Pseudomonas paraversuta sp. nov. isolated from refrigerated dry-aged beef.

A polyphasic approach was applied to investigate the diversity of microbiota that evolved during cold storage beef ripening. Isolate V4/DAB/S4/2aT with a unique BOX-rep-PCR fingerprint profile revealed more than 99 % nucleotide identities upon pairwise comparisons of 16S rDNA sequences from the type strains Pseudomonas versuta DSM 101070T, Pseudomonas saxonica DSM 108989T, Pseudomonas deceptionensis DSM 26521T and Pseudomonas weihenstephanensis DSM 29166T, placing it within the Pseudomonas fragi / lundensis branch of the genus Pseudomonas. Additional rpoB based comparison revealed P. versuta DSM 101070T as the nearest relative, with 98.5 % nucleotide identity. Calculation of ANIb values of the V4/DAB/S4/2aT draft genome identified P. versuta DSM 101070T with 90.1 %, P. deceptionensis DSM 26521T with 85.1 %, P. fragi DSM 3456T with 84.4 %, Pseudomonas psychrophila DSM 17535T and Pseudomonas bubulae DSM 107389T with 84.2 % similarities each. Pairwise genome-to-genome distance calculations [digital DNA-DNA hybridization (dDDH)] resulted in values of 47.1, 35.1, 34.8, 34.2 and 34.1 %, respectively. A second isolate was detected years later in ground beef and showed ANIb values of 99.3 % and dDDH of 96.1 % relatedness to V4/DAB/S4/2aT. The DNA G+C content was 58.6 mol% for both isolates. The predominant cellular fatty acids of V4/DAB/S4/2aT were C16 : 0, C18 : 1ω7c, C17 : 0 cyclo and a summed feature containing C16 : 1ω7c and/or C15 : 0 iso 2-OH. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol, the major respiratory quinone was Q9, with a small portion of Q8. The combined data on genotypic and phenotypic features support the proposal of a novel species, for which the name Pseudomonas paraversuta sp. nov. is proposed. The type strain is V4/DAB/S4/2aT (=DSM 111361T=LMG 31844T) and a second isolate is UBT376 (=DSM 111360=LMG 31845).

Pseudomonas paracarnis sp. nov., isolated from refrigerated beef.

During a project focusing on the diversity of meat microbiota associated with beef ripening, a Pseudomonas strain was isolated exhibiting high 16S rRNA gene sequence similarities (>99 %) to Pseudomonas carnis DSM 107652T, P. lactis DSM 29167T, P. paralactis DSM 29164T and P. azotoformans DSM 18862T. Phylogenetic analysis of the complete rpoB gene sequences of the isolate V5/DAB/2/5T indicated a separate branch with about 99.0 % nucleotide identities to the closest relatives P. carnis DSM 107652T, P. lactis DSM 29167T and P. paralactis DSM 29164T, while average nucleotide identities (ANIb) calculated from the draft genomes were 94.8, 94.2 and 90.2 %, respectively. Pairwise genome-to-genome distance calculations (GGDC) resulted in values of 67.7, 63.5 and 45.7 %, respectively, lying below the actual species demarcation line as well. A second isolate, UBT403, was detected some years later by using matrix-assisted laser desorption ionization-time of flight MS of the microbiota of minced beef. The fatty acid profile of V5/DAB/2/5T consisted of C16 : 0, summed feature C 16 : 1 ω7c/iso-C15 : 0 2-OH, C18 : 1 ω7c, C17 : 0 cyclo, C12 : 0, C12 : 0 3-OH, C10 : 0 3-OH and C12 : 0 2-OH. The major cellular lipids were aminopholipids, phospholipids, phosphatidylethanolamine and phosphatidylglycerol; the major quinone was Q9 with a minor proportion of Q8. Based on phenotypic and chemotaxonomic characterizations, the isolates can be considered as representing a novel species, for which the name Pseudomonas paracarnis sp. nov. is proposed. The type strain is V5/DAB/2/5T (=DSM 111363T=LMG 31846T); a second strain is UBT403 (=DSM 111362=LMG 31847).

Pseudomonas carnis sp. nov., isolated from meat.

During investigations of spoilage-associated meat microbiota, Pseudomonas isolates were found in two different laboratories showing highest similarities to Pseudomonas lactis DSM 29167T, Pseudomonas paralactis DSM 29164T and Pseudomonas azotoformans DSM 18862T based on 16S rRNA gene sequence comparisons. Phylogenetic analysis of the complete rpoB gene sequences of isolates B4-1T and SpeckC indicated a separate branch with 99.0 and 99.1 % identity, respectively, to their closest relative (P. lactis DSM 29167T). Further phenotypic and chemotaxonomic characterizations, as well as average nucleotide identity (ANIb) values obtained from the draft genomes, revealed that these isolates could be considered as representing a novel species, with ANIb values of around 94 and 90 % with their closest relatives P. lactis and P. paralactis. Other related species showed ANIb values below 90 %, including Pseudomonas libanensis DSM 17149T, Pseudomonas synxantha DSM 18928T, Pseudomonas orientalis DSM 17489T, Pseudomonas veronii DSM 11331T and P. azotoformans DSM 18862T. Genome-to-genome distance calculations between B4-1T and its closest relative, P. lactis DSM 29167T, showed 62.6 % relatedness. The G+C contents of B4-1T and SpeckC were 59.8 and 59.9 mol%, respectively. The major cellular lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol; the major quinone was Q9. Based on these data, the new species Pseudomonas carnis sp. nov. is proposed, the type strain is B4-1T (=DSM 107652T=LMG 30892T); a second strain is SpeckC (=DSM 107651=LMG 30893).

Pseudomonas bubulae sp. nov., isolated from beef.

Two Pseudomonas isolates derived independently from raw refrigerated processing meat of bovine origin intended for the manufacture of Bologna-type cooked sausage could be distinguished from other known species in subsequent phylogenetic analyses. Comparison of the complete rpoB gene sequences in combination with nearly complete 16S rRNA gene sequences revealed a separate branch within the Pseudomonas fragi group. In further analyses, comprising phenotypic and chemotaxonomic characterization as well as average nucleotide identity (ANI) values obtained from the draft genome assemblies, the two isolates could be distinguished from all so far published closely related species. The closest relative was P. fragi DSM 3456T with ANI values of about 90.2 %. Other close Pseudomonas neighbours were P. psychrophila DSM 17535T (86.5 %), P. deceptionensis DSM 26521T (86.4 %), P. versuta DSM 101070T (83.8 %), P. taetrolens DSM 21104T (83.2 %), P. weihenstephanensis DSM 29166T (82.3 %), P. helleri DSM 29165T (82.7 %) and P. lundensis DSM 6252T (81.9 %). The G+C contents of isolates TH39T and TH26 were both 58.2 mol%. The major cellular lipids of strain TH39T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol; the major quinone was Q9 with small amounts of Q8. Based on these data, the isolates TH39T and TH26 (=DSM 107389=LMG 30831) represent a novel species within the genus Pseudomonas , for which the name Pseudomonas bubulae sp. nov. is proposed. The type strain is TH39T (=DSM 107390T=LMG 30830T).

Lactobacillus insicii sp. nov., isolated from fermented raw meat.

The analysis of the bacterial microbiota of retain samples of pork salami revealed an isolate (strain TMW 1.2011T) that could neither be assigned to typical genera of starter organisms nor to any other known meat-associated species. Cells were Gram-stain-positive, short, straight rods occurring singly, in pairs or short chains. Phylogenetic analysis of the 16S rRNA gene sequence and specific phenotypic characteristics showed that strain TMW 1.2011T belonged to the phylogenetic Lactobacillus alimentarius group, and the closest neighbours were Lactobacillus nodensis JCM 14932T (97.8 % 16S rRNA gene sequence similarity), Lactobacillus tucceti DSM 20183T (97.4 %), 'Lactobacillus ginsenosidimutans' EMML 3041 (97.3 %), Lactobacillus versmoldensis DSM 14857T (96.9 %) and Lactobacillus furfuricola JCM 18764T (97.2 %). Similarities using partial gene sequences of the alternative chronometers pheS, dnaK and rpoA also support these relationships. DNA-DNA relatedness between the novel isolate and L. nodensis JCM 14932T, L. versmoldensis DSM 14857T and L. tucceti DSM 20183T, L. furfuricola JCM 18764T and 'L. ginsenosidimutans' EMML 3041 were below 70 % and the DNA G+C content was 36.3 mol%. The cell-wall peptidoglycan type is l-Lys-Gly-d-Asp. Based on phylogenetic, chemotaxonomic and physiological evidence, strain TMW 1.2011T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus insicii sp. nov. is proposed. The type strain is TMW 1.2011T ( = CECT 8802T = DSM 29801T).

Analysis of the sakacin P gene cluster from Lactobacillus sake Lb674 and its expression in sakacin-negative Lb. sake strains.

Sakacin P is a small, heat-stable, ribosomally synthesized peptide produced by certain strains of Lactobacillus sake. It inhibits the growth of several Gram-positive bacteria, including Listeria monocytogenes. A 7.6 kb chromosomal DNA fragment from Lb. sake Lb674 encompassing all genes responsible for sakacin P production and immunity was sequenced and introduced into Lb. sake strains Lb790 and Lb706X which are bacteriocin-negative and sensitive to sakacin P. The transformants produced sakacin P in comparable amounts to the parental strain, Lb674. The sakacin P gene cluster comprised six consecutive genes: sppK, sppR, sppA, spiA, sppT and sppE, all transcribed in the same direction. The deduced proteins SppK and SppR resembled the histidine kinase and response regulator proteins of bacterial two-component signal transducing systems of the AgrB/AgrA-type. The genes sppA and spiA encoded the sakacin P preprotein and the putative immunity protein, respectively. The predicted proteins SppT and SppE showed strong similarities to the proposed transport proteins of several other bacteriocins and to proteins implicated in the signal-sequence-independent export of Escherichia coli haemolysin A. Deletion and frameshift mutation analyses showed that sppK, sppT and sppE were essential for sakacin P production in Lb706X. The putative SpiA peptide was shown to be involved in immunity to sakacin P. Analogues of sppR and spiA were found on the chromosomes of Lb. sake Lb706X and Lb790, indicating the presence of an incomplete spp gene cluster in these strains.