. nov. is proposed. The type strain is 2DT(T) (= UCM B-5715T = CIP 106778T).

Evolution of the lactic acid bacterial community during malt whisky fermentation: a polyphasic study.

The development of the lactic acid bacterial community in a commercial malt whisky fermentation occurred in three broad phases. Initially, bacteria were inhibited by strong yeast growth. Fluorescence microscopy and environmental scanning electron microscopy revealed, in this early stage, both cocci and rods that were at least partly derived from the wort and yeast but also stemmed from the distillery plant. Denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA genes and sequence analysis revealed cocci related to Streptococcus thermophilus or Saccharococcus thermophilus, Lactobacillus brevis, and Lactobacillus fermentum. The middle phase began 35 to 40 h after yeast inoculation and was characterized by exponential growth of lactobacilli and residual yeast metabolism. Lactobacillus casei or Lactobacillus paracasei, L. fermentum, and Lactobacillus ferintoshensis were detected in samples of fermenting wort examined by DGGE during this stage. Bacterial growth was accompanied by the accumulation of acetic and lactic acids and the metabolism of residual maltooligosaccharides. By 70 h, two new PCR bands were detected on DGGE gels, and the associated bacteria were largely responsible for the final phase of the fermentation. The bacteria were phylogenetically related to Lactobacillus acidophilus and Lactobacillus delbrueckii, and strains similar to the former had previously been recovered from malt whisky fermentations in Japan. These were probably obligately homofermentative bacteria, required malt wort for growth, and could not be cultured on normal laboratory media, such as MRS. Their metabolism during the last 20 to 30 h of fermentation was associated with yeast death and autolysis and further accumulation of lactate but no additional acetate.

Characterization of lactobacilli from Scotch malt whisky distilleries and description of Lactobacillus ferintoshensis sp. nov., a new species isolated from malt whisky fermentations.

Sixty-four strains of Lactobacillus were isolated from fermentation samples from 23 malt whisky distilleries located in the major whisky producing regions of Scotland. The strains were assigned to 26 ribotype patterns. Strains of some ribotype patterns were widely distributed and recovered from distilleries throughout Scotland, while strains representing other ribotypes were particular to a specific region or even a certain distillery. Repeated sampling of a single distillery over a 12 month period showed that the range of bacteria present, as indicated by ribotyping, was stable, but was influenced by changes in malt supply and the period of closure for annual maintenance. Partial 16S rDNA sequence analysis of ribotype representatives revealed Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus paracasei and Lactobacillus pentosus as the major species present in the distilleries; however, four isolates could not be identified by this procedure. Determination of the full 16S rDNA gene sequence from one of these isolates (strain R7-84) revealed >98.5% similarity to Lactobacillus buchneri and its phylogenetic neighbours. DNA from two other strains showed greater than 70% hybridization to DNA from R7-84 under non-stringent renaturation conditions and DNA from strain R7-84 shared less than 65% hybridization with members of the L. buchneri group. It is proposed that these three strains should be placed in a new species for which the name Lactobacillus ferintoshensis represented by the type strain R7-84(T) is suggested.

Anaerobic metabolism in Bacillus licheniformis NCIB 6346.

SUMMARYThe products of anaerobic metabolism of glucose and its derivatives sorbitol, gluconate and glucuronate by Bacillus licheniformis have been determined by proton NMR. Glucose was fermented through mixed-acid fermentation pathways to acetate, 2,3-butanediol, ethanol, formate, lactate, succinate and pyruvate. However, the bacterium was incapable of fermenting the three glucose derivatives. When B. licheniformis cells were incubated anaerobically with glucose in the presence of nitrate, the reduced products and formate did not appear and acetate was formed as the major metabolite. Growth and formation of acetate was also observed when B. licheniformis cells were incubated anaerobically with each of the three glucose derivatives, in the presence of nitrate. A formate-nitrate oxido-reductase system was induced under anaerobic conditions, with increased activities when nitrate was added to the anaerobic growth medium. However no activity was detected when cell; were grown in the presence of molecular oxygen. Formate-nitrate oxido-reductase activity was absent in chlorate-resistant mutants isolated spontaneously or following Tn917 insertional mutagenesis. The spontaneous mutants fermented glucose in the presence of nitrate suggesting that they were incapable of nitrate respiration, due to a deficiency in one or more components of the formate-nitrate oxido-reductase system. Two insertional mutants exhibited elevated ß-galactosidase activity when grown in the presence of nitrate.