Fergus G. Priest

Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test

Comparative 16S rRNA sequence analysis has demonstrated that the genusBacillus consists of at least five phyletic lines. rRNA group 3 bacilli of Ash, Farrow, Wallbanks and Collins (1991) comprisingBacillus polymyxa and close relatives is phylogenetically so removed fromBacillus subtilis, the type species of the genus and other aerobic, endospore-forming bacilli that they warrant reclassification in a new genusPaenibacillus. The genusPaenibacillus can be readily distinguished from otherBacillus groups using a battery of phenotypic characters and a highly specific gene probe based on 16S rRNA.

Phenetic diversity of alkaliphilic Bacillus strains : proposal for nine new species

Summary: One hundred and nineteen strains of alkaliphilic and alkalitolerant, aerobic endospore-forming bacteria were examined for 47 physiological and biochemical characters, and DNA base composition. Numerical analysis (S J and S SM/UPGMA clustering) revealed 11 clusters that comprised three or more strains. Most of the phena were further characterized by analysis of carbohydrate utilization profiles using the API 50CH system, but strains of two taxa could not be cultured by this method. DNA reassociation studies showed that nine of the phena were homogeneous, but strains of phenon 4 and phenon 8 were each subdivided into two DNA hydridization groups. The strains could therefore be classified into 13 taxa plus a number of unassigned single-membered clusters. Two taxa were equated with Bacillus cohnii and B. alcalophilus and nine of the remainder are proposed as new species with the following names: B. agaradhaerens sp. nov., B. clarkii sp. nov., B. clausii sp. nov., B. gibsonii sp. nov., B. halmapalus sp. nov., B. halodurans comb, nov., B. horikoshii sp. nov., B. pseudalcalophilus sp. nov. and B. pseudofirmus sp. nov. Two taxa were insufficiently distinct to allow confident identification and these have therefore not been proposed as new species.

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.

Bacillus sporothermodurans, a New Species Producing Highly Heat-Resistant Endospores

Bacteria that differentiate into highly heat-resistant endospores (HHRS strains) may survive ultrahigh-temperature treatment of milk and germinate in the final product. They do not noticeably spoil the milk and are nonpathogenic. The complete (>96%) 16S rRNA genes from three HHRS strains were identical, and phylogenetic analysis placed them alongside Bacillus firmus in the B. megaterium group of the genus Bacillus. Moreover, the approximately 550 nucleotides between regions U2 and U5 were invariant for seven HHRS strains. However, three cloned 16S rRNA genes from one HHRS strain, M215, showed marked size and sequence variations within the V1 and V2 regions. DNA reassociation assays confirmed the distinction between a reference HHRS strain and closely related members of the B. megaterium group, notably, B. firmus (30%), B. benzoevorans (28%), and B. circulans (20%). Ribotyping and pyrolysis mass spectrometry both indicated that the HHRS strains belong to a homogeneous, species-ranked taxon, an exception being strain TP1248, which is slightly atypical. The HHRS strains are unusual in that they grow poorly, if at all, on nutrient agar; good growth is obtained on brain heart infusion agar. On subculture, most HHRS strains form long, filamentous rods which stain unevenly in the Gram reaction. They are strictly aerobic and do not produce acid from sugars. We propose the name Bacillus sporothermodurans for these bacteria, which are phenotypically and phylogenetically distinct from other Bacillus species. The type strain is M215 (= DSMZ 10599).

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.

Decarboxylation of Substituted Cinnamic Acids by Lactic Acid Bacteria Isolated during Malt Whisky Fermentation

ABSTRACT Seven strains of Lactobacillus isolated from malt whisky fermentations and representing Lactobacillus brevis,L. crispatus, L. fermentum, L. hilgardii, L. paracasei, L. pentosus, andL. plantarum contained genes for hydroxycinnamic acid (p-coumaric acid) decarboxylase. With the exception ofL. hilgardii, these bacteria decarboxylatedp-coumaric acid and/or ferulic acid, with the production of 4-vinylphenol and/or 4-vinylguaiacol, respectively, although the relative activities on the two substrates varied between strains. The addition of p-coumaric acid or ferulic acid to cultures ofL. pentosus in MRS broth induced hydroxycinnamic acid decarboxylase mRNA within 5 min, and the gene was also induced by the indigenous components of malt wort. In a simulated distillery fermentation, a mixed culture of L. crispatus and L. pentosus in the presence of Saccharomyces cerevisiae decarboxylated added p-coumaric acid more rapidly than the yeast alone but had little activity on added ferulic acid. Moreover, we were able to demonstrate the induction of hydroxycinnamic acid decarboxylase mRNA under these conditions. However, in fermentations with no additional hydroxycinnamic acid, the bacteria lowered the final concentration of 4-vinylphenol in the fermented wort compared to the level seen in a pure-yeast fermentation. It seems likely that the combined activities of bacteria and yeast decarboxylate p-coumaric acid and then reduce 4-vinylphenol to 4-ethylphenol more effectively than either microorganism alone in pure cultures. Although we have shown that lactobacilli participate in the metabolism of phenolic compounds during malt whisky fermentations, the net result is a reduction in the concentrations of 4-vinylphenol and 4-vinylguaiacol prior to distillation.

Evolution of the Lactic Acid Bacterial Community during Malt Whisky Fermentation: a Polyphasic Study

ABSTRACT 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 Streptococcusthermophilus or Saccharococcusthermophilus, Lactobacillusbrevis, and Lactobacillusfermentum. The middle phase began 35 to 40 h after yeast inoculation and was characterized by exponential growth of lactobacilli and residual yeast metabolism. Lactobacilluscasei or Lactobacillusparacasei, L. fermentum, and Lactobacillusferintoshensis 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 Lactobacillusacidophilus and Lactobacillusdelbrueckii, 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.

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.

Characterization of Bacillus thuringiensis and related bacteria by ribosomal RNA gene restriction fragment length polymorphisms

Ribosomal RNA gene restriction patterns have been determined for 43 strains of Bacillus thuringiensis representing 10 serovars and eight reference strains of B. anthracis, B. cereus and B. mycoides. Strains within a B. thuringiensis serovar produced highly related or identical ribotype patterns: in particular, 12 strains of serovar israelensis, five strains of serovar kurstaki, two strains of serovar galleriae and three strains of serovar aizawa produced ribotype patterns consistent with serotype designations. Moreover, variety tenebrionis (serotype 8a8b), a coleopteran pathogen, could be distinguished from the more common lepidopteran pathogens of this serotype (serovar morrisoni) by ribotyping. The correlation of ribotype patterns with serotype suggests a clonal population structure for B. thuringiensis.

On the industrial use of Bacillus licheniformis: a review

We document here that in those rare cases where disease has been related to Bacillus licheniformis, infection was associated with bypassing the normal biological protective barriers or severely debililated patients. No case suggests any invasive properties of this bacterium. B. licheniformis can therefore be considered non-pathogenic to humans in general. Food-borne illness caused by possible B. licheniformis toxins have been reported, but only in a very few cases and only in connection with consumption of inappropriately prepared food. Considerable experience concerning the industrial use of recombinant B. licheniformis strains has now accumulated and authorities in the United States, Europe and Japan have approved production with and products from recombinant B. licheniformis strains. We conclude that B. licheniformis is a safe host for the production of harmless, industrial products.