Liesbeth Lebbe

Virgibacillus : a new genus to accommodate Bacillus pantothenticus (Proom and Knight 1950). Emended description of Virgibacillus pantothenticus

Twelve strains named Bacillus pantothenticus, at least 29 Bacillus strains representing 16 species belonging to rRNA groups 1 and 2, one Bacillus dipsosauri strain, and 38 strains of Amphibacillus, Aneurinibacillus, Brevibacillus, Halobacillus, Paenibacillus, Sporosarcina and Marinococcus, were characterized genotypically using amplified rDNA restriction analysis (ARDRA), and phenotypically using routine diagnostic characters comprising 61 biochemical tests in the API System and 15 observations of vegetative cell and sporangial morphology. The B. pantothenticus strains were also characterized by fatty acid methyl ester analysis and SDS-PAGE of whole-cell proteins. ARDRA revealed that strains of B. pantothenticus formed a cluster quite separate from other species in rRNA group 1, supporting the recognition of the former as a separate genus, for which the name Virgibacillus is proposed. The polyphasic data also indicate the presence of an as yet undescribed new species within this genus. The species Virgibacillus pantothenticus and related organisms comprising this new genus can be distinguished from members of Bacillus rRNA group 1 (Bacillus sensu stricto), and from members of Paenibacillus and other aerobic endospore-forming bacteria by routine phenotypic tests.

Aerobic endospore-forming bacteria from geothermal environments in northern Victoria Land, Antarctica, and Candlemas island, south Sandwich archipelago, with the proposal of Bacillus fumarioli sp. nov.

Aerobic endospore-forming bacteria were isolated from soils taken from active fumaroles on Mount Rittmann and Mount Melbourne in northern Victoria Land, Antarctica, and from active and inactive fumaroles on Candlemas Island, South Sandwich archipelago. The Mt Rittmann and Mt Melbourne soils yielded a dominant, moderately thermophilic and acidophilic, aerobic endospore-former growing at pH 5.5 and 50 degrees C, and further strains of the same organism were isolated from a cold, dead fumarole at Clinker Gulch, Candlemas Island. Amplified rDNA restriction analysis, SDS-PAGE and routine phenotypic tests show that the Candlemas Island isolates are not distinguishable from the Mt Rittmann strains, although the two sites are 5600 km apart, and 16S rDNA sequence comparisons and DNA relatedness data support the proposal of a new species, Bacillus fumarioli, the type strain of which is LMG 17489T.

Acidovorax caeni sp. nov., a denitrifying species with genetically diverse isolates from activated sludge.

Four Gram-negative, rod-shaped, non-spore-forming, denitrifying isolates were obtained from the activated sludge of an aerobic-anaerobic wastewater treatment plant in Belgium. Analysis of repetitive sequence-based PCR showed that the four isolates were genetically different from each other. Results of 16S rRNA gene sequence analysis and DNA-DNA hybridization experiments indicated that these four isolates were affiliated to the genus Acidovorax and could be differentiated from all recognized species of the genus. Analysis of whole-cell proteins and results of physiological and biochemical tests allowed differentiation of the new isolates from their closest phylogenetic neighbours. These new isolates therefore represent a novel species of the genus Acidovorax, for which the name Acidovorax caeni sp. nov. is proposed. The type strain is R-24608(T) (=LMG 24103(T) =DSM 19327(T)).

Occurrence of Bacillus sporothermodurans and other aerobic spore-forming species in feed concentrate for dairy cattle.

Aims: To determine the aerobic spore composition and presence of Bacillus sporothermodurans spores in feed concentrate for dairy cattle.

Polyphasic characterization of Bacillus coagulans strains, illustrating heterogeneity within this species, and emended description of the species.

Because of its food spoiling capacity on the one hand and its significant role in the production of industrially valuable products on the other, Bacillus coagulans is of economic concern. Several studies have revealed a great deal of diversity within the species and this has led to a number of taxonomic adjustments. The present study aims to clarify the diversity within Bacillus coagulans sensu stricto and determine the taxonomic status of the species. Therefore, a polyphasic study was performed on a set of B. coagulans strains from diverse habitats. Techniques as ARDRA, SDS-PAGE of whole cell proteins, FAME analysis, routine phenotypic tests and rep-PCR illustrate considerable intra-species heterogeneity, while 16S rDNA sequence comparison and DNA-DNA relatedness support the accommodation of these strains in one species. Although most techniques demonstrate appreciable heterogeneity among the Bacillus coagulans strains, the intraspecies groupings are not consistent throughout all the methods applied and are not supported by any economic, historic or practical traits. Therefore, a division in subspecies seems inappropriate. In attempt to achieve a better species delineation, an emended description of Bacillus coagulans is included.

Polyphasic identification of Bacillus and Brevibacillus strains from clinical, dairy and industrial specimens and proposal of Brevibacillus invocatus sp nov.

Thirty-three clinical, dairy and industrial isolates of aerobic endospore-forming bacteria which were unreactive in routine identification tests were characterized genotypically by using amplified rDNA restriction analysis (ARDRA), 16S rDNA sequencing and DNA-DNA reassociation, and phenotypically by using fatty acid methyl ester (FAME) analysis, SDS-PAGE of whole-cell proteins, API Biotype 100 assimilation tests and 16 other routine phenotypic tests. Three isolates were identified as strains of Bacillus badius, 12 as Brevibacillus agri, including 3 strains associated with an outbreak of waterborne illness, 4 as Brevibacillus centrosporus and 2 as Brevibacillus parabrevis; 12 strains contaminating an antibiotic production plant were recognized as members of a new species, for which the name Brevibacillus invocatus is proposed, with the type strain LMG 18962T (= B2156T = CIP 106911T = NCIMB 13772T).

Proposal of Virgibacillus proomii sp. nov. and emended description of Virgibacillus pantothenticus (Proom and Knight 1950) Heyndrickx et al. 1998

A polyphasic study of strains originally received as Bacillus (now Virgibacillus) pantothenticus, along with strains representing species belonging to Bacillus, Halobacillus and Paenibacillus, was undertaken using amplified rDNA restriction analysis (ARDRA), fatty acid methyl ester (FAME) analysis, SDS-PAGE of whole-cell proteins and routine diagnostic characters comprising 61 biochemical tests in the API system and 15 observations of vegetative cell and sporangial morphology. It revealed the presence within Virgibacillus of an as yet undescribed new species, for which the name Virgibacillus proomii is proposed; V. proomii can be distinguished from V. pantothenticus and members of Bacillus sensu stricto, and from members of Paenibacillus and other aerobic endospore-forming bacteria, by routine phenotypic tests. The type strain of Virgibacillus proomii is LMG 12370T.

A Polyphasic Reassessment of the Genus Aneurinibacillus, Reclassification of Bacillus thermoaerophilus (Meier-Stauffer et al. 1996) as Aneurinibacillus thermoaerophilus comb. nov., and Emended Descriptions of A. aneurinilyticus corrig., A. migulanus, and A. thermoaerophilus

Fifty-three strains representing 25 species of aerobic endospore-forming bacteria, of which 11 (37 strains) belong to the genera Aneurinibacillus and Brevibacillus (Bacillus rRNA group 4 of Ash et al. [Lett. Appl. Microbiol. 13:202-206, 1991]), were characterized genotypically by amplified ribosomal DNA restriction analysis (ARDRA) and/or phenotypically by fatty acid methyl ester analysis, sodium dodecylsulfate-polyacrylamide gel electrophoresis of whole-cell proteins, pyrolysis mass spectrometry, 99 API Biotype 100 assimilation tests, and 16 other routine phenotypic tests. ARDRA revealed that Aneurinibacillus aneurinilyticus, Aneurinibacillus migulunus, and Bacillus thermoaerophilus formed a cluster quite separate from Brevibacillus species, supporting the distinction of both genera and the transfer of B. thermoaerophilus to the genus Aneurinibacillus. Two of the species, A. aneurinilyticus (the type species) and A. migulanus, are phenotypically and genotypically quite similar but can be distinguished from each other by several phenotypic characters. Phenotypic differentiation at the generic level is also described.

Diversity of activated sludge bacteria receiving the 3-chloroaniline-degradative plasmid pC1gfp

Plasmid pC1 of Delftia acidovorans CA28, encoding the oxidative deamination of 3-chloroaniline (3-CA), was tagged with a mini-Tn5 transposon containing the gfp gene. The labelled plasmid, designated pC1gfp, was subsequently transferred to Pseudomonas putida UWC3 and the plasmid transfer from this donor to the bacterial community in activated sludge was studied. Mating experiments were performed on both Luria-Bertani agar plates and directly in liquid activated sludge. Green fluorescent colonies appearing on mineral medium containing 3-CA as sole nitrogen source were picked up and verified to be true pC1gfp-harbouring transconjugants. REP- and BOX-PCR genomic fingerprinting revealed a large diversity in the transconjugant collection, indicating the occurrence of multiple plasmid transfer events. Remarkably, Luria-Bertani agar plate matings yielded a different set of transconjugants compared to plasmid transfers directly in liquid activated sludge. From the plate matings, mainly Aeromonas sp. was isolated, whereas D. acidovorans strains were predominant in the transconjugant collection from liquid activated sludge. The majority of the isolates showed 3-CA deamination, probably to chlorocatechol, but several pC1gfp-harbouring transconjugants performed a rapid and complete 3-CA degradation.

High diversity of Bradyrhizobium strains isolated from several legume species and land uses in Brazilian tropical ecosystems

The genus Bradyrhizobium stands out among nitrogen-fixing legume-nodulating bacteria because it predominates among the efficient microsymbionts of forest, forage, and green manure legume species, as well as important species of grain legumes, such as soybean, cowpea, and peanut. Therefore, the diversity of Bradyrhizobium strains is a relevant resource from environmental and economic perspectives, and strains isolated from diverse legume species and land uses in Brazilian tropical ecosystems were assessed in this study. To accomplish this, sequences of four housekeeping genes (atpD, dnaK, gyrB, and recA) were individually analysed, with the first three also being considered using multilocus sequence analysis (MLSA). The sensitivity of the strains to different antibiotics, their tolerance to different levels of salinity, and their ability to nodulate soybean plants were also measured. The phylogenetic trees based on each individual gene, and on the concatenated housekeeping genes, revealed several strain clusters separated from any currently described species. The Bradyrhizobium strains studied were generally resistant to antibiotics. All strains were able to grow at salinity levels of up to 0.5% NaCl, whereas only strains UFLA03-142, UFLA03-143, UFLA03-145, and UFLA03-146 grew in the presence of 1% NaCl. Together, the results indicated that some of the strains studied were potential novel species, indicating that the various soils and ecosystems in Brazil may harbour an as yet unknown diversity of rhizobia.