Pseudomonas kulmbachensis sp. nov. and Pseudomonas paraveronii sp. nov., originating from chilled beef and chicken breast.

By investigating wet and dry age-related ripening of beef, Pseudomonas strains V3/3/4/13T and V3/K/3/5T were isolated. Strain V3/3/4/13T exhibited more than 99 % 16S rRNA gene-based similarity to Pseudomonas fragi and other members of this group, while isolate V3/K/3/5T was very close to Pseudomonas veronii and a number of relatives within the Pseudomonas fluorescens group. Additional comparisons of complete rpoB sequences and draft genomes allowed us to place isolate V3/3/4/13T close to Pseudomonas deceptionensis DSM 26521T. In the case of V3/K/3/5T the closest relative was P. veronii DSM 11331T. Average nucleotide identity (ANIb) and digital DNA-DNA hybridization (dDDH) values calculated from the draft genomes of V3/3/4/13T and P. deceptionensis DSM 26521T were 88.5 and 39.8 %, respectively. For V3/K/3/5T and its closest relative P. veronii DSM 11331T, the ANIb value was 95.1 % and the dDDH value was 60.7 %. The DNA G+C contents of V3/3/4/13T and V3/K/3/5T were 57.4 and 60.8 mol%, respectively. Predominant fatty acids were C16 : 0, C18 : 1 ω7c, C17 : 0 cyclo and summed feature C16 : 1 ω7ct/C15 : 0 iso 2OH. The main respiratory quinones were Q9, with minor proportions of Q8 and, in the case of V3/K/3/5T, additional Q10. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and, in the case of V3/K/3/5T, additional phosphatidylcholine. Based on the combined data, isolates V3/3/4/13T and V3/K/3/5T should be considered as representatives of two novel Pseudomonas species. The type strain of the newly proposed Pseudomonas kulmbachensis sp. nov. is V3/3/4/13T (=DSM 113654T=LMG 32520T), a second strain belonging to the same species is FLM 004-28 (=DSM 113604=LMG 32521); the type strain for the newly proposed Pseudomonas paraveronii sp. nov. is V3/K/3/5T (=DSM 113573T=LMG 32518T) with a second isolate FLM 11 (=DSM 113572=LMG 32519).

Rathayibacter rubneri sp. nov. isolated from Allium cepa var. Rijnsburger, an onion landrace.

The novel, aerobic, Gram-stain-positive, rod-shaped bacterial strain, ZW T2_19T, was isolated from an onion sample (Allium cepa var. Rijnsburger). Analyses of the 16S rRNA gene sequence revealed that ZW T2_19T represented a member of the genus Rathayibacter but may represent a novel species of this genus. Analyses of the whole draft genome sequences, i.e. digital DNA-DNA hybridisation (dDDH) and average nucleotide identity (ANI) of ZW T2_19T and all type strains of species of the genus Rathayibacter confirmed that ZW T2_19T represents a novel species of the genus Rathayibacter. The genome size of ZW T2_19T is 4.01 Mbp and the DNA G+C content is 71.8 mol%. Glucose, mannose, rhamnose and ribose were detected as whole-cell sugars of ZW T2_19T. The major respiratory quinone of ZW T2_19T is menaquinone MK-10, at 78.9 %. The detected peptidoglycan type in ZW T2_19T is a variant of type B2γ with {Gly} [l-diaminobutyric acid (l-DAB)/l-homoserine (l-Hse)] d-Glu-l-DAB. Polar lipids in ZW T2_19T consisted of one diphosphatidylglycerol, one phosphatidylglycerol, seven glycolipids, one phospholipid and one lipid. The fatty acid profile of ZW T2_19T predominantly consisted of anteiso-C15 : 0 (53 %), iso-C16 : 0 (21 %) and anteiso-C17 : 0 (18 %). In addition, API 20NE, API 50CH, API Coryne, API ZYM, antibiotic susceptibility, haemolysis and growth at different temperatures and with different supplements was investigated. On the basis of the results obtained using this polyphasic approach, including molecular, phenotypic and biochemical analyses, we propose the novel species Rathayibacter rubneri with the type and only strain ZW T2_19T (= DSM 114294T = LMG 32700T).

Pseudomonas rustica sp. nov., isolated from bulk tank raw milk at a German dairy farm.

Here we present the description of a novel Pseudomonas species, designated Pseudomonas rustica sp. nov., which was isolated from raw milk samples obtained from Germany. Results of initial 16S rRNA gene sequence analysis assigned the strain into the genus Pseudomonas and showed Pseudomonas helmanticensis, Pseudomonas neuropathica and Pseudomonas atagonensis to be its closest relatives. Further studies including sequence analysis of the rpoB gene, multi-gene phylogenetic tree reconstruction, whole-genome sequence comparisons, cellular fatty acid analysis and chemotaxonomic characterization showed a clear separation from the known Pseudomonas species. Isolate MBT-4T was closely related to Pseudomonas helmanticensis, 'Pseudomonas crudilactis' and Pseudomonas neuropathica with average nucleotide identities based on blast values of 88.8, 88.8 and 88.6%, respectively. Therefore, the strain can be classified into the Pseudomonas koreensis subgroup of the Pseudomonas fluorescens group. The G+C content of strain MBT-4T was 58.9 mol%. The strain was catalase- and oxidase-positive, while the ß-galactosidase reaction was negative. Growth occurred between 4 and 30 °C and at pH values from pH 6.0 to 8.0. In conclusion, strain MBT-4T belongs to a novel species, for which the name Pseudomonas rustica sp. nov. is proposed. The type strain is MBT-4T (=DSM 112348T=LMG 32241T) and strain MBT-17 is also a representative of this species.

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).

Genome analysis reveals that the correct name of type strain Adlercreutzia caecicola DSM 22242T is Parvibacter caecicola Clavel et al. 2013.

The strain Adlercreutzia caecicola DSM 22242T (=CCUG 57646T=NR06T) was taxonomically described in 2013 and named as Parvibacter caecicola Clavel et al. 2013. In 2018, the name of the strain DSM 22242T was changed to Adlercreutzia caecicola (Clavel et al. 2013) Nouioui et al. 2018 due to taxonomic investigations of the closely related genera Adlercreutzia, Asaccharobacter and Enterorhabdus within the phylum Actinobacteria. However, the first whole draft genome of strain DSM 22242T was published by our group in 2019. Therefore, the genome was not available within the study of Nouioui et al. (2018). The results of the polyphasic approach within this study, including phenotypic and biochemical analyses and genome-based taxonomic investigations [genome-wide average nucleotide identity (gANI), alignment fraction (AF), average amino acid identity (AAI), percentage of orthologous conserved proteins (POCP) and genome blast distance phylogeny (GBDP) tree], indicated that the proposed change of the name Parvibacter caecicola to Adlercreutzia caecicola was not correct. Therefore, it is proposed that the correct name of Adlercreutzia caecicola (Clavel et al. 2013) Nouioui et al. 2018 strain DSM 22242T is Parvibacter caecicola Clavel et al. 2013.

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 kielensis sp. nov. and Pseudomonas baltica sp. nov., isolated from raw milk in Germany.

In this study, nine Gram-negative, motile and rod-shaped bacteria were isolated during a Germany-wide investigation of raw milk microbiota. The strains could be differentiated from their closest relatives by phenotypic and chemotaxonomic characterization and average nucleotide identity (ANIb) values calculated from draft genome assemblies. Strains MBT-1T, MBT-8, MBT-9, MBT-10, MBT-11 and MBT-12 were related to the Pseudomonas chlororaphis subgroup. Isolates MBT-2T, MBT-13 and MBT-14 were closely related to Pseudomonas rhizosphaerae DSM 16299T with an ANIb of 88.2 % and a genome-to-genome distance result of 36.0 %. The G+C content of the DNA of strains MBT-1T and MBT-2T was 60.84 and 62.48 mol%, respectively. The major fatty acids were C16 : 1 ω7c (summed feature 3), C16 : 0 and C18 : 1 ω7c (summed feature 8). The strains were catalase-positive, while production of urease, ß-galactosidase and indole were negative. Growth occurred at 4-30 °C and at pH values of pH 6.0-8.0. Based on these results, we conclude that the strains belong to two novel species, for which the names Pseudomonas kielensis sp. nov. and Pseudomonas baltica sp. nov. are proposed. The type strains are MBT-1T (=DSM 111668 T= LMG 31954T) and MBT-2T (=DSM 111761 T=LMG 31955T).

Coping with anoxia: a comprehensive proteomic and transcriptomic survey of denitrification.

Ralstonia eutropha H16 is a denitrifying microorganism able to use nitrate and nitrite as terminal electron acceptors under oxygen deprivation. To identify proteins showing an altered expression pattern in response to oxygen supply, R. eutropha cells grown aerobically and anaerobically were compared in a comprehensive proteome and transcriptome approach. Nearly 700 proteins involved in several processes including respiration, formation of cell appendages, and DNA and cofactor biosynthesis were found to be differentially expressed. A combination of 1D gel-LC and conventional 2D gel analysis of six consecutive sample points covering the entire denitrification sequence revealed a detailed view on the shifting abundance of the key proteins of denitrification. Denitrification- or anaerobiosis-induced alterations of the respiratory chain included a distinct expression pattern for multiple terminal oxidases. Alterations in the central carbon metabolism were restricted to a few key functions including the isoenzymes for aconitase and isocitrate dehydrogenase. Although R. eutropha is a strictly respiratory bacterium, the abundance of certain fermentation enzymes was increased. This work represents a comprehensive survey of denitrification on the proteomic and transcriptomic levels and provides unique insight into how R. eutropha adapts its metabolism to low oxygen conditions.

A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum--solvent stress caused by a transient n-butanol pulse.

The main product of the anaerobic fermentative bacterium Clostridium acetobutylicum is n-butanol, an organic solvent with severe toxic effects on the cells. Therefore, the identification of the molecular factors related to n-butanol stress constitutes a major strategy for furthering the understanding of the biotechnological production of n-butanol, an important industrial biofuel. Previous reports concerning n-butanol stress in C. acetobutylicum dealt exclusively with batch cultures. In this study, for the first time a comprehensive transcriptional analysis of n-butanol-stressed C. acetobutylicum was conducted using stable steady state acidogenic chemostat cultures. A total of 358 differentially expressed genes were significantly affected by n-butanol stress. Similarities, such as the upregulation of general stress genes, and differences in gene expression were compared in detail with earlier DNA microarrays performed in batch cultivation experiments. The main result of this analysis was the observation that genes involved in amino acid and nucleotide biosynthesis, as well as genes for specific transport systems were upregulated by n-butanol. Our results exclude any transcriptional response triggered by exogenous pH changes or solventogenic n-butanol formation. Finally, our data suggest that metabolic flux through the glycerolipid biosynthetic pathway increases, confirming that C. acetobutylicum modifies the cytoplasmic membrane composition in response to n-butanol stress.

A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum--cellular behavior in adaptation to n-butanol.

To gain more insight into the butanol stress response of Clostridium acetobutylicum the transcriptional response of a steady state acidogenic culture to different levels of n-butanol (0.25-1%) was investigated. No effect was observed on the fermentation pattern and expression of typical solvent genes (aad, ctfA/B, adc, bdhA/B, ptb, buk). Elevated levels of butanol mainly affected class I heat-shock genes (hrcA, grpE, dnaK, dnaJ, groES, groEL, hsp90), which were upregulated in a dose- and time-dependent manner, and genes encoding proteins involved in the membrane composition (fab and fad or glycerophospholipid related genes) and various ABC-transporters of unknown specificity. Interestingly, fab and fad genes were embedded in a large, entirely repressed cluster (CAC1988-CAC2019), which inter alia encoded an iron-specific ABC-transporter and molybdenum-cofactor synthesis proteins. Of the glycerophospholipid metabolism, the glycerol-3-phosphate dehydrogenase (glpA) gene was highly upregulated, whereas a glycerophosphodiester ABC-transporter (ugpAEBC) and a phosphodiesterase (ugpC) were repressed. On the megaplasmid, only a few genes showed differential expression, e.g. a rare lipoprotein (CAP0058, repressed) and a membrane protein (CAP0102, upregulated) gene. Observed transcriptional responses suggest that C. acetobutylicum reacts to butanol stress by induction of the general stress response and changing its cell envelope and transporter composition, but leaving the central catabolism unaffected.

Global transcriptional changes of Clostridium acetobutylicum cultures with increased butanol:acetone ratios.

Artificial electron carriers have been widely used to shift the solvent ratio toward butanol in acetone-butanol-ethanol (ABE) fermentation of solventogenic clostridia according to decreased hydrogen production. In this study, first insights on the molecular level were gained to explore the effect of methyl viologen addition to cultures of Clostridium acetobutylicum. Employing batch fermentation in mineral salts medium, the butanol:acetone ratio was successively increased from 2.3 to 12.4 on a 100-ml scale in serum bottles and from 1.4 to 16.5 on a 1300-ml scale in bioreactors, respectively. The latter cultures were used for DNA microarray analyses to provide new information on the transcriptional changes referring to methyl viologen exposure and thus, exhibit gene expression patterns according to the manipulation of the cellular redox balance. Methyl viologen-exposed cultures revealed lower expression levels of the sol operon (CAP0162-0164) and the adjacent adc gene (CAP0165) responsible for solvent formation as well as iron and sulfate transporters and the CAC0105-encoded ferredoxin. On the contrary, genes for riboflavin biosynthesis, for the butyrate/butanol metabolic pathway and genes coding for sugar transport systems were induced. Interestingly, the adhE2-encoded bifunctional NADH-dependent aldhehyde/alcohol-dehydrogenase (CAP0035) was upregulated up to more than 100-fold expression levels as compared to the control culture without methyl viologen addition. The data presented here indicate a transcriptional regulation for decreased acetone biosynthesis and the redox-dependent substitution of adhE1 (CAP0162) by adhE2.

Genome-wide gene expression analysis of the switch between acidogenesis and solventogenesis in continuous cultures of Clostridium acetobutylicum.

Clostridium acetobutylicum is able to switch from acidogenic growth to solventogenic growth. We used phosphate-limited continuous cultures that established acidogenic growth at pH 5.8 and solventogenic growth at pH 4.5. These cultures allowed a detailed transcriptomic study of the switch from acidogenesis to solventogenesis that is not superimposed by sporulation and other growth phase-dependent parameters. These experiments led to new insights into the physiological role of several genes involved in solvent formation. The adc gene for acetone decarboxylase is upregulated well before the rest of the sol locus during the switch, and pyruvate decarboxylase is induced exclusively for the period of this switch. The aldehyde-alcohol dehydrogenase gene adhE1 located in the sol operon is regulated antagonistically to the paralog adhE2 that is expressed during acidogenic conditions. A similar antagonistic pattern can be seen with the two paralogs of thiolase genes, thlA and thlB. Interestingly, the genes coding for the putative cellulosome in C. acetobutylicum are exclusively transcribed throughout solventogenic growth. The genes for stress response are only induced during the shift but not in the course of solventogenesis when butanol is present in the culture. Finally, the data clearly indicate that solventogenesis is independent from sporulation.

Transcriptional analysis of catabolite repression in Clostridium acetobutylicum growing on mixtures of D-glucose and D-xylose.

Clostridium acetobutylicum is a strict anaerobic organism that is used for biotechnological butanol fermentation. It ferments various hexoses and pentoses to solvents but prefers glucose presumably using a catabolite repression mechanism. Accordingly during growth on a mixture of D-glucose and D-xylose a typical diauxic growth pattern was observed. We used DNA microarrays and real-time RT-PCR to study gene expression during growth on D-glucose, D-xylose mixtures on a defined minimal medium together with monitoring substrate consumption and product formation. We identified two putative operons involved in D-xylose degradation. The first operon (CAC1344-CAC1349) includes a transporter, a xylulose-kinase, a transaldolase, a transketolase, an aldose-1-epimerase and a putative xylose isomerase that has been annotated as an arabinose isomerase. This operon is induced by D-xylose but was catabolite repressed by D-glucose. A second operon (CAC2610-CAC2612) consists of a xylulose-kinase, a hypothetical protein and a gene that has been annotated as a L-fucose isomerase that might in fact code for a xylose isomerase. This operon was induced by D-xylose but was not subject to catabolite repression. In accordance with these results we identified a CRE site in the catabolite repressed operon but not in the operon that was not subject to catabolite repression.