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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 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).

In conventional absorption spectrometers, the range of accessible concentrations of analytes in aqueous solution is significantly limited by the dynamic range of the measurement system. Here we introduce the concept of multiscale spectroscopy allowing extending that range by orders of magnitude within one single device. The concept relies on using multiple light-sample interaction lengths, boosting the accessible concentration range by a particular extension factor. We experimentally implement our concept by a liquid core waveguide having multiple fiber ports side-wise attached to the waveguide, thus probing the light propagating inside the core at predefined distances from the input. This configuration provides three orders of magnitude of interaction length in one device. To verify the concept we exemplarily determine the concentrations of nitrate and of Rhodamine 6G in water, showing one hundred times improved measurement capabilities. The multiscale spectrometer uses the entire sample volume and allows the simultaneous measurement of fluorescence and attenuance. Due to its integrated design and the extended measurements capabilities, we anticipate application of our device in many application-relevant areas such as water quality analysis or environmental science., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We present a novel design for a compact flow-through fluorescence detector for flow analyses applications and prove its functionality in the experiment. The detector operates by detecting the diffusely emitted fluorescence in a glass capillary, which is a measure for the concentration of the analyte to be detected. The fluorescence is excited via an axially coupled fibre providing LED light and is collected by a photodiode. As model analyte we used dissolved reactive phosphate. The determination of the phosphate concentration is based on the reaction of molybdate to phosphomolybdate, which quenches the fluorescence of Rhodamine 6G. The experiments rely on a reversed flow injection analysis system especially designed for decoupling the analytical setup from environmental pressure for in situ applications. By combining the optics part and the fluidic setup a measuring range of 0-40 µg L(-1) PO4-P with detection limits of 0.22 µg L(-1) PO4-P (7 nmol L(-1)) for water and of 0.45 µg L(-1) PO4-P (14.5 nmol L(-1)) for seawater have been obtained. Our novel system has a sampling frequency of up to 300 samples per hour and achieves a repeatability between 0.6% (RSD) for the blank value signal (n=15) and 4.6% (RSD) for a phosphate concentration of 20.8 µg L(-1) PO4-P (n=15)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

Sakacin 674, a bacteriocin produced by Lactobacillus sake Lb764 and which inhibits the growth of Listeria monocytogenes, was purified to homogeneity by ammonium sulphate precipitation and sequential ion exchange, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence of sakacin 674 was determined by Edman degradation. The bacteriocin consisted of 43 amino acid residues and had a calculated molecular mass of 4436.6 Da, which is in good agreement with the molecular mass of 4437.2 as determined by mass spectrometry. The structural gene encoding sakacin 674 (sakR) was located on the chromosome. This gene was cloned and sequenced. It encoded a primary translation product of 61 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin 674. Sakacin 674 resembled other known bacteriocins and was very similar to sakacin P.

A comparative analysis of cotranscribed gene clusters comprising the structural genes mcrA, mcrB, mcrC, mcrD, and mcrG was carried out in three species of methanogens. mcrA, mcrB, and mcrG are the structural genes for the three subunits of methyl coenzyme M reductase, while the two other genes encode polypeptides of unknown functions. The degree of conservation of the mcr gene products among different species of methanogens varies. No correlation was found between the conservation of the G+C contents of the homologous genes and of the amino acid sequences of their products among the different bacteria. The comparison of RNA polymerase core subunit genes of Methanobacterium thermoautotrophicum as evolutionary markers with their equivalents in Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster showed that homologous polypeptide domains are encoded by different numbers of genes suggesting gene fusion of adjacent genes in the course of evolution. The archaebacterial subunits exhibit much stronger homology with their eukaryotic than with their eubacterial equivalents on the polypeptide sequence level. All the analyzed genes are preceded by ribosome binding sites of eubacterial type. In addition to known putative promoter sequences, conserved structural elements of the DNA were detected surrounding the transcription initiation sites of the mcr genes.

The sequence of the genes encoding the four largest subunits of the RNA polymerase of the archaebacterium Methanobacterium thermoautotrophicum was determined and putative translation signals were identified. The genes are more strongly homologous to eukaryotic than to eubacterial RNA polymerase genes. Analysis of the polypeptide sequences revealed colinearity of two pairs of adjacent archaebacterial genes encoding the B" and B' or A and C genes, respectively, with two eubacterial and two eukaryotic genes each encoding the two largest RNA polymerase subunits. This difference in sequence organization is discussed in terms of gene fusion in the course of evolution. The degree of conservation is much higher between the archaebacterial and the eukaryotic polypeptides than between the archaebacterial and the eubacterial enzyme. Putative functional domains were identified in two of the subunits of the archaebacterial enzyme.

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)