Taxonomic revision of the genus Geobacillus: emendation of Geobacillus, G. stearothermophilus, G. jurassicus, G. toebii, G. thermodenitrificans and G. thermoglucosidans (nom. corrig., formerly 'thermoglucosidasius'); transfer of Bacillus thermantarcticus to the genus as G. thermantarcticus comb. nov.; proposal of Caldibacillus debilis gen. nov., comb. nov.; transfer of G. tepidamans to Anoxybacillus as A. tepidamans comb. nov.; and proposal of Anoxybacillus caldiproteolyticus sp. nov.

Sixty-two strains of thermophilic aerobic endospore-forming bacteria were subjected to polyphasic taxonomic study including 16S rRNA gene sequence analysis, polar lipid and fatty acid analysis, phenotypic characterization, and DNA-DNA hybridization experiments. Distinct clusters of the species Geobacillus stearothermophilus, Geobacillus thermodenitrificans, Geobacillus toebii and Geobacillus thermoglucosidasius were formed, allowing their descriptions to be emended, and the distinctiveness of the poorly represented species Geobacillus jurassicus, Geobacillus subterraneus and Geobacillus caldoxylosilyticus was confirmed. It is proposed that the name Geobacillus thermoglucosidasius be corrected to Geobacillus thermoglucosidans nom. corrig. Bacillus thermantarcticus clustered between Geobacillus species on the basis of 16S rRNA gene sequence analysis, and its transfer to the genus Geobacillus as Geobacillus thermantarcticus comb. nov. (type strain LMG 23032(T)=DSM 9572(T)=strain M1(T)=R-35644(T)) is proposed. The above-mentioned species, together with Geobacillus thermoleovorans and Geobacillus thermocatenulatus, form a monophyletic cluster representing the genus Geobacillus. The distinctiveness of 'Geobacillus caldoproteolyticus' was confirmed and it is proposed that it be accommodated, along with Geobacillus tepidamans, in the genus Anoxybacillus as Anoxybacillus caldiproteolyticus sp. nov. (type strain DSM 15730(T)=ATCC BAA-818(T)=LMG 26209(T)=R-35652(T)) and Anoxybacillus tepidamans comb. nov. (type strain LMG 26208(T)=ATCC BAA-942(T)=DSM 16325(T)=R-35643(T)), respectively. The type strain of Geobacillus debilis was not closely related to any members of the genera Anoxybacillus and Geobacillus, and it is proposed that this species be placed in the new genus Caldibacillus as Caldibacillus debilis gen. nov. comb. nov. The type strain of the type species, Caldibacillus debilis, is LMG 23386(T) (=DSM 16016(T)=NCIMB 13995(T)=Tf(T)=R-35653(T)).

Lysinibacillus macroides sp. nov., nom. rev.

'Bacillus macroides' ATCC 12905(T) ( = DSM 54(T) = LMG 18474(T)), isolated in 1947 from cow dung, was not included in the Approved Lists of Bacterial Names and so it lost standing in bacteriological nomenclature. Reinvestigation of the strain, including DNA-DNA relatedness experiments, revealed that 'Bacillus macroides' is genomically distinct from its closest relatives Lysinibacillus xylanilyticus, Lysinibacillus boronitolerans and Lysinibacillus fusiformis (as determined by 16S rRNA gene sequence analysis, with pairwise similarity values of 99.2, 98.8 and 98.5 %, respectively, with the type strains of these species). Further analysis showed that 'Bacillus macroides' shares the A4α L-Lys-D-Asp peptidoglycan type with other members of the genus Lysinibacillus and can thus be attributed to this genus. These results, combined with additional phenotypic data, justify the description of strain LMG 18474(T) ( = DSM 54(T) = ATCC 12905(T)) as Lysinibacillus macroides sp. nov., nom. rev.

Emended descriptions of Geobacillus thermoleovorans and Geobacillus thermocatenulatus.

Nineteen thermophilic, aerobic, endospore-forming bacterial strains were subjected to 16S rRNA gene sequence analysis. Eight of these strains had been received as cultures of Geobacillus kaustophilus, G. lituanicus, G. stearothermophilus, 'G. thermoleovorans subsp. stromboliensis', G. vulcani, 'Bacillus caldolyticus', 'B. caldotenax' and 'B. caldovelox', but they showed close relationships with the type strain of G. thermoleovorans, as did two other strains received as G. thermoleovorans. All strains underwent further taxonomic analysis by API and other phenotypic tests and fatty acid methyl ester analysis, and selected strains were analysed for their polar lipids and for DNA relatedness. The 11 strains that formed the G. thermoleovorans 16S rRNA cluster also showed some phenotypic similarities, and DNA relatedness data support the reassignment of the strains received as G. kaustophilus, G. lituanicus, 'G. thermoleovorans subsp. stromboliensis', G. vulcani, 'B. caldolyticus', 'B. caldotenax' and 'B. caldovelox', and one of the G. stearothermophilus strains, as members of the species G. thermoleovorans. Four other strains received as G. kaustophilus were misnamed; two were identified as G. stearothermophilus and two appeared to be closely related to Anoxybacillus rupiensis. One strain received as G. stearothermophilus remained unidentified. On the basis of a single strain, Geobacillus thermocatenulatus was shown to represent a distinct species, but study of the type strain of Geobacillus gargensis showed this species to be a later heterotypic synonym of Geobacillus thermocatenulatus. Emended descriptions of Geobacillus thermoleovorans and Geobacillus thermocatenulatus are therefore presented.

Bacillus thermolactis sp. nov., isolated from dairy farms, and emended description of Bacillus thermoamylovorans.

A polyphasic taxonomic study was performed on 22 thermotolerant, aerobic, endospore-forming bacteria from dairy environments. Seventeen isolates were retrieved from raw milk, one from a filter cloth and four from grass, straw or milking equipment. These latter four isolates (R-6546, R-7499, R-7764 and R-7440) were identified as Bacillus thermoamylovorans based on DNA-DNA hybridizations (values above 70 % with Bacillus thermoamylovorans LMG 18084(T)) but showed discrepancies in characteristics with the original species description, so an emended description of this species is given. According to 16S rRNA gene sequence analysis and DNA-DNA hybridization experiments, the remaining 18 isolates (R-6488(T), R-28193, R-6491, R-6492, R-7336, R-33367, R-6486, R-6770, R-31288, R-28160, R-26358, R-7632, R-26955, R-26950, R-33520, R-6484, R-26954 and R-7165) represented one single species, most closely related to Bacillus thermoamylovorans (93.9 % 16S rRNA gene sequence similarity), for which the name Bacillus thermolactis is proposed. Cells were Gram-stain-positive, facultatively anaerobic, endospore-forming rods that grew optimally at 40-50 °C. The cell wall peptidoglycan type of strain R-6488(T), the proposed type strain, was A1γ based on meso-diaminopimelic acid. Major fatty acids of the strains were C(16 : 0) (28.0 %), iso-C(16 : 0) (12.1 %) and iso-C(15 : 0) (12.0 %). MK-7 was the predominant menaquinone, and major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and some unidentified phospholipids. DNA G+C content was 35.0 mol%. Phenotypic properties allowed discrimination from other thermotolerant species of the genus Bacillus and supported the description of the novel species Bacillus thermolactis, with strain R-6488(T) ( = LMG 25569(T)  = DSM 23332(T)) as the proposed type strain.