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A polyphasic approach was applied to investigate the diversity of microbiota that evolved during cold storage beef ripening. Isolate V4/DAB/S4/2a T 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 101070 T , Pseudomonas saxonica DSM 108989 T , Pseudomonas deceptionensis DSM 26521 T and Pseudomonas weihenstephanensis DSM 29166 T , placing it within the Pseudomonas fragi / lundensis branch of the genus Pseudomonas . Additional rpoB based comparison revealed P. versuta DSM 101070 T as the nearest relative, with 98.5 % nucleotide identity. Calculation of ANIb values of the V4/DAB/S4/2a T draft genome identified P. versuta DSM 101070 T with 90.1 %, P. deceptionensis DSM 26521 T with 85.1 %, P. fragi DSM 3456 T with 84.4 %, Pseudomonas psychrophila DSM 17535 T and Pseudomonas bubulae DSM 107389 T 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/S4/2a T . The DNA G+C content was 58.6 mol% for both isolates. The predominant cellular fatty acids of V4/DAB/S4/2a T were C 16 : 0 , C 18 : 1 ω7 c , C 17 : 0 cyclo and a summed feature containing C 16 : 1 ω7 c and/or C 15 : 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/S4/2a T (=DSM 111361 T =LMG 31844 T ) and a second isolate is UBT376 (=DSM 111360=LMG 31845).

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 107652 T , P. lactis DSM 29167 T , P. paralactis DSM 29164 T and P. azotoformans DSM 18862 T . Phylogenetic analysis of the complete rpoB gene sequences of the isolate V5/DAB/2/5 T indicated a separate branch with about 99.0 % nucleotide identities to the closest relatives P. carnis DSM 107652 T , P. lactis DSM 29167 T and P. paralactis DSM 29164 T , 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/2/5 T consisted of C 16 : 0 , summed feature C 16 : 1 ω 7 c /iso-C 15 : 0 2-OH, C 18 : 1 ω7 c, C 17 : 0 cyclo, C 12 : 0 , C 12 : 0 3-OH, C 10 : 0 3-OH and C 12 : 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/2/5 T (=DSM 111363 T =LMG 31846 T ); a second strain is UBT403 (=DSM 111362=LMG 31847).

During investigations of spoilage-associated meat microbiota, Pseudomonas isolates were found in two different laboratories showing highest similarities to Pseudomonas lactis DSM 29167 T , Pseudomonas paralactis DSM 29164 T and Pseudomonas azotoformans DSM 18862 T based on 16S rRNA gene sequence comparisons. Phylogenetic analysis of the complete rpoB gene sequences of isolates B4-1 T and SpeckC indicated a separate branch with 99.0 and 99.1 % identity, respectively, to their closest relative ( P. lactis DSM 29167 T ). 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 17149 T , Pseudomonas synxantha DSM 18928 T , Pseudomonas orientalis DSM 17489 T , Pseudomonas veronii DSM 11331 T and P. azotoformans DSM 18862 T . Genome-to-genome distance calculations between B4-1 T and its closest relative, P. lactis DSM 29167 T , showed 62.6 % relatedness. The G+C contents of B4-1 T 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 B4-1 T (=DSM 107652 T =LMG 30892 T ); a second strain is SpeckC (=DSM 107651=LMG 30893).

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 rpo B 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 3456 T with ANI values of about 90.2 %. Other close Pseudomonas neighbours were P. psychrophila DSM 17535 T (86.5 %), P. deceptionensis DSM 26521 T (86.4 %), P. versuta DSM 101070 T (83.8 %), P. taetrolens DSM 21104 T (83.2 %), P. weihenstephanensis DSM 29166 T (82.3 %), P. helleri DSM 29165 T (82.7 %) and P. lundensis DSM 6252 T (81.9 %). The G+C contents of isolates TH39 T and TH26 were both 58.2 mol%. The major cellular lipids of strain TH39 T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol; the major quinone was Q9 with small amounts of Q8. Based on these data, the isolates TH39 T 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 TH39 T (=DSM 107390 T =LMG 30830 T ).

The recombinant Pseudomonas putida strain CB1-9, which acquired the ability to grow on chlorobenzenes, contains a 33-kilobase (kb) plasmid (pKFL3) which lacked homology to an indigenous 15-kb plasmid (pKFL1) in Pseudomonas alcaligenes C-0 parent but was homologous to a 55-kb plasmid (pKFL2) from the P. putida R5-3 parent. Chromosomal DNA of P. alcaligenes C-0 hybridized to probes prepared from pKFL3 but not to probes prepared from pKFL2. A single clone from a genomic library of P. alcaligenes C-0 hybridized to EcoRI-digested pKFL3. Southern blot hybridization with the insert DNA from that clone identified homology with specific restriction enzyme fragments in pKFL3. The ability of the recombinant to utilize 3-chlorobenzoate, chlorobenzene, and 1,4-dichlorobenzene as well as its loss of utilization of xylenes and methylbenzoates appears to be associated with the transfer and integration of chromosomal DNA from P. alcaligenes into a Tol-like plasmid of P. putida R5-3.

Separate continuous cultures of Pseudomonas putida R5-3, grown on toluene, and Pseudomonas alcaligenes C-O, grown on benzoate, were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors. A recombinant strain, P. putida CB1-9, was isolated in less than 1 month. P. alcaligenes C-0 grew on benzoate and 3-chlorobenzoate but not on toluene, P. putida R5-3 grew on benzoate and toluene but not on 3-chlorobenzoate, and neither strain grew on chlorobenzene or 1,4-dichlorobenzene; however, the recombinant P. putida CB1-9 grew on all of these substrates. Chlorobenzene-utilizing strains were not found in continuous cultures run at the lowest growth rate (0.05/h) or in the absence of the donor strain, P. alcaligenes C-0. Chloride was released in stoichiometric amounts when P. putida CB1-9 was grown on either chlorobenzene or 1,4-dichlorobenzene. The recombinant strain was related to P. putida R5-3, phenotypically and genetically. Restriction enzyme digests of the single 57-kilobase (kb) plasmid in R5-3 and of the single 33-kb plasmid in CB1-9 were similar, but also indicated rearrangement of plasmid DNA. Coincidental or causal to the loss of the 24-kb fragment was the observation that the recombinant--unlike its parent, R5-3--did not grow on xylenes or methylbenzoates. Although both ortho-pyrocatechase (OP) and meta-pyrocatechase (MP) were found in CB1-9 and R5-3, MP activity was 20- to 50-fold higher in R5-3 cells grown on 4-methylbenzoate than in the same cells grown on benzene.(ABSTRACT TRUNCATED AT 250 WORDS)