Massilia umbonata sp. nov., able to accumulate poly-ß-hydroxybutyrate, isolated from a sewage sludge compost-soil microcosm.

A bacterial strain, designated strain LP01(T), was isolated from a laboratory-scale microcosm packed with a mixture of soil and sewage sludge compost designed to study the evolution of microbial biodiversity over time. The bacterial strain was selected for its potential ability to store polyhydroxyalkanoates (PHAs) as intracellular granules. The cells were aerobic, Gram-stain-negative, non-endospore-forming motile rods. Phylogenetically, the strain was classified within the genus Massilia, as its 16S rRNA gene sequence had similarity of 99.2 % with respect to those of Massilia albidiflava DSM 17472(T) and M. lutea DSM 17473(T). DNA-DNA hybridization showed low relatedness of strain LP01(T) to the type strains of other, phylogenetically related species of the genus Massilia. It contained Q-8 as the predominant ubiquinone and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) as the major fatty acid(s). It was found to contain small amounts of the fatty acids C18 : 0 and C14 : 0 2-OH, a feature that served to distinguish it from its closest phylogenetic relatives within the genus Massilia. The DNA G+C content was 66.0 mol%. Phylogenetic, phenotypic and chemotaxonomic data obtained in this study suggest that strain LP01(T) represents a novel species of the genus Massilia, for which the name Massilia umbonata sp. nov. is proposed. The type strain is LP01(T) ( = CECT 7753(T) = DSM 26121(T)).

Emended descriptions of Bacillus sporothermodurans and Bacillus oleronius with the inclusion of dairy farm isolates of both species.

Bacillus sporothermodurans is an industrially important micro-organism because of its ability to produce endospores which resist ultra-high temperature (UHT) and industrial sterilization processes. It was described by Pettersson et al. (1996) [Pettersson, B., Lembke, F., Hammer, P., Stackebrandt, E. & Priest, F. G. (1996). Int J Syst Bacteriol 46, 759-764] based on seven genetically homogeneous isolates all from UHT milk. Bacillus oleronius, the closest phylogenetic neighbour of B. sporothermodurans, was described by Kuhnigk et al. (1995) [Kuhnigk, T., Borst, E.-M., Breunig, A., König, H., Collins, M. D., Hutson, R. A. & Kämpfer, P. (1995). Can J Microbiol 41, 699-706] based on a single strain, isolated from the hindgut of the termite Reticulitermes santonensis. A polyphasic study of a heterogeneous collection of B. sporothermodurans and B. oleronius strains isolated from various sources and geographical origins led to an emended description of both species. Additional data presented are the results of fatty acid, quinone and/or cell wall (polar lipid) analyses. DNA-DNA hybridization confirmed 3 subgroups of strains obtained after SDS-PAGE analysis of cellular proteins as B. sporothermodurans. One named B. sporothermodurans strain (R-7489) was reclassified as a Bacillus fordii strain. The phenotypic profiles of both species were rather heterogeneous, sometimes different from the original descriptions and did not differ in a large number of characteristics, although B. oleronius generally gave stronger reactions in its positive tests than did B. sporothermodurans; the variable and weak reactions for both organisms with some substrates blurred the distinction between the two. However, differences in polar lipid, SDS-PAGE and menaquinone profiles clearly allow distinction between the two species.

Selection and identification of bacterial strains with methyl-tert-butyl ether, ethyl-tert-butyl ether, and tert-amyl methyl ether degrading capacities.

Nine bacterial strains isolated from two hydrocarbon-contaminated soils were selected because of their capacity for growth in culture media amended with 200 mg/L of one of the following gasoline oxygenates: Methyl-tert-butyl ether (MTBE), ethyl-tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). These strains were identified by amplification of their 16S rRNA gene, using fDl and rD1 primers, and were tested for their capacity to grow and biotransform these oxygenates in both mineral and cometabolic media. The isolates were classified as Bacillus simplex, Bacillus drentensis, Arthrobacter sp., Acinetobacter calcoaceticus, Acinetobacter sp., Gordonia amicalis (two strains), Nocardioides sp., and Rhodococcus ruber. Arthrobacter sp. (strain MG) and A. calcoaceticus (strain M10) consumed 100 (cometabolic medium) and 82 mg/L (mineral medium) of oxygenate TAME in 21 d, respectively, under aerobic conditions. Rhodococcus ruber (strain E10) was observed to use MTBE and ETBE as the sole carbon and energy source, whereas G. amicalis (strain T3) used TAME as the sole carbon and energy source for growth. All the bacterial strains transformed oxygenates better in the presence of an alternative carbon source (ethanol) with the exception of A. calcoaceticus (strain M10). The capacity of the selected strains to remove MTBE, ETBE, and TAME looks promising for application in bioremediation technologies.

Bacillus ruris sp. nov., from dairy farms.

Four novel ellipsoidal spore-forming Bacillus isolates with swollen sporangia, isolated from raw milk and feed concentrate, showed a high level of similarity in SDS-PAGE, fatty acid methyl esters and routine phenotypic tests. However, 16S rRNA gene sequence comparisons showed that this taxon was different from other related Bacillus species, and only a low level of DNA relatedness was found with the closest phylogenetic and phenotypic relative, Bacillus galactosidilyticus. This taxon could be differentiated from B. galactosidilyticus on the basis of morphological differences, stronger acid reactions with a wide range of substrates after 48 h incubation, and qualitative and quantitative differences in fatty acid content. On the basis of these data, a novel species, Bacillus ruris sp. nov., is proposed, with LMG 22866T (=DSM 17057T) as the type strain.

Paenibacillus wynnii sp. nov., a novel species harbouring the nifH gene, isolated from Alexander Island, Antarctica.

Soil taken from 12 different locations at Mars Oasis on Alexander Island, Antarctica, yielded unidentified isolates of endospore-forming bacteria. Soil from four of the locations contained Gram-negative, facultatively anaerobic, motile rods that were able to grow at 4 degrees C and which formed ellipsoidal spores that lay paracentrally or subterminally in swollen or slightly swollen sporangia. All of the strains harboured the nitrogenase gene nifH. Phenotypic tests, amplified rDNA restriction analysis (ARDRA), fatty acid analysis and SDS-PAGE analysis suggested that the isolates represented a novel taxon of Paenibacillus. 16S rRNA gene sequence comparison supported the proposal of a novel species, Paenibacillus wynnii sp. nov. (type strain, LMG 22176(T)=CIP 108306(T)).

Bacillus arenosi sp. nov., Bacillus arvi sp. nov. and Bacillus humi sp. nov., isolated from soil.

A group of nine Gram-positive endospore-forming bacteria was isolated from soil of the Drentse A agricultural research area in The Netherlands. Using (GTG)5-PCR genomic fingerprinting and fatty acid analysis, the nine isolates were divided into three consistent groups. On the basis of 16S rRNA gene sequence similarity of representative strains, the nine isolates were shown to belong to the genus Bacillus. The first group of four isolates was most closely related to Bacillus carboniphilus (95.5 %) and Bacillus sporothermodurans (95.5 %). The second and third groups of three and two isolates, respectively, showed highest sequence similarity to Bacillus neidei (97.0 and 97.1 %, respectively) and Bacillus pycnus (both 96.7 %). A DNA-DNA relatedness study confirmed the consistency of the three groups delineated by (GTG)5-PCR and fatty acid analysis. A small number of phenotypic characters allowed differentiation of the three groups of isolates. The three groups therefore represent novel species, for which the names Bacillus humi, Bacillus arenosi and Bacillus arvi are proposed, with LMG 22167T (=DSM 16318T), LMG 22166T (=DSM 16319T) and LMG 22165T (=DSM 16317T) as the respective type strains.

Study of mural painting isolates, leading to the transfer of 'Bacillus maroccanus' and 'Bacillus carotarum' to Bacillus simplex, emended description of Bacillus simplex, re-examination of the strains previously attributed to 'Bacillus macroides' and description of Bacillus muralis sp. nov.

A group of 24 strains was isolated from deteriorated mural paintings situated in Spain (necropolis of Carmona) and Germany (church of Greene-Kreiensen). (GTG)5-PCR genomic fingerprinting was performed on these strains to assess their genomic variability and the strains were delineated into four groups. Representatives were studied by 16S rRNA gene sequencing and were found to be closely related to Bacillus simplex and the species 'Bacillus macroides' (strain NCIMB 8796) and 'Bacillus maroccanus' (names not validly published) according to a fasta search. The close similarity between B. simplex, 'B. macroides' NCIMB 8796, 'B. maroccanus' and the mural painting isolates was confirmed by additional (GTG)5-PCR, ARDRA, FAME and SDS-PAGE analyses. Furthermore, these techniques revealed that strains of 'Bacillus carotarum', another name that has not been validly published, also showed high similarity to this group of organisms. On the other hand, it was shown that the strains labelled 'B. macroides' in different collections do not all belong to the same species. Strain NCIMB 8796 can be allocated to B. simplex, while strain DSM 54 (=ATCC 12905) shares the highest 16S rRNA gene sequence similarity with Bacillus sphaericus and Bacillus fusiformis (both around 98.6 %). On the basis of further DNA-DNA hybridization data and the study of phenotypic characteristics, one group of five mural painting strains was attributed to a novel species in the genus Bacillus, for which the name Bacillus muralis sp. nov. is proposed. Finally, the remaining mural painting strains, one (LMG 18508=NCIMB 8796) of two strains belonging to 'B. macroides' and strains belonging to 'B. maroccanus' and 'B. carotarum' are allocated to the species B. simplex and an emended description of B. simplex is given.

Paenibacillus cineris sp. nov. and Paenibacillus cookii sp. nov., from Antarctic volcanic soils and a gelatin-processing plant.

Seven strains of aerobic, endospore-forming bacteria were found in soil taken from an active fumarole on Lucifer Hill, Candlemas Island, South Sandwich archipelago, Antarctica, and four strains were from soil of an inactive fumarole at the foot of the hill. Amplified rDNA restriction analysis, 16S rDNA sequence comparisons, SDS-PAGE and routine phenotypic tests support the proposal of two novel species of Paenibacillus, Paenibacillus cineris sp. nov. and Paenibacillus cookii sp. nov., the type strains of which are LMG 18439T (=CIP 108109T) and LMG 18419T (=CIP 108110T), respectively. A further strain, isolated from a gelatin-production process, showed more than 99% 16S rDNA sequence similarity to the proposed P. cookii type strain and, although the gelatin isolate was atypical when compared with the fumarole isolates by repeated element primed-PCR, SDS-PAGE and phenotypic analyses, it was shown by DNA-DNA reassociation studies to belong to the same species. Strains of P. cookii produce spreading growth with motile microcolonies. Both species produce swollen sporangia that are typical for the genus, they both show 97.6% 16S rDNA sequence similarity to Paenibacillus azoreducens, they have 51.5-51.6 mol% G+C in their DNA and their major fatty acid is anteiso-C(15 : 0); however, fatty acids C(16 : 0) and anteiso-C(17 : 0) represent, respectively, 18 and 10 % of the total in P. cineris, but 11 and 20% in P. cookii.

Bacillus farraginis sp. nov., Bacillus fortis sp. nov. and Bacillus fordii sp. nov., isolated at dairy farms.

Forty-eight bacterial strains were isolated at dairy farms from raw milk, the milking apparatus, green fodder or feed concentrate after a heat treatment of 30 min at 100 degrees C. In this way, spore-forming bacteria with a very high intrinsic heat resistance were selected for. The aerobic spore-forming isolates were subjected to a polyphasic taxonomical study, including repetitive element sequence-based PCR typing, whole-cell protein profiling, 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base composition, fatty acid analysis, and morphological and biochemical characteristics. A comparison of the REP- and (GTG)5-PCR and whole-cell protein SDS-PAGE profiles resulted in three clusters of similar strains. Analysis of the 16S rDNA sequences and DNA-DNA relatedness data showed that these clusters represented three novel species. The highest 16S rDNA similarity to a recognized species found for the three groups was around 94% with Bacillus lentus and Bacillus sporothermodurans. Further phenotypic characterization supported the proposal of three novel species in the genus Bacillus, Bacillus farraginis, Bacillus fortis and Bacillus fordii. The respective type strains are R-6540T (=LMG 22081T=DSM 16013T), R-6514T (=LMG 22079T=DSM 16012T) and R-7190T (=LMG 22080T=DSM 16014T); their G+C DNA base contents are 43.7, 44.3 and 41.9 mol%, respectively. Although in variable amounts, a predominance of the branched fatty acids iso-C(15 : 0) and anteiso-C(15 : 0) was observed in all three novel species.

Genomic and phenotypic comparison of Bacillus fumarioli isolates from geothermal Antarctic soil and gelatine.

Bacillus fumarioli was originally isolated from geothermal soils in continental and maritime Antarctica, and recently, it has been shown to be a frequent contaminant of gelatine extracts obtained from European and American production plants. These habitats are geographically widely separated, share similar temperature and pH conditions, but have substantially different organic loads. Because of the prevalence in gelatine extracts and the dissimilarity of this habitat to geothermal soil, a comparative study was performed to assess the diversity among B. fumarioli strains and reveal possible intraspecies differences that might correspond to their niches of origin. Genomic (rep-PCR, 16S rDNA sequencing, DNA-DNA hybridisations) and phenotypic techniques (analysis of fatty acid content, total cellular proteins, metabolic and morphological traits) illustrate the very close relationship between isolates from the two niches. An abundant protein band was demonstrated for gelatine isolates only. This band was shown to result from a protein with high similarity to a stress response protein. Furthermore, subtractive hybridisation revealed genomic differences between Antarctic and gelatine isolates that may indicate adaptive evolution to a specific environment.

Paenibacillus lactis sp. nov., isolated from raw and heat-treated milk.

Endospore-forming bacteria were recovered from individual packages from different processing lines in a dairy plant during a tenacious periodical contamination of their UHT-milk production. Two colony types were seen, one of which was identified as Bacillus sporothermodurans. Analysis of the 16S rRNA gene of the second colony type placed these isolates within the genus Paenibacillus, with Paenibacillus lautus as the closest known relative. Moreover, over 99 % similarity was observed to the 16S rDNA sequence of MB 2035, a strain isolated previously from raw milk during a survey at dairy farms for very heat-resistant spore-forming bacteria. Nine other potentially closely related strains among the dairy farm isolates were found using rep-PCR typing. The taxonomic positions of these 19 isolates were further investigated using 16S rRNA gene sequencing and DNA-DNA hybridizations of representative strains. All 19 isolates shared a high degree of phenotypic similarity and were easily distinguished from closely related members of the genus. Anteiso-C(15 : 0), C(16 : 0) and iso-C(15 : 0) were among the major fatty acids and the genomic DNA G+C content was 51.6-51.7 mol%. Therefore, based on their phenotypic, phylogenetic and genomic distinctiveness, these 19 strains, isolated from both raw and heat-treated milk, are placed in the genus Paenibacillus as Paenibacillus lactis sp. nov. The type strain is MB 1871(T) (=LMG 21940(T)=DSM 15596(T)).

Anoxybacillus contaminans sp. nov. and Bacillus gelatini sp. nov., isolated from contaminated gelatin batches.

Aerobic, endospore-forming bacteria that are attributed to the genus Bacillus or related genera constitute a hazard to the quality of gelatin. During repetitive extragenic palindromic DNA (rep)-PCR screening of gelatin isolates, a group of five isolates (group 1) and a group of 66 isolates (group 2) that did not match any pattern in our database were found. On the basis of 16S rDNA sequence analysis, representative strains of the different rep-PCR fingerprint types of group 1 were shown to be related most closely to Anoxybacillus species, but with sequence similarity of <97 %. Likewise, representative strains of group 2 were shown to be related most closely to Bacillus species, with 16S rDNA sequence similarity of <97 %. DNA-DNA reassociation values of isolates that displayed the most divergent rep-PCR profiles revealed that strains within each group belonged to a single species, according to recommendations for species delineation. A mean fatty acid profile could be calculated for each group. Isolates within a single group had similar patterns of results in API and other phenotypic tests; no correlation of patterns of results with rep-PCR groups was seen. Physiological characterization of group 1 isolates allows their distinction from other Anoxybacillus species. Despite the weak reaction of group 2 isolates in API tests, physiological characterization allows distinction between Bacillus species that react weakly in API tests. Two novel species are therefore proposed, with the names Anoxybacillus contaminans sp. nov. (type strain, LMG 21881(T)=DSM 15866(T)) and Bacillus gelatini sp. nov. (type strain, LMG 21880(T)=DSM 15865(T)).

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.

Bacillus shackletonii sp. nov., from volcanic soil on Candlemas Island, South Sandwich archipelago.

A sample of mossy soil taken from the eastern lava flow of northern Candlemas Island, South Sandwich archipelago, yielded six isolates of aerobic, endospore-forming bacteria. Miniaturized routine phenotypic tests and other observations, amplified rDNA restriction analysis and SDS-PAGE analysis suggested that the strains represent a novel taxon. 16S rDNA sequence comparisons support the proposal of a novel species, Bacillus shackletonii sp. nov., the type strain of which is LMG 18435(T) (=CIP 107762(T)).

Bacillus galactosidilyticus sp. nov., an alkali-tolerant beta-galactosidase producer.

A novel Bacillus isolate from raw milk and four strains from diverse origins that were identified previously as Bacillus lentus, Bacillus firmus and Bacillus circulans showed a high degree of similarity in amplified rDNA restriction analysis, SDS-PAGE and routine phenotypic tests, whilst 16S rDNA sequence comparisons and DNA relatedness data showed that this taxon was different from related Bacillus species. On the basis of these data, Bacillus galactosidilyticus sp. nov. is proposed, with the type strain LMG 17892(T) (=DSM 15595(T)=Logan B2188(T)=MB 800(T)).

Bacillus novalis sp. nov., Bacillus vireti sp. nov., Bacillus soli sp. nov., Bacillus bataviensis sp. nov. and Bacillus drentensis sp. nov., from the Drentse A grasslands.

A group of 42 isolates were isolated from the soil of several disused hay fields, in the Drentse A agricultural research area (The Netherlands), that were taken out of production at different times. The group represents hitherto-uncultured Bacillus lineages that have previously been found, by a non-cultural method, to be predominant in soil. The strains were subjected to a polyphasic taxonomic study, including (GTG)5-PCR, 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base-ratio determination, fatty acid analysis and morphological and biochemical characterization. By comparing the groupings obtained by (GTG)5-PCR and 16S rDNA sequence analysis, six clusters of similar strains could be recognized. A DNA-DNA relatedness study showed that these clusters represented five novel genospecies. Further analysis supported the proposal of five novel species in the genus Bacillus, namely Bacillus novalis sp. nov. (type strain IDA3307T=R-15439T=LMG 21837T=DSM 15603T), Bacillus vireti sp. nov. (type strain IDA3632T=R-15447T=LMG 21834T=DSM 15602T), Bacillus soli sp. nov. (type strain IDA0086T=R-16300T=LMG 21838T=DSM 15604T), Bacillus bataviensis sp. nov. (type strain IDA1115T=R-16315T=LMG 21833T=DSM 15601T) and Bacillus drentensis sp. nov. (type strain IDA1967T=R-16337T=LMG 21831T=DSM 15600T).

Bacillus decolorationis sp. nov., isolated from biodeteriorated parts of the mural paintings at the Servilia tomb (Roman necropolis of Carmona, Spain) and the Saint-Catherine chapel (Castle Herberstein, Austria).

Microbial growths causing discoloration on the Roman wall paintings of the Servilia tomb at the necropolis of Carmona (Spain) and the medieval wall paintings of the Saint-Catherine chapel at Castle Herberstein (Austria) were investigated and from four different samples, a group of ten strains with similar characteristics was isolated. The isolates were characterized in a polyphasic taxonomic study, including 16S rDNA sequence analysis, (GTG)5-PCR genomic fingerprinting, DNA-DNA hybridization, DNA base ratio, fatty acid analysis, morphological and biochemical characterization. The data obtained attribute the isolates to a novel species of the genus Bacillus, for which the name Bacillus decolorationis sp. nov. is proposed. The type strain is strain LMG 19507T (=DSM 14890T).

Virgibacillus carmonensis sp. nov., Virgibacillus necropolis sp. nov. and Virgibacillus picturae sp. nov., three novel species isolated from deteriorated mural paintings, transfer of the species of the genus salibacillus to Virgibacillus, as Virgibacillus marismortui comb. nov. and Virgibacillus salexigens comb. nov., and emended description of the genus Virgibacillus.

A group of 13 strains was isolated from samples of biofilm formation on the mural paintings of the Servilia tomb (necropolis of Carmona, Spain) and the Saint-Catherine chapel (castle at Herberstein, Austria). The strains were subjected to a polyphasic taxonomic study, including (GTG)5-PCR, 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base ratio determination, analysis of fatty acids, polar lipids and menaquinones and morphological and biochemical characterization. In a phylogenetic tree based on neighbour-joining of 16S rDNA sequences, the strains are divided in two major groups, representing three novel species according to DNA-DNA relatedness, that are positioned at approximately equal distances from Virgibacillus and Salibacillus. After comparison of the novel results with existing data, the transfer of the species of Salibacillus to Virgibacillus is proposed, with the resulting new combinations Virgibacillus marismortui comb. nov. and Virgibacillus salexigens comb. nov. Additionally, three novel species are described, for which the names Virgibacillus carmonensis sp. nov., Virgibacillus necropolis sp. nov. and Virgibacillus picturae sp. nov. are proposed. The respective type strains are LMG 20964T (=DSM 14868T), LMG 19488T (=DSM 14866T) and LMG 19492T (= DSM 14867T). Finally, an emended description of the genus Virgibacillus is given.

Bacillus luciferensis sp. nov., from volcanic soil on Candlemas Island, South Sandwich archipelago.

Aerobic, endospore-forming bacteria were found in soil taken from an active fumarole on Lucifer Hill, Candlemas Island, South Sandwich archipelago. Amplified rDNA restriction analysis, SDS-PAGE, repetitive element primed-PCR (rep-PCR) and routine phenotypic tests suggested that six of the isolates represent a novel taxon, and 16S rDNA sequence comparisons support the proposal of a novel species, Bacillus luciferensis sp. nov., the type strain of which is strain LMG 18422(T) (= CIP 107105(T)).