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Two strains of a Gram-staining-positive species were isolated from German bulk tank milk. On the basis of their 16S rRNA sequences they were affiliated to the genus Facklamia but could not be assigned to any species with a validly published name. Facklamia miroungae ATCC BAA-466 T (97.3 % 16S rRNA sequence similarity), Facklamia languida CCUG 37842 T (96.9 %), and Facklamia hominis CCUG 36813 T (96.6 %) are the closest relatives. In the 16S rRNA phylogeny and in the core-genome phylogeny strains WS 5301 T and WS 5302 form a well-supported, separate lineage. Pairwise average nucleotide identity calculated using MUMmer (ANIm) between WS 5301 T and type strains of other Facklamia species is well below the species cut-off (95 %) and ranges from 83.4 to 87.7 %. The DNA G+C content of the type strain is 36.4 mol% and the assembly size of the genome is 2.2 Mb. Cells of WS 5301 T are non-motile, non-endospore-forming, oxidase-negative, catalase-negative and facultatively anaerobic cocci. The fastidious species grows at 10-40 °C and with up to 7.0 % (w/v) NaCl in BHI supplemented with 5 g l -1 yeast extract. Major polar lipids are phosphatidylglycerol, diphosphatidylglycerol and two glycolipids. Predominant fatty acids are C 16 : 1 ω9 c and C 18 : 1 ω9 c . On the basis of their genomic, physiological and chemotaxonomic characteristics the strains examined in this study represent the same, hitherto unknown species. We propose the name Facklamia lactis sp. nov. for which WS 5301 T (=DSM 111018 T =LMG 31861 T ) is the type strain and WS 5302 (=DSM 111019=LMG 31862) is an additional strain of this novel species.

During a study investigating the microbiota of raw milk and its semi-finished products, strains WS 5106 T and WS 5096 were isolated from cream and skimmed milk concentrate. They could be assigned to the genus Pseudomonas by their 16S rRNA sequences, but not to any validly named species. In this work, a polyphasic approach was used to characterize the novel strains and to investigate their taxonomic status. Examinations based on the topology of core genome phylogenomy as well as average nucleotide identity (ANIm) comparisons suggested a novel Pseudomonas species within the Pseudomonas fluorescens subgroup. With pairwise ANIm values of 90.1 and 89.8 %, WS 5106 T was most closely related to Pseudomonas nabeulensis CECT 9765 T and Pseudomonas kairouanensis CECT 9766 T . The G+C content of strain WS 5106 T was 60.1 mol%. Morphologic analyses revealed Gram-stain-negative, aerobic, catalase and oxidase positive, rod-shaped and motile cells. Proteolysis on skimmed milk agar as well as lipolysis on tributyrin agar occurred at both 28 and 6 °C. Tolerated growth conditions were temperatures between 4 and 34 °C, pH values between 6.0 and 8.0, and salt concentrations of up to 5 %. Fatty acid profiles showed a pattern typical for Pseudomonas , with C 16 : 0 as the dominant component. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol and the dominating quinone was Q-9. Based on these results, it is proposed to classify the strains as a novel species, Pseudomonas cremoris sp. nov., with WS 5106 T (=DSM 111143 T =LMG 31863 T ) as type strain and WS 5096 (=DSM 111129=LMG 31864) as an additional strain.

Three strains of a Gram-stain-positive, catalase-negative, facultative anaerobic, and coccoid species were isolated from German bulk tank milk. Phylogenetic analyses based on the 16S rRNA gene sequences indicated that the three strains (WS4937 T , WS4759 and WS5303) constitute an independent phylogenetic lineage within the family Aerococcaceae with Facklamia hominis CCUG 36813 T (93.7-94.1 %) and Eremococcus coleocola M1831/95/2 T (93.5 %) as most closely related type species. The unclassified strains demonstrated variable growth with 6.5 % (w/v) NaCl and tolerated pH 6.5-9.5. Growth was observed from 12 to 39 °C. Their cell-wall peptidoglycan belongs to the A1 α type (l-Lys-direct) consisting of alanine, glutamic acid and lysine. The predominant fatty acids were C 16 : 1  ω9 c , C 16 : 0 and C 18 : 1  ω9 c and in the polar lipids profile three glycolipids, a phospholipid, phosphatidylglycerol, phosphoglycolipid and diphosphatidylglycerol were found. The G+C content of strain WS4937 T was 37.4 mol% with a genome size of ~3.0 Mb. Based on phylogenetic, phylogenomic and biochemical characterizations, the isolates can be demarcated from all other genera of the family Aerococcaceae and, therefore, the novel genus Fundicoccus gen. nov. is proposed. The type species of the novel genus is Fundicoccus ignavus gen. nov., sp. nov. WS4937 T (=DSM 109652 T =LMG 31441 T ).

Eight facultatively anaerobic rod-shaped bacteria were isolated from raw milk and two other dairy products. Results of phylogenetic analyses based on 16S rRNA gene sequences showed that the isolates are placed in a distinct lineage within the family Propionibacteriaceae with Propioniciclava sinopodophylli and Propioniciclava tarda as the closest relatives (94.6 and 93.5 % similarity, respectively). The cell-wall peptidoglycan contained meso -diaminopimelic acid, alanine and glutamic acid and was of the A1γ type ( meso -DAP-direct). The major cellular fatty acid was anteiso-C 15 : 0 and the major polar lipids were diphosphatidylglycerol, phosphatidyglycerol and three unidentified glycolipids. The quinone system contained predominantly menaquinone MK-9(H 4 ). The G+C content of the genomic DNA of strain VG341 T was 67.7 mol%. The whole-cell sugar pattern contained ribose, rhamnose, arabinose and galactose. On the basis of phenotypic and genetic data, eight strains (VG341 T , WS4684, WS4769, WS 4882, WS4883, WS4901, WS4902 and WS4904) are proposed to be classified as members of a novel species in a new genus of the family Propionibacteriaceae , for which the name Brevilactibacter flavus gen. nov., sp. nov. is proposed. The type strain is VG341 T (=WS4900 T =DSM 100885 T =LMG 29089 T ) and seven additional strains are WS4684, WS4769, WS4882, WS4883, WS4901, WS4902 and WS4904. Furthermore, we propose the reclassification of P. sinopodophylli as Brevilactibacter sinopodophylli comb. nov.

Two strains, WS 5063 T and WS 5067, isolated from raw cow's milk and skimmed milk concentrate, could be affiliated as members of the same, hitherto unknown, Pseudomonas species by 16S rRNA and rpoD gene sequences. Multilocus sequence and average nucleotide identity (ANIm) analyses based on draft genome sequences confirmed the discovery of a novel Pseudomonas species. It was most closely related to Pseudomonas synxantha DSM 18928 T with an ANIm of 91.4 %. The DNA G+C content of WS 5063 T was 60.0 mol %. Phenotypic characterizations showed that the isolates are rod-shaped, motile, catalase- and oxidase-positive, and aerobic. Growth occurred at 4-34 °C and at pH values of pH 5.5-8.0. Both strains showed strong β-haemolysis on blood agar. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The dominant quinone was Q-9 (90 %), but noticeable amounts of Q-8 (9 %) and traces of Q-7 were also detected. Fatty acid profiles were typical for Pseudomonas species and exhibited C 16 : 0 as a major component. Based on these results, we conclude that both strains belong to a novel species, for which the name Pseudomonas haemolytica sp. nov. is proposed. The type strain is WS 5063 T (=DSM 108987 T =LMG 31232 T ) and an additional strain is WS 5067 (=DSM 108988=LMG 31233).

A polyphasic approach was used to investigate the taxonomic status of two bacterial strains, WS 5072 T and WS 5092, isolated from skimmed milk concentrate and raw cow's milk. The 16S rRNA and rpoD gene sequences affiliated the strains to the same, hitherto unknown, Pseudomonas species. Further examinations of the draft genomes based on multilocus sequence analysis and average nucleotide identity confirmed the presence of a novel Pseudomonas species. It was most closely related to Pseudomonas fragi DSM 3456 T with 86.3 % ANIm. The DNA G+C content of strain WS 5072 T was 56.3 mol%. Cells were aerobic, Gram-negative, catalase and oxidase positive, rod-shaped and motile. Growth occurred at 4-34 °C, pH 5.5-8.0 and with salt concentrations of up to 7 %. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The dominating quinone was Q-9 with 94 %, with noticeable amounts of Q-8 (5 %) and traces of Q-7 and Q-10. Fatty acid profiles showed a composition common for Pseudomonas with the major component C 16 : 0 . Based on these results, the novel species Pseudomonas saxonica sp. nov. is proposed, with the type strain WS 5072 T (=DSM 108989 T =LMG 31234 T ) and the additional strain WS 5092 (=DSM 108990=LMG 31235).

Five strains, designated WS 4672T, WS 4998, WS 4992T, WS 4997 and WS 5000, isolated from bovine raw milk formed two individual groups in a phylogenetic analysis. The most similar species on the basis of 16S rRNA gene sequences were Pseudomonas azotoformans IAM 1603T, Pseudomonas gessardii CIP 105469T and Pseudomonas libanensis CIP 105460T showing 99.7-99.6 % similarity. Using rpoD gene sequences Pseudomonas veronii LMG 17761T (93.3 %) was most closely related to strain WS 4672T and Pseudomonas libanensis CIP 105460T to strain WS 4992T (93.3 %). The five strains could be differentiated from their closest relatives and from each other by phenotypic and chemotaxonomic characterization and ANIb values calculated from draft genome assemblies. ANIb values of strains WS 4992T and WS4671T to the closest relatives are lower than 90 %. The major cellular polar lipids of both strains are phosphatidylethanolamine, phosphatidylglycerol, a phospholipid and diphosphatidylglycerol, and their major quinone is Q-9. The DNA G+C content of strains WS 4992T and WS 4672T were 60.0  and 59.7  mol%, respectively. Based on these genotypic and phenotypic traits two novel species of the genus Pseudomonas are proposed: Pseudomonas lactis sp. nov. [with type strain WS 4992T (=DSM 29167T=LMG 28435T) and the additional strains WS 4997 and WS 5000], and Pseudomonasparalactis sp. nov. [with type strain WS 4672T (=DSM 29164T=LMG 28439T) and additional strain WS 4998].

Analysis of the microbiota of raw cow's milk and semi-finished milk products yielded seven isolates assigned to the genus Pseudomonas that formed two individual groups in a phylogenetic analysis based on partial rpoD and 16S rRNA gene sequences. The two groups could be differentiated from each other and also from their closest relatives as well as from the type species Pseudomonas aeruginosa by phenotypic and chemotaxonomic characterization and average nucleotide identity (ANIb) values calculated from draft genome assemblies. ANIb values within the groups were higher than 97.3 %, whereas similarity values to the closest relatives were 85 % or less. The major cellular lipids of strains WS4917T and WS4993T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol; the major quinone was Q-9 in both strains, with small amounts of Q-8 in strain WS4917T. The DNA G+C contents of strains WS4917T and WS4993T were 58.08 and 57.30 mol%, respectively. Based on these data, strains WS4917T, WS4995 ( = DSM 29141 = LMG 28434), WS4999, WS5001 and WS5002 should be considered as representatives of a novel species of the genus Pseudomonas, for which the name Pseudomonas helleri sp. nov. is proposed. The type strain of Pseudomonas helleri is strain WS4917T ( = DSM 29165T = LMG 28433T). Strains WS4993T and WS4994 ( = DSM 29140 = LMG 28438) should be recognized as representing a second novel species of the genus Pseudomonas, for which the name Pseudomonas weihenstephanensis sp. nov. is proposed. The type strain of Pseudomonas weihenstephanensis is strain WS4993T ( = DSM 29166T = LMG 28437T).

The facultative anaerobic bacterium Listeria monocytogenes encounters microaerophilic or anaerobic conditions in various environments, e.g. in soil, in decaying plant material, in food products and in the host gut. To elucidate the adaptation of Listeria monocytogenes to variations in oxygen tension, global transcription analyses using DNA microarrays were performed. In total, 139 genes were found to be transcribed differently during aerobic and anaerobic growth; 111 genes were downregulated and 28 genes were upregulated anaerobically. The oxygen-dependent transcription of central metabolic genes is in agreement with results from earlier physiological studies. Of those genes more strongly expressed under lower oxygen tension, 20 were knocked out individually. Growth analysis of these knock out mutants did not indicate an essential function for the respective genes during anaerobiosis. However, even if not essential, transcriptional induction of several genes might optimize the bacterial fitness of Listeria monocytogenes in anaerobic niches, e.g. during colonization of the gut. For example, expression of the anaerobically upregulated gene lmo0355, encoding a fumarate reductase α chain, supported growth on 10 mM fumarate under anaerobic but not under aerobic growth conditions. Genes essential for anaerobic growth were identified by screening a mutant library. Eleven out of 1360 investigated mutants were sensitive to anaerobiosis. All 11 mutants were interrupted in the atp locus. These results were further confirmed by phenotypic analysis of respective in-frame deletion and complementation mutants, suggesting that the generation of a proton motive force via F1F0-ATPase is essential for anaerobic proliferation of Listeria monocytogenes.

A novel red-pigmented bacterial strain, designated WS 4628(T), was isolated from a pharmaceutical clean room of a vaccine-producing company and was investigated in a taxonomic study using a polyphasic approach. The strain was Gram-stain-positive, strictly aerobic, motile, catalase-positive and produced spherical to slightly ellipsoidal endospores in rods. The genomic DNA G+C content was 44.1 mol%. The major fatty acids were anteiso-C15:0, iso-C15:0 and anteiso-C17:0 and the predominant quinone was MK-6. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, an unidentified phosphoglycolipid and an unidentified phospholipid. meso-diaminopimelic acid (type A1γ) was present in the cell-wall peptidoglycan and the major whole-cell sugars were glucose and ribose. The closest phylogenetic neighbours were identified as Bacillus badius ATCC 14574(T) (95.8% 16S rRNA gene sequence similarity), Bacillus indicus Sd/3(T) (94.8%), Jeotgalibacillus alimentarius YKJ-13(T) (94.8%) and Bacillus cibi JG-30(T) (94.8%). Phylogenetic, physiological, biochemical and morphological differences between strain WS 4628(T) and its closest relatives in the families Bacillaceae and Planococcaceae suggest that this strain represents a novel species in a new genus in the family Bacillaceae for which the name Domibacillus robiginosus gen. nov., sp. nov. is proposed; the type strain of the type species is WS 4628(T) (=DSM 25058(T)=LMG 26645(T)).

A gram-positive, oxidase- and catalase-positive, strictly aerobic and motile bacterium, designated WS 4626(T), was isolated from a medical practice. Spherical endospores were formed terminally in swollen rods. The genomic DNA G+C content was 37.1 mol%. Cells contained iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 1ω10c, anteiso-C17 : 0 and iso-C17 : 0 as the predominant cellular fatty acids and MK-7 and MK-6 as the major isoprenoid quinones. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol, the cell-wall peptidoglycan was type A4α, L-Lys-D-Asp and the major cell-wall sugar was xylose. The closest phylogenetic relatives were Lysinibacillus xylanilyticus XDB9(T) (96.7 % 16S rRNA gene sequence similarity) and Lysinibacillus odysseyi 34hs-1(T) (96.5 %). DNA-DNA relatedness between the isolate and L. odysseyi DSM 18869(T) was very low (6 %). On the basis of the data presented, strain WS 4626(T) represents a novel species of the genus Lysinibacillus, for which the name Lysinibacillus meyeri sp. nov. is proposed. The type strain is WS 4626(T) ( = DSM 25057(T) = LMG 26643(T)).

A novel Gram-staining-positive, rod-shaped, motile, strictly aerobic, endospore-forming bacterium, designated WCC 4585(T), was isolated from a pharmaceutical production line. The organism grew optimally at 30 °C, at pH 8 and in the presence of 0.5 % (w/v) NaCl. Oval endospores were formed subterminally and terminally in swollen sporangia. The cell-wall diamino acid was meso-diaminopimelic acid (type A1γ) and the genomic DNA G+C content was 38.7 mol%. The major menaquinone was MK-7. The cellular fatty acid profile contained major amounts of iso-C15 : 0, anteiso-C15 : 0 and anteiso-C17 : 0, and the cellular phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and aminophospholipid. The isolate was most closely related to Bacillus oceanisediminis H2(T), Bacillus infantis SMC 4352-1(T), Bacillus firmus NCIMB 9366(T), Bacillus circulans ATCC 4513(T) and Bacillus horneckiae DSM 23495(T) with which it shared less than 98.0 % 16S rRNA gene sequence similarity. DNA-DNA relatedness values between strain WCC 4585(T) and five type strains of related species were ≤27 % and sequence similarity values based on groEL sequences were ≤88.7 %. On the basis of the characteristics presented, strain WCC 4585(T) is proposed to represent a novel species, Bacillus gottheilii sp. nov. The type strain is WCC 4585(T)( = DSM 23668(T) = CCUG 59876(T) = LMG 25856(T)).

The phylogenetic position and phenotypic characteristics of two non-spore-forming bacilli similar to members of the genus Listeria were studied. The gram-reaction-positive, slightly motile, facultatively anaerobic strains were isolated from the water plant Lemna trisulca sampled from a freshwater pond in Bavaria, Germany. Although no identification was possible employing the API Listeria test (bioMérieux), 16S rRNA sequence analysis confirmed a close phylogenetic similarity to Listeria rocourtiae DSM 22097(T) (99.0 % sequence similarity) and a more distant relationship to other Listeria species (96.0 % to Listeria monocytogenes DSM 20600(T) and 95.0 % similarity to Listeria grayi DSM 20601(T)). DNA-DNA hybridization analysis between the isolates and Listeria rocourtiae DSM 22097(T) yielded a similarity of 22.5 %. Analysis of partial sequences of sigB, prs, recA and HSP60 were studied and compared with those of other members of the genus Listeria and Brochothrix thermosphacta DSM 20171(T) supporting the relationships indicated by 16S rRNA gene sequences. The studied isolates were non-haemolytic and were not associated with cases of human or animal disease. While the results demonstrate that the strains belong to the genus Listeria, phenotypic and genotypic differences from Listeria rocourtiae DSM 22097(T) suggest that the strains represent a novel species for which the name Listeria weihenstephanensis sp. nov. is proposed; the type strain is WS 4560(T) ( = DSM 24698(T) = LMG 26374(T)), with WS 4615 ( = DSM 24699 = LMG 26375) as a second strain of the species.

Three Gram-reaction-positive bacteria, isolated from the air in a medical practice (strains WS4601(T), WS4602) or a pharmaceutical clean room (strain WS4599), were characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA and recA gene sequences of the three novel strains showed that they formed a distinct lineage within the genus Micrococcus, sharing 16S rRNA gene sequence similarities of 96.1-98.0 % with other species of this genus. Chemotaxonomic features also supported the classification of the three novel strains within the genus Micrococcus. The major cellular fatty acids of strain WS4601(T) were anteiso-C(15 : 0) and iso-C(15 : 0), the cell-wall peptidoglycan was of type A3α (L-Lys-L-Ala), and the predominant respiratory quinones were MK-7(H(2)) and MK-8(H(2)). The polar lipid profile contained diphosphatidylglycerol and phosphatidylglycerol, but no phosphatidylinositol. The G+C content of the genomic DNA was 70.4 mol%. Numerous physiological properties were found that clearly distinguished strains WS4599, WS4601(T) and WS4602 from established members of the genus Micrococcus. Based on the phenotypic and phylogenetic data, strains WS4599, WS4601(T) and WS4602 are considered to represent three different strains of a novel species of the genus Micrococcus, for which the name Micrococcus cohnii sp. nov. is proposed. The type strain is WS4601(T) (=DSM 23974(T)=LMG 26183(T)).

Four Gram-stain-positive, aerobic bacterial strains isolated from a pharmaceutical clean room (strain WS4616(T)), a dessert milk product (strain WS4617) and from raw milk (strains WS4623 and WS4624) were characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA and recA gene sequences showed that they formed a distinct lineage within the family Propionibacteriaceae. Similarity values between 16S rRNA gene sequences of the four novel strains and the type species of all genera belonging to the family Propionibacteriaceae were 89.2-94.1%. The major cellular fatty acid was anteiso-C(15:0) and the major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. Respiratory quinones were MK-8(H(4)) and MK-9(H(4)). The cell-wall peptidoglycan of type A3γ contained ll-diaminopimelic acid, alanine, glycine and glutamic acid. The G+C content of the genomic DNA of strain WS4616(T) was 67.7 mol%. The whole-cell sugar pattern contained ribose, mannose, arabinose, glucose and galactose. On the basis of phenotypic and genetic data, strains WS4616(T), WS4617, WS4623 and WS4624 are classified as members of a novel species in a new genus of the family Propionibacteriaceae, for which the name Naumannella halotolerans gen. nov., sp. nov. is proposed. The type strain is WS4616(T) ( = DSM 24323(T) = LMG 26184(T)) and three additional strains are WS4617, WS4623 and WS4624.

A novel halophilic, Gram-reaction-negative, strictly aerobic, non-motile, rod-shaped and oxidase- and catalase-positive bacterial strain, designated WCC 4520(T), was isolated from a semi-hard, Raclette-type cheese. The colonies were yellow-orange; flexirubin-type pigments were not found. The strain hydrolysed gelatin, hippurate, tyrosine and Tweens 20 and 80. Optimal growth was observed with 6-8% (w/v) NaCl, at pH 7-8 and at 27-30 °C. The genomic DNA G+C content was 33.6 mol%. In phylogenetic analysis based on 16S rRNA gene sequences, strain WCC 4520(T) appeared to be a member of the family Flavobacteriaceae and the closest phylogenetic neighbours were identified as Psychroflexus gondwanensis DSM 5423(T) (94.0% 16S rRNA gene sequence similarity) and Psychroflexus salinarum CCUG 56752(T) (94.0%). The predominant cellular fatty acids were iso-C(15:0), anteiso-C(15:0), iso-C(15:1) G and iso-C(17:0) 3-OH. The only detected quinone was MK-6 and the major polar lipids were phosphatidylethanolamine, an unidentified aminolipid and an unidentified polar lipid. Minor polar lipids and traces of polyamines were also detected. On the basis of the data presented, strain WCC 4520(T) represents a novel species of the genus Psychroflexus, for which the name Psychroflexus halocasei sp. nov. is proposed. The type strain is WCC 4520(T) ( = LMG 25857(T) = CCUG 59705(T)).

Four non-fermenting, rod-shaped, Gram-staining-negative bacterial strains, designated WCC 4512(T), WS 4555, WCC 4521(T) and WS 4556, were isolated from raw milk and the dairy environment. Phylogenetic analyses based on 16S rRNA and groEL gene sequences demonstrated the affiliation of the four strains to two distinct clusters within the class Sphingobacteriia, phylum 'Bacteroidetes'. Strains WCC 4512(T) and WS 4555 showed the highest 16S rRNA gene sequence similarity to the type strain of S. daejeonense (97.3 and 97.2%, respectively), whereas strains WCC 4521(T) and WS 4556 were most closely related to S. composti LMG 23401(T) (97.6% 16S rRNA gene sequence similarity). The DNA G+C contents of strains WCC 4512(T) and WCC 4521(T) were 44.2 and 39.3 mol%, respectively. The major cellular fatty acids and the presence of menaquinone MK-7 as the predominant quinone for both strains WCC 4512(T) and WCC 4521(T) supported their affiliation to the genus Sphingobacterium. DNA-DNA hybridization experiments between strain WCC 4512(T) and S. daejeonense LMG 23402(T) and between strain WCC 4521(T) and S. composti LMG 23401(T) revealed DNA relatedness values of 2% (repetition, 3%) and 8% (repetition, 17%), respectively. On the basis of phenotypic and genetic properties, as well as phylogenetic distinctiveness, it is suggested that the four strains represent two novel Sphingobacterium species with strain WCC 4512(T) (=DSM 22361(T)=LMG 25272(T)) as the type strain of Sphingobacterium lactis sp. nov. (WS 4555 is a reference strain of S. lactis) and strain WCC 4521(T) (=DSM 22362(T)=LMG 25273(T)) as the type strain of Sphingobacterium alimentarium sp. nov. (WS 4556 is a reference strain of S. alimentarium).

Three Gram-staining-positive, strictly aerobic, motile, catalase-positive, endospore-forming rods, designated WCC 4582(T), WCC 4581 and WCC 4583, were isolated from two different food sources and a pharmaceuticals production site. The three isolates were highly similar in their 16S rRNA gene sequences (100 % similarity) and groEL sequences (99.2-100 % similarity), Fourier-transform infrared spectroscopic fingerprints and other features tested. The isolates were most closely related to Bacillus horneckiae; the isolates and the type strain of B. horneckiae shared 97.6 % and 89.6 % 16S rRNA gene and groEL sequence similarities, respectively. The organisms grew optimally at 30 °C, at pH 7 and in the presence of 0.5 % (w/v) NaCl. The cell-wall peptidoglycan of WCC 4582(T) contained meso-diaminopimelic acid (A1γ) and the genomic DNA G+C content was 36.4 mol%. DNA-DNA relatedness between strain WCC 4582(T) and B. horneckiae NRRL B-59162(T) was 17 %. The three isolates are considered to represent a novel species of the genus Bacillus, for which the name Bacillus kochii sp. nov. is proposed. The type strain is WCC 4582(T) ( = DSM 23667(T) = CCUG 59877(T) = LMG 25855(T)).

Three Gram-negative, rod-shaped, catalase- and oxidase-positive, facultatively anaerobic and motile bacteria, strains WS 4538, WS 4539T and WS 4540, were isolated from the surfaces of two fully ripened French red smear soft cheeses. Based on 16S rRNA gene sequence similarity, all three strains were shown to belong to the genus Vibrio. They are most closely related to Vibrio rumoiensis S-1T (96.3% similarity) and Vibrio litoralis MANO22DT (95.9%). DNA-DNA hybridization confirmed that all three isolates belong to the same species and clearly separated strain WS 4539T from V. rumoiensis DSM 19141T (38-42% relatedness) and V. litoralis DSM 17657(T) (28-37%). In contrast to their nearest relatives, the strains exhibited beta-galactosidase and aesculin hydrolase activities. A 14 bp insertion in the 16S rRNA gene sequence forms an elongated structure at helix 10 in the rRNA molecule and provides a tool for PCR-based identification of the novel species. Partial sequences of the housekeeping genes atpA, recA, rpoA and pyrH supported the conclusion that the three isolates constitute a separate species within the genus Vibrio. The name Vibrio casei sp. nov. is proposed for the novel taxon. Strain WS 4539T (=DSM 22364T =LMG 25240T; DNA G+C content 41.8 mol%) is the type strain and WS 4540 (=DSM 22378 =LMG 25241) is a reference strain.

The phylogenetic position and physiological characters of six hitherto-unknown lactic acid bacterial isolates, which form part of the surface microbiota of German red smear soft cheese, are reported. The coccoid cells are aerotolerant, Gram-positive, catalase-negative and non-motile. The cell-wall peptidoglycan contains alanine, glutamic acid, lysine and aspartic acid and is of the A4alpha type (l-Lys-d-Asp). The sequences of the 16S rRNA, groEL and rpoB genes of the six isolates are identical and reveal that these isolates represent an independent lineage within the radiation of the family Enterococcaceae in the phylum Firmicutes. Their closest phylogenetic neighbour is the lactic acid bacterium Atopobacter phocae M1590/94/2(T), with which they share 94.9 % 16S rRNA gene sequence similarity; representatives of other genera such as Granulicatella, Carnobacterium and Trichococcus are more distantly related. DNA-DNA hybridization studies reveal that the six isolates are members of a single species, and this is confirmed by similarities in biochemical characteristics. The six isolates were assigned four different groups by Fourier-transform infrared and randomly amplified polymorphic DNA typing. Therefore, it is formally proposed that these isolates should be classified in a single novel species of a novel genus and be named Bavariicoccus seileri gen. nov., sp. nov. The type strain of Bavariicoccus seileri is WCC 4188(T) (=DSM 19936(T) =CCUG 55508(T)).

Cereulide, a depsipeptide structurally related to the antibiotic valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that cereulide is produced non-ribosomally by the plasmid-encoded peptide synthetase Ces. Using deletion mutants of the emetic reference strain B. cereus F4810/72, the influence of the well-known transcription factors PlcR, Spo0A and AbrB on cereulide production and on the transcription of the cereulide synthetase gene cluster was investigated. Our data demonstrate that cereulide synthesis is independent of the B. cereus specific virulence regulator PlcR but belongs to the Spo0A-AbrB regulon. Although cereulide production turned out to be independent of sporulation, it required the activity of the sporulation factor Spo0A. The sigma(A)-promoted transcription of spo0A was found to be crucial for cereulide production, while the sigma(H)-driven transcription of spo0A did not affect cereulide synthesis. Overexpression of the transition state factor AbrB in B. cereus F4810/72 resulted in a non-toxic phenotype. Moreover, AbrB was shown to bind efficiently to the main promoter region of the ces operon, indicating that AbrB acts as a repressor of cereulide production by negatively affecting ces transcription.

Twelve Yersinia enterocolitica mutants carrying luxCDABE-transposon insertions in motility and chemotaxis genes were isolated on the basis of strong low-temperature induction. Two transposons were located within an 11.2 kb enteric flagellar cluster 2 (Flag-2) of Y. enterocolitica biotype 2, serotype O : 9 strain W22703. The Flag-2 gene cluster is absent from the corresponding genomic location of the sequenced strain Y. enterocolitica biotype 1B, serotype O : 8 strain 8081. Evidence for the functionality of the O : 9 Flag-2 genes, probably located within the plasticity zone of the genome, is provided by swarming assays. PCR analysis of 49 strains revealed the presence of Flag-2 genes in biotypes 2-5, but not in biotypes 1A or 1B. Bioluminescence, measured between 6 and 37 degrees C, showed that the expression of all genes located in Flag-2 and in the known flagellar cluster, Flag-1, was highest at approximately 20 degrees C, and that expression of two Flag-2 genes is FlhC-dependent. In a motility assay, a non-motile and a hyper-motile phenotype resulted from knockout mutations of the Flag-1 genes fliS1 and fliT, respectively. Complemented strains validated these results, confirming the regulatory role of FliT.

A Gram-positive, aerobic, rod-shaped, motile, endospore-forming bacterium was isolated from pasteurized milk from Bavaria, Germany. 16S rRNA gene sequence similarities indicated that strain WSBC 24001(T) was most closely related to Virgibacillus species (95.3-96.1 %), Oceanobacillus species (95.6-95.7 %), Bacillus firmus IAM 12464(T) (95.5 %) and Bacillus niacini IFO 15566(T) (95.2 %). However, strain WSBC 24001(T) showed the highest level of sequence similarity to an unnamed strain, MB-9(T) (97.6 %), which was isolated from coastal surface sediments in California. Hence, this strain was included in our study. The genomic DNA G + C contents of strains WSBC 24001(T) and MB-9(T) were 36.4 mol and 40.8 mol%, respectively. The major respiratory quinone of both strains was menaquinone MK-7 and the peptidoglycan type was A4beta (L-orn<--D-Asp). The polar lipid profiles of these strains contained a predominance of diphosphatidylglycerol and moderate to minor amounts of phosphatidylglycerol, an unknown phospholipid and an unknown aminophospholipid. However, strain WSBC 24001(T) could be distinguished from strain MB-9(T) by the presence of an unknown lipid. The fatty acid profiles of the two strains comprised mainly iso- and anteiso-branched acids, but showed some significant quantitative differences in the amounts of certain acids. The DNA-DNA relatedness value (15.5 %) clearly demonstrated that strains WSBC 24001(T) and MB-9(T) are representatives of two different species. On the basis of their phylogenetic position and morphological, physiological and chemotaxonomic properties, a novel genus is proposed, Ornithinibacillus gen. nov., with two novel species, the type species Ornithinibacillus bavariensis sp. nov. (type strain WSBC 24001(T) = DSM 15681(T) = CCM 7096(T)) and Ornithinibacillus californiensis sp. nov. (type strain MB-9(T) = DSM 16628(T) = CCM 7237(T)).

An in-depth polyphasic approach was applied to study the population structure of the human pathogen Bacillus cereus. To assess the intraspecific biodiversity of this species, which is the causative agent of gastrointestinal diseases, a total of 90 isolates from diverse geographical origin were studied by genetic [M13-PCR, random amplification of polymorphic DNA (RAPD), multilocus sequence typing (MLST)] and phenetic [Fourier transform Infrared (FTIR), protein profiling, biochemical assays] methods. The strain set included clinical strains, isolates from food remnants connected to outbreaks, as well as isolates from diverse food environments with a well documented strain history. The phenotypic and genotypic analysis of the compiled panel of strains illustrated a considerable diversity among B. cereus connected to diarrhoeal syndrome and other non-emetic food strains, but a very low diversity among emetic isolates. Using all typing methods, cluster analysis revealed a single, distinct cluster of emetic B. cereus strains. The isolates belonging to this cluster were neither able to degrade starch nor could they ferment salicin; they did not possess the genes encoding haemolysin BL (Hbl) and showed only weak or no haemolysis. In contrast, haemolytic-enterotoxin-producing B. cereus strains showed a high degree of heterogeneity and were scattered over different clusters when different typing methods were applied. These data provide evidence for a clonal population structure of cereulide-producing emetic B. cereus and indicate that emetic strains represent a highly clonal complex within a potentially panmictic or weakly clonal background population structure of the species. It may have originated only recently through acquisition of specific virulence factors such as the cereulide synthetase gene.

The intraspecific diversity of 31 strains of Brevibacterium linens, 27 strains of Corynebacterium glutamicum and 29 strains of Rhodococcus erythropolis was determined by partial 16S rDNA sequence analysis and Fourier-transform infrared (FT-IR) spectroscopy. As a prerequisite for the analyses, 27 strains derived from culture collections which had carried invalid or wrong species designations were reclassified in accordance with polyphasic taxonomical data. FT-IR spectroscopy proved to be a rapid and reliable method for screening for similar isolates and for identifying these actinomycetes at the species level. Two main conclusions emerged from the analyses. (1) Comparison of intraspecific 16S rDNA similarities suggested that R. erythropolis strains have a very low diversity, B. linens displays high diversity and C. glutamicum occupies an intermediate position. (2) No correlation of FT-IR spectral similarity and 16S rDNA sequence similarity below the species level (i.e. between strains of one species) was observed. Therefore, diversification of 16S rDNA sequences and microevolutionary change of the cellular components detected by FT-IR spectroscopy appear to be de-coupled.

An extensive Fourier-transform infrared (FT-IR) spectroscopy database for the identification of bacteria from the two suborders Micrococcineae and Corynebacterineae (Actinomycetales, Actinobacteria) as well as other morphologically similar genera was established. The database consists of averaged IR spectra from 730 reference strains, covering 220 different species out of 46 genera. A total of 192 species are represented by type strains. The identity of 352 reference strains was determined by comparative 16S rDNA sequence analysis and, if necessary, strains were reclassified accordingly. FT-IR frequency ranges, weights and reproducibility levels were optimized for this section of high-G+C gram-positive bacteria. In an internal validation, 98.1% of 208 strains were correctly identified at the species level. A simulated external validation which was carried out using 544 strains from 54 species out of 16 genera resulted in a correct identification of 87.3% at the species level and 95.4% at the genus level. The performance of this identification system is well within the range of those having been reported in the literature for the identification of coryneform bacteria by phenotypical methods. Coryneform and related taxa display a certain degree of overlapping distribution of different taxonomical markers, leading to a limited differentiation capacity of non-genotypical identification methods in general. However, easy handling, rapid identification within 25 h starting from a single colony, a satisfactory differentiation capacity and low cost, render FT-IR technology clearly superior over other routine methods for the identification of coryneform bacteria and related taxa.

Different approaches were used to examine the function of teichoic acids (TA) as phage receptors among selected Listeria strains, and to identify and characterize specific receptor structures of host cells belonging to different serovars. This included successive removal of cell wall constituents, preparation and purification of TA, and GLC analysis of TA components. Adsorption of Listeria monocytogenes bacteriophages could be inhibited by polyvalent antisera, specific lectins and addition of purified TA. The results confirmed the necessity of TA in general and of rhamnose and glucosamine in particular for adsorption of Listeria phage A118, which is a temperate Siphovirus (morphotype B1), attacking predominantly serovars 1/2. Host binding of siphoviral phage A500 (predominantly lysing serovars 4b), was also dependent on cell wall TA. A phage-resistant L. monocytogenes strain was shown to lack glucosamine in its TA. These results support the view that TA substituents may play an important role not only in antigenicity of Listeria cells, but also in specificity of host recognition by two temperate Listeria phages. In contrast, the broad-host-range virulent phage A511 (Myovirus, morphotype A1) uses the listerial peptidoglycan as primary receptor. This corresponds well with the observation that A511 is capable of lysing the majority of L. monocytogenes strains.

Monocins in Listeria were induced by UV-irradiation of liquid cultures, and defective phage particles were purified from the lysates. Electron microscopy showed flexible, non-contractile bacteriophage-tail-like particles, consisting of specific proteins of molecular mass 20-45 kDa and pI 4.6-6.7. These particles were able to lyse listerial cells. DNA sequence homologies between chromosomal DNA of monocin-producing strains and labelled Listeria phage DNAs were inferred from DNA/DNA hybridizations, suggesting that most of the prophage DNA is still present in the listerial chromosome. An endolysin gene cpl2438 was cloned from listerial chromosomal DNA and was identified by its expression of lytic activity against Listeria cells in a bioassay. The gene consists of 864 nt encoding a protein of 287 aa with a calculated molecular mass of 32975 Da (CPL2438). This is in good agreement with the size of a protein observed in SDS-PAGE after overexpression of the lytic protein in Escherichia coli. The nucleotide sequence of a putative holin gene (hol2438, 291 nt) upstream of cpl2438 was determined after PCR-amplification of listerial DNA and it shows typical features common to the holin gene family. Expression of the encoded protein (HOL2438, 95 aa, 10.1 kDa) in E. coli was found to be lethal for the host cells. The results underline the close relationship between monocins and intact Listeria bacteriophages, indicating that monocins are incompletely assembled phage particles derived from cryptic prophages of Listeria, probably including the phage lysin.

The major structural proteins of 13 temperate and one virulent Listeria typing bacteriophages were analysed and compared using isoelectric focusing in immobilized pH gradients (IPG), ultrathin-layer two-dimensional electrophoresis, amino acid analysis and N-terminal amino acid sequences of selected proteins. Isoelectric points for major capsid and tail proteins of the 12 members of the siphoviridae family included in this study ranged from 4.70 to 5.92, whereas one of the two myoviridae investigated (B054) showed structural proteins in the 6.1 to 6.3 range. In comparison to protein profiles from one-dimensional SDS gels, the IPG technique gave better resolution and improved discrimination of phage proteins. Combination of this technique and SDS gel electrophoresis made it possible to correlate M(r) and isoelectric points of major structural proteins. Tail polypeptides of all siphoviridae are smaller and, with one exception, more acidic than their corresponding capsid counterparts. We also determined the amino acid composition of capsid and tail proteins. When compared with an average protein, they were found to be fairly rich in acidic and short-chain hydrophobic amino acids, as well as in lysine. In addition, the N-terminal amino acid sequences of major capsid and tail proteins of four representative listeriaphages were compared. The base composition of listeriaphage DNAs was between 37% and 39% G + C, reflecting that of their bacterial hosts. Each phage had a distinct restriction endonuclease pattern, and genome sizes ranged from 35 to 116 kb. DNA-DNA hybridization permitted the identification of five DNA homology groups. The two myoviruses studied (A511 and B054) showed no DNA homology to other phages, confirming their unique nature. The 12 siphoviruses were classified into three DNA homology groups with little cross-homology. Furthermore, phage A006 was found to share little DNA homology with the other investigated members of species 2671. Therefore, a new species (A006) is proposed. With respect to phage classification and taxonomy, a good correlation between the various approaches was observed, mostly corresponding to particle morphology.