Andean soil-derived lignocellulolytic bacterial consortium as a source of novel taxa and putative plastic-active enzymes.

An easy and straightforward way to engineer microbial environmental communities is by setting up liquid enrichment cultures containing a specific substrate as the sole source of carbon. Here, we analyzed twenty single-contig high-quality metagenome-assembled genomes (MAGs) retrieved from a microbial consortium (T6) that was selected by the dilution-to-stimulation approach using Andean soil as inoculum and lignocellulose as a selection pressure. Based on genomic metrics (e.g., average nucleotide and amino acid identities) and phylogenomic analyses, 15 out of 20 MAGs were found to represent novel bacterial species, with one of those (MAG_26) belonging to a novel genus closely related to Caenibius spp. (Sphingomonadaceae). Following the rules and requirements of the SeqCode, we propose the name Andeanibacterium colombiense gen. nov., sp. nov. for this taxon. A subsequent functional annotation of all MAGs revealed that MAG_7 (Pseudobacter hemicellulosilyticus sp. nov.) contains 20, 19 and 16 predicted genes from carbohydrate-active enzymes families GH43, GH2 and GH92, respectively. Its lignocellulolytic gene profile resembles that of MAG_2 (the most abundant member) and MAG_3858, both of which belong to the Sphingobacteriaceae family. Using a database that contains experimentally verified plastic-active enzymes (PAZymes), twenty-seven putative bacterial polyethylene terephthalate (PET)-active enzymes (i.e., alpha/beta-fold hydrolases) were detected in all MAGs. A maximum of five putative PETases were found in MAG_3858, and two PETases were found to be encoded by A. colombiense. In conclusion, we demonstrate that lignocellulose-enriched liquid cultures coupled with genome-resolved metagenomics are suitable approaches to unveil the hidden bacterial diversity and its polymer-degrading potential in Andean soil ecosystems.

The Restriction-Modification Systems of Clostridium carboxidivorans P7.

Clostridium carboxidivorans P7 (DSM 15243) is a bacterium that converts syngas (a mixture of CO, H2, and CO2) into hexanol. An optimized and scaled-up industrial process could therefore provide a renewable source of fuels and chemicals while consuming industry waste gases. However, the genetic engineering of this bacterium is hindered by its multiple restriction-modification (RM) systems: the genome of C. carboxidivorans encodes at least ten restriction enzymes and eight methyltransferases (MTases). To gain insight into the complex RM systems of C. carboxidivorans, we analyzed genomic methylation patterns using single-molecule real-time (SMRT) sequencing and bisulfite sequencing. We identified six methylated sequence motifs. To match the methylation sites to the predicted MTases of C. carboxidivorans, we expressed them individually in Escherichia coli for functional characterization. Recognition motifs were identified for all three Type I MTases (CAYNNNNNCTGC/GCAGNNNNNRTG, CCANNNNNNNNTCG/CGANNNNNNNNTGG and GCANNNNNNNTNNCG/CGNNANNNNNNNTGC), two Type II MTases (GATAAT and CRAAAAR), and a single Type III MTase (GAAAT). However, no methylated recognition motif was found for one of the three Type II enzymes. One recognition motif that was methylated in C. carboxidivorans but not in E. coli (AGAAGC) was matched to the remaining Type III MTase through a process of elimination. Understanding these enzymes and the corresponding recognition sites will facilitate the development of genetic tools for C. carboxidivorans that can accelerate the industrial exploitation of this strain.

Multi-omics analysis reveals the molecular response to heat stress in a "red tide" dinoflagellate.

BACKGROUND: "Red tides" are harmful algal blooms caused by dinoflagellate microalgae that accumulate toxins lethal to other organisms, including humans via consumption of contaminated seafood. These algal blooms are driven by a combination of environmental factors including nutrient enrichment, particularly in warm waters, and are increasingly frequent. The molecular, regulatory, and evolutionary mechanisms that underlie the heat stress response in these harmful bloom-forming algal species remain little understood, due in part to the limited genomic resources from dinoflagellates, complicated by the large sizes of genomes, exhibiting features atypical of eukaryotes. RESULTS: We present the de novo assembled genome (~ 4.75 Gbp with 85,849 protein-coding genes), transcriptome, proteome, and metabolome from Prorocentrum cordatum, a globally abundant, bloom-forming dinoflagellate. Using axenic algal cultures, we study the molecular mechanisms that underpin the algal response to heat stress, which is relevant to current ocean warming trends. We present the first evidence of a complementary interplay between RNA editing and exon usage that regulates the expression and functional diversity of biomolecules, reflected by reduction in photosynthesis, central metabolism, and protein synthesis. These results reveal genomic signatures and post-transcriptional regulation for the first time in a pelagic dinoflagellate. CONCLUSIONS: Our multi-omics analyses uncover the molecular response to heat stress in an important bloom-forming algal species, which is driven by complex gene structures in a large, high-G+C genome, combined with multi-level transcriptional regulation. The dynamics and interplay of molecular regulatory mechanisms may explain in part how dinoflagellates diversified to become some of the most ecologically successful organisms on Earth.

Thiorhodovibrio frisius and Trv. litoralis spp. nov., Two Novel Members from a Clade of Fastidious Purple Sulfur Bacteria That Exhibit Unique Red-Shifted Light-Harvesting Capabilities.

In the pursuit of cultivating anaerobic anoxygenic phototrophs with unusual absorbance spectra, a purple sulfur bacterium was isolated from the shoreline of Baltrum, a North Sea island of Germany. It was designated strain 970, due to a predominant light harvesting complex (LH) absorption maximum at 963-966 nm, which represents the furthest infrared-shift documented for such complexes containing bacteriochlorophyll a. A polyphasic approach to bacterial systematics was performed, comparing genomic, biochemical, and physiological properties. Strain 970 is related to Thiorhodovibrio winogradskyi DSM 6702T by 26.5, 81.9, and 98.0% similarity via dDDH, ANI, and 16S rRNA gene comparisons, respectively. The photosynthetic properties of strain 970 were unlike other Thiorhodovibrio spp., which contained typical LH absorbing characteristics of 800-870 nm, as well as a newly discovered absorption band at 908 nm. Strain 970 also had a different photosynthetic operon composition. Upon genomic comparisons with the original Thiorhodovibrio strains DSM 6702T and strain 06511, the latter was found to be divergent, with 25.3, 79.1, and 97.5% similarity via dDDH, ANI, and 16S rRNA gene homology to Trv. winogradskyi, respectively. Strain 06511 (=DSM 116345T) is thereby described as Thiorhodovibrio litoralis sp. nov., and the unique strain 970 (=DSM 111777T) as Thiorhodovibrio frisius sp. nov.

Ongoing diversification of the global fish pathogen Piscirickettsia salmonis through genetic isolation and transposition bursts.

The management of bacterial pathogens remains a key challenge of aquaculture. The marine gammaproteobacterium Piscirickettsia salmonis is the etiological agent of piscirickettsiosis and causes multi-systemic infections in different salmon species, resulting in considerable mortality and substantial commercial losses. Here, we elucidate its global diversity, evolution, and selection during human interventions. Our comprehensive analysis of 73 closed, high quality genome sequences covered strains from major outbreaks and was supplemented by an analysis of all P. salmonis 16S rRNA gene sequences and metagenomic reads available in public databases. Genome comparison showed that Piscirickettsia comprises at least three distinct, genetically isolated species of which two showed evidence for continuing speciation. However, at least twice the number of species exist in marine fish or seawater. A hallmark of Piscirickettsia diversification is the unprecedented amount and diversity of transposases which are particularly active in subgroups undergoing rapid speciation and are key to the acquisition of novel genes and to pseudogenization. Several group-specific genes are involved in surface antigen synthesis and may explain the differences in virulence between strains. However, the frequent failure of antibiotic treatment of piscirickettsiosis outbreaks cannot be explained by horizontal acquisition of resistance genes which so far occurred only very rarely. Besides revealing a dynamic diversification of an important pathogen, our study also provides the data for improving its surveillance, predicting the emergence of novel lineages, and adapting aquaculture management, and thereby contributes towards the sustainability of salmon farming.

Nocardia australiensis sp. nov. and Nocardia spumae sp. nov., isolated from sea foam in Queensland, Australia.

Strains USC-21046T and USC-21048T were isolated from foaming coastal marine waters on the Sunshine Coast, Queensland, Australia. Both strains displayed growth and morphological characteristics typical for members belonging to the genus Nocardia. The major polar lipids were diphosphatidylglycerol and phosphatidylethanolamine, and the major fatty acids were C16 : 0, C18 : 1 ω9c, C18 : 0 and C18 : 0 10-methyl. The mycolic acids of strains USC-21046T and USC-21048T consisted of chain lengths between 50-64 and 56-68, respectively. Moreover, both of those strains contained meso-diaminopimelic acid and ribose, arabinose, glucose and galactose as whole cell sugars. Based on the phylogenomic results, both strains belonged to the genus Nocardia with strain USC-21046T showing an 80.4 % genome similarity to N. vinacea NBRC 16497T and N. pseudovaccinii NBRC 100343T, whereas USC-21048T strain showed an 83.6 % genome similarity to N. aobensis NBRC 100429T. Both strains were delineated from their closely related relatives based on physiological (e.g. growth on sole carbon source) and chemotaxonomic (e.g. cellular fatty composition) differences. The digital DNA-DNA hybridization (dDDH) values between USC-21046T and USC-21048T and their closely related relatives were below the dDDH threshold value of ≤70 % used for the taxonomic classification of novel species status. The genome length of strains USC-21046T and USC-21048T were 6 878 863 and 7 066 978 bp, with G+C contents of 65.2 and 67.8 mol%, respectively. For the novel isolates, we propose the names Nocardia australiensis sp. nov. with the type strain USC-21046T (=DSM 111727T=NCCB 100867T) and Nocardia spumae sp. nov. with the type strain USC-21048T (=DSM 111726T=NCCB 100868T).

Naturally induced biphasic phytoplankton spring bloom reveals rapid and distinct substrate and bacterial community dynamics.

Phytoplankton spring blooms are typical features in coastal seas and provide heterotrophic bacteria with a rich blend of dissolved substrates. However, they are difficult to study in coastal seas in-situ. Here, we induced a phytoplankton spring bloom and followed its fate for 37 days in four 600 L-mesocosms. To specifically investigate the significance of phytoplankton-born dissolved organic carbon (DOC) we used artificial seawater with low DOC background and inoculated it with a 100 µm-prefiltered plankton community from the North Sea. A biphasic bloom developed, dominated by diatoms and Phaeocystis globosa respectively. In between, bacterial numbers peaked, followed by a peak in virus-like particles, implying that virus infection caused the collapse. Concentrations of dissolved free amino acids exhibited rapid changes, in particular during the diatom bloom and until the peak in bacterial abundance. Dissolved combined amino acids and neutral monosaccharides accumulated continuously, accounting for 22% of DOC as a mean and reaching levels as high as 44%. Bacterial communities were largely dominated by Bacteroidetes, especially the NS3a marine group (family Flavobacteriaceae), but Rhodobacteraceae and Gammaproteobacteria were also prominent members. Our study shows rapid organic matter and community composition dynamics that are hard to trace in natural coastal ecosystems.

Rare Halophilic Nocardiopsis from Algerian Saharan Soils as Tools for Biotechnological Processes in Pharmaceutical Industry.

The Sahara Desert, one of the most extreme ecosystems in the planet, constitutes an unexplored source of microorganisms such as mycelial bacteria. In this study, we investigated the diversity of halophilic actinobacteria in soils collected from five regions of the Algerian Sahara. A total of 23 halophilic actinobacterial strains were isolated by using a humic-vitamin agar medium supplemented with 10% NaCl. The isolated halophilic strains were subjected to taxonomic analysis using a polyphasic approach, which included morphological, chemotaxonomic, physiological (numerical taxonomy), and phylogenetic analyses. The isolates showed abundant growth in CMA (complex medium agar) and TSA (tryptic soy agar) media containing 10% NaCl, and chemotaxonomic characteristics were consistent with their assignment to the genus Nocardiopsis. Analysis of the 16S rRNA sequence of 23 isolates showed five distinct clusters and a similarity level ranging between 98.4% and 99.8% within the Nocardiopsis species. Comparison of their physiological characteristics with the nearest species showed significant differences with the closely related species. Halophilic Nocardiopsis isolated from Algerian Sahara soil represents a distinct phyletic line suggesting a potential new species. Furthermore, the isolated strains of halophilic Nocardiopsis were screened for their antagonistic properties against a broad spectrum of microorganisms by the conventional agar method (agar cylinders method) and found to have the capacity to produce bioactive secondary metabolites. Except one isolate (AH37), all isolated Nocardiopsis showed moderate to high biological activities against Pseudomonas syringae and Salmonella enterica, and some isolates showed activities against Agrobacterium tumefaciens, Serratia marcescens, and Klebsiella pneumoniae. However, no isolates were active against Bacillus subtilis, Aspergillus flavus, or Aspergillus niger. The obtained finding implies that the unexplored extreme environments such as the Sahara contain many new bacterial species as a novel drug source for medical and industrial applications.

Vibrio syngnathi sp. nov., a fish pathogen, isolated from the Kiel Fjord.

A new Vibrio strain, K08M4T, was isolated from the broad-nosed pipefish Syngnathus typhle in the Kiel Fjord. Infection experiments revealed that K08M4T was highly virulent for juvenile pipefish. Cells of strain K08M4T were Gram-stain-negative, curved rod-shaped and motile by means of a single polar flagellum. The strain grew aerobically at 9-40° C, at pH 4-10.5 and it tolerated up to 12 % (w/v) NaCl. The most prevalent (>10 %) cellular fatty acids of K08M4T were C16 : 1 ω7c and C16 : 0. Whole-genome comparisons revealed that K08M4T represents a separate evolutionary lineage that is distinct from other Vibrio species and falls within the Splendidus clade. The genome is 4,886,292 bp in size, consists of two circular chromosomes (3,298,328 and 1, 587,964 bp) and comprises 4,178 protein-coding genes and 175 RNA genes. In this study, we describe the phenotypic features of the new isolate and present the annotation and analysis of its complete genome sequence. Based on these data, the new isolate represents a new species for which we propose the name Vibrio syngnathi sp. nov. The type strain is K08M4T (=DSM 109818T=CECT 30086T).

Arcobacter roscoffensis sp. nov., a marine bacterium isolated from coastal seawater.

A novel Gram-negative, aerobic, motile, rod-shaped, beige-pigmented bacterium, strain ARW1-2F2T, was isolated from a seawater sample collected from Roscoff, France. Strain ARW1-2F2T was catalase-negative and oxidase-positive, and grew under mesophilic, neutrophilic and halophilic conditions. The 16S rRNA sequences revealed that strain ARW1-2F2T was closely related to Arcobacter lekithochrous LFT 1.7T and Arcobacter caeni RW17-10T(95.8 and 95.5 % gene sequence similarity, respectively). The genome of strain ARW1-2F2T was sequenced and had a G+C content of 28.7%. Two different measures of genome similarity, average nucleotide identity based on blast and digital DNA-DNA hybridization, indicated that strain ARW1-2F2T represents a new Arcobacter species. The predominant fatty acids were C16 : 1 ω7c/C16 : 1 ω6c and C18 : 1 ω7c/C18 : 1 ω6c. The results of a polyphasic analysis supported the description of strain ARW1-2F2T as representing a novel species of the genus Arcobacter, for which the name Arcobacter roscoffensis sp. nov. is proposed with the type strain ARW1-2F2T (DSM 29169T=KCTC 52423T).

Genomics of the "tumorigenes" clade of the family Rhizobiaceae and description of Rhizobium rhododendri sp. nov.

Tumorigenic members of the family Rhizobiaceae, known as agrobacteria, are responsible for crown and cane gall diseases of various crops worldwide. Tumorigenic agrobacteria are commonly found in the genera Agrobacterium, Allorhizobium, and Rhizobium. In this study, we analyzed a distinct "tumorigenes" clade of the genus Rhizobium, which includes the tumorigenic species Rhizobium tumorigenes, as well as strains causing crown gall disease on rhododendron. Here, high-quality, closed genomes of representatives of the "tumorigenes" clade were generated, followed by comparative genomic and phylogenomic analyses. Additionally, the phenotypic characteristics of representatives of the "tumorigenes" clade were analyzed. Our results showed that the tumorigenic strains isolated from rhododendron represent a novel species of the genus Rhizobium for which the name Rhizobium rhododendri sp. nov. is proposed. This species also includes additional strains originating from blueberry and Himalayan blackberry in the United States, whose genome sequences were retrieved from GenBank. Both R. tumorigenes and R. rhododendri contain multipartite genomes, including a chromosome, putative chromids, and megaplasmids. Synteny and phylogenetic analyses indicated that a large putative chromid of R. rhododendri resulted from the cointegration of an ancestral megaplasmid and two putative chromids, following its divergence from R. tumorigenes. Moreover, gene clusters specific for both species of the "tumorigenes" clade were identified, and their biological functions and roles in the ecological diversification of R. rhododendri and R. tumorigenes were predicted and discussed.

Structural genome variants of Pseudomonas aeruginosa clone C and PA14 strains.

Plasticity of Pseudomonas aeruginosa chromosomes is mainly driven by an extended accessory genome that is shaped by insertion and deletion events. Further modification of the genome composition can be induced by chromosomal inversion events which lead to relocation of genes in the affected genomic DNA segments, modify the otherwise highly conserved core genome synteny and could even alter the location of the replication terminus. Although the genome of the first sequenced strain, PAO1, displayed such a large genomic inversion, knowledge on such recombination events in the P. aeruginosa population is limited. Several large inversions had been discovered in the late 1990s in cystic fibrosis isolates of the major clonal lineage C by physical genome mapping, and subsequent work on these examples led to the characterization of the DNA at the recombination breakpoints and a presumed recombination mechanism. Since then, the topic was barely addressed in spite of the compilation of thousands of P. aeruginosa genome sequences that are deposited in databases. Due to the use of second-generation sequencing, genome contig assembly had usually followed synteny blueprints provided by the existing reference genome sequences. Inversion detection was not feasible by these approaches, as the respective read lengths did not allow reliable resolution of sequence repeats that are typically found at the borders of inverted segments. In this study, we applied PacBio and MinION long-read sequencing to isolates of the mentioned clone C collection. Confirmation of inversions predicted from the physical mapping data demonstrated that unbiased sequence assembly of such read datasets allows the detection of genomic inversions and the resolution of the recombination breakpoint regions. Additional long-read sequencing of representatives of the other major clonal lineage, PA14, revealed large inversions in several isolates, from cystic fibrosis origin as well as from other sources. These findings indicated that inversion events are not restricted to strains from chronic infection background, but could be widespread in the P. aeruginosa population and contribute to genome plasticity. Moreover, the monitored examples emphasized the role of small mobile DNA units, such as IS elements or transposons, and accessory DNA elements in the inversion-related recombination processes.

Comparative Genomics of Legionella pneumophila Isolates from the West Bank and Germany Support Molecular Epidemiology of Legionnaires' Disease.

Legionella pneumophila is an environmental bacterium and clinical pathogen that causes many life-threating outbreaks of an atypical pneumonia called Legionnaires' disease (LD). Studies of this pathogen have focused mainly on Europe and the United States. A shortage in L. pneumophila data is clearly observed for developing countries. To reduce this knowledge gap, L. pneumophila isolates were studied in two widely different geographical areas, i.e., the West Bank and Germany. For this study, we sequenced and compared the whole genome of 38 clinical and environmental isolates of L. pneumophila covering different MLVA-8(12) genotypes in the two areas. Sequencing was conducted using the Illumina HiSeq 2500 platform. In addition, two isolates (A194 and H3) were sequenced using a Pacific Biosciences (PacBio) RSII platform to generate complete reference genomes from each of the geographical areas. Genome sequences from 55 L. pneumophila strains, including 17 reference strains, were aligned with the genome sequence of the closest strain (L. pneumophila strain Alcoy). A whole genome phylogeny based on single nucleotide polymorphisms (SNPs) was created using the ParSNP software v 1.0. The reference genomes obtained for isolates A194 and H3 consisted of circular chromosomes of 3,467,904 bp and 3,691,263 bp, respectively. An average of 36,418 SNPs (min. 8569, max. 70,708 SNPs) against our reference strain L. pneumophila str. Alcoy, and 2367 core-genes were identified among the fifty-five strains. An analysis of the genomic population structure by SNP comparison divided the fifty-five L. pneumophila strains into six branches. Individual isolates in sub-lineages in these branches differed by less than 120 SNPs if they had the same MLVA genotype and were isolated from the same location. A bioinformatics analysis identified the genomic islands (GIs) for horizontal gene transfer and mobile genetic elements, demonstrating that L. pneumophila showed high genome plasticity. Four L. pneumophila isolates (H3, A29, A129 and L10-091) contained well-defined plasmids. On average, only about half of the plasmid genes could be matched to proteins in databases. In silico phage findings suggested that 43 strains contained at least one phage. However, none of them were found to be complete. BLASTp analysis of proteins from the type IV secretion Dot/Icm system showed those proteins highly conserved, with less than 25% structural differences in the new L. pneumophila isolates. Overall, we demonstrated that whole genome sequencing provides a molecular surveillance tool for L. pneumophila at the highest conceivable discriminatory level, i.e., two to eight SNPs were observed for isolates from the same location but several years apart.

Molecular characterization of Arcobacter butzleri isolates from poultry in rural Ghana.

In recent years, Arcobacter butzleri has gained clinical significance as an emerging diarrheagenic pathogen associated with poultry and water reservoirs. The full clinical significance of Arcobacter remains rather speculative due to variable virulence and antibiotic susceptibility of individual strains. The aims of the present study were (i) to identify antibiotic resistance genes (ARGs) in the genome sequences of two multidrug-resistant A. butzleri isolates, (ii) to use multilocus-sequence typing (MLST) to generate a guiding phylogeny of A. butzleri isolates collected in Kumasi, Ghana, (iii) to examine the distribution of ARGs in the test cohort, and (iv) to assess the strain's virulence and possible antibiotic treatment options for arcobacteriosis based on the genome sequences and the ARG distribution. A total of 48 A. butzleri isolates obtained from poultry were included in the analysis. These isolates were genotyped by MLST and the antibiotic susceptibilities of isolates to ampicillin, ciprofloxacin, tetracycline, gentamicin, and erythromycin were tested by disk diffusion. Whole genome sequence data of two multidrug-resistant (MDR) A. butzleri isolates were obtained by a combination of single-molecule real-time (SMRT) and Illumina sequencing technology. A total of 14 ARGs were identified in the two generated genome sequences. For all 48 isolates, the frequency of these 14 ARGs was investigated by PCR or amplicon sequencing. With 44 different sequence types found among 48 isolates, strains were phylogenetically heterogeneous. Four of 48 isolates showed an ARG constellation indicating a multidrug-resistant phenotype. The virulence genes in the two A. butzleri genomes showed that the species might be characterized by a somewhat lower virulence as Campylobacter species. The phenotypic susceptibility data combined with the distribution of the particular ARGs especially oxa-464 and the T81I point mutation of the quinolone resistance determining region (QRDR) in a significant percentage of isolates indicated that macrolides and tetracycline can be recommended for calculated antibiotic treatment of arcobacteriosis in Ghana, but not ampicillin and quinolones.

Aminithiophilus ramosus gen. nov., sp. nov., a sulphur-reducing bacterium isolated from a pyrite-forming enrichment culture, and taxonomic revision of the family Synergistaceae.

A novel sulphur-reducing bacterium was isolated from a pyrite-forming enrichment culture inoculated with sewage sludge from a wastewater treatment plant. Based on phylogenetic data, strain J.5.4.2-T.3.5.2T could be affiliated with the phylum Synergistota. Among type strains of species with validly published names, the highest 16S rRNA gene sequence identity value was found with Aminiphilus circumscriptus ILE-2T (89.2 %). Cells of the new isolate were Gram-negative, non-spore-forming, straight to slightly curved rods with tapered ends. Motility was conferred by lateral flagella. True branching of cells was frequently observed. The strain had a strictly anaerobic, asaccharolytic, fermentative metabolism with peptides and amino acids as preferred substrates. Sulphur was required as an external electron acceptor during fermentative growth and was reduced to sulphide, whereas it was dispensable during syntrophic growth with a Methanospirillum species. Major fermentation products were acetate and propionate. The cellular fatty acid composition was dominated by unsaturated and branched fatty acids, especially iso-C15 : 0. Its major polar lipids were phosphatidylglycerol, phosphatidylethanolamine and distinct unidentified polar lipids. Respiratory lipoquinones were not detected. Based on the obtained data we propose the novel species and genus Aminithiophilus ramosus, represented by the type strain J.5.4.2-T.3.5.2T (=DSM 107166T=NBRC 114655T) and the novel family Aminithiophilaceae fam. nov. to accommodate the genus Aminithiophilus. In addition, we suggest reclassifying certain members of the Synergistaceae into new families to comply with current standards for the classification of higher taxa. Based on phylogenomic data, the novel families Acetomicrobiaceae fam. nov., Aminiphilaceae fam. nov., Aminobacteriaceae fam. nov., Dethiosulfovibrionaceae fam. nov. and Thermovirgaceae fam. nov. are proposed.

Complete Genome Sequence of Nocardia iowensis DSM 45197T (= NRRL 5646T).

We report the complete genome sequence and predicted functional profile of Nocardia iowensis DSM 45197T. N. iowensis DSM 45197T is a spore-forming, mesophilic, Gram-positive bacterium that was isolated from garden soil in Osceola, Iowa, USA. This organism has been exploited for its production of glycocinnamolyspermidine antibiotics and biotransformation of xenobiotic substances. Other significant features of N. iowensis DSM 45197T include the first fully characterized carboxylic acid reductase (CAR) and the first bacterial nitric oxide synthase system. The genome sequence of N. iowensis DSM 45197T can facilitate further understanding of its function, as well as the pathogenesis of Nocardia spp. N. iowensis DSM 45197T has a genome size of 8.95 Mbp; about 46% of the coding sequences have no known homologues and were labeled hypothetical proteins. This finding implies further potential for biomedical and biotechnological research applications.

The novel oligopeptide utilizing species Anaeropeptidivorans aminofermentans M3/9T, its role in anaerobic digestion and occurrence as deduced from large-scale fragment recruitment analyses.

Research on biogas-producing microbial communities aims at elucidation of correlations and dependencies between the anaerobic digestion (AD) process and the corresponding microbiome composition in order to optimize the performance of the process and the biogas output. Previously, Lachnospiraceae species were frequently detected in mesophilic to moderately thermophilic biogas reactors. To analyze adaptive genome features of a representative Lachnospiraceae strain, Anaeropeptidivorans aminofermentans M3/9T was isolated from a mesophilic laboratory-scale biogas plant and its genome was sequenced and analyzed in detail. Strain M3/9T possesses a number of genes encoding enzymes for degradation of proteins, oligo- and dipeptides. Moreover, genes encoding enzymes participating in fermentation of amino acids released from peptide hydrolysis were also identified. Based on further findings obtained from metabolic pathway reconstruction, M3/9T was predicted to participate in acidogenesis within the AD process. To understand the genomic diversity between the biogas isolate M3/9T and closely related Anaerotignum type strains, genome sequence comparisons were performed. M3/9T harbors 1,693 strain-specific genes among others encoding different peptidases, a phosphotransferase system (PTS) for sugar uptake, but also proteins involved in extracellular solute binding and import, sporulation and flagellar biosynthesis. In order to determine the occurrence of M3/9T in other environments, large-scale fragment recruitments with the M3/9T genome as a template and publicly available metagenomes representing different environments was performed. The strain was detected in the intestine of mammals, being most abundant in goat feces, occasionally used as a substrate for biogas production.

Two New Rhizobiales Species Isolated from Root Nodules of Common Sainfoin (Onobrychis viciifolia) Show Different Plant Colonization Strategies.

Root nodules of legume plants are primarily inhabited by rhizobial nitrogen-fixing bacteria. Here, we propose two new Rhizobiales species isolated from root nodules of common sainfoin (Onobrychis viciifolia), as shown by core-gene phylogeny, overall genome relatedness indices, and pan-genome analysis. Mesorhizobium onobrychidis sp. nov. actively induces nodules and achieves atmospheric nitrogen and carbon dioxide fixation. This species appears to be depleted in motility genes and is enriched in genes for direct effects on plant growth performance. Its genome reveals functional and plant growth-promoting signatures, like a large unique chromosomal genomic island with high density of symbiotic genetic traits. Onobrychidicola muellerharveyae gen. nov. sp. nov. is described as a type species of the new genus Onobrychidicola in Rhizobiaceae. This species comprises unique genetic features and plant growth-promoting traits (PGPTs), which strongly indicate its function in biotic stress reduction and motility. We applied a newly developed bioinformatics approach for in silico prediction of PGPTs (PGPT-Pred), which supports the different lifestyles of the two new species and the plant growth-promoting performance of M. onobrychidis in the greenhouse trial. IMPORTANCE The intensive use of chemical fertilizers has a variety of negative effects on the environment. Increased utilization of biological nitrogen fixation (BNF) is one way to mitigate those negative impacts. In order to optimize BNF, suitable candidates for different legume species are required. Despite intensive search for new rhizobial bacteria associated with legumes, no new rhizobia have recently been identified from sainfoin (Onobrychis viciifolia). Here, we report on the discovery of two new rhizobial species associated with sainfoin, which are of high importance for the host and may help to increase sustainability in agricultural practices. We employed the combination of in silico prediction and in planta experiments, which is an effective way to detect promising plant growth-promoting bacteria.

The Sixth Element: a 102-kb RepABC Plasmid of Xenologous Origin Modulates Chromosomal Gene Expression in Dinoroseobacter shibae.

The model organism Dinoroseobacter shibae and many other marine Rhodobacterales (Roseobacteraceae, Alphaproteobacteria) are characterized by a multipartite genome organization. Here, we show that the original isolate (Dshi-6) contained six extrachromosomal replicons (ECRs), whereas the strain deposited at the DSMZ (Dshi-5) lacked a 102-kb plasmid. To determine the role of the sixth plasmid, we investigated the genomic and physiological differences between the two strains. Therefore, both genomes were (re)sequenced, and gene expression, growth, and substrate utilization were examined. For comparison, we included additional plasmid-cured strains in the analysis. In the Dshi-6 population, the conjugative 102-kb RepABC-9 plasmid was present in only about 50% of the cells, irrespective of its experimentally validated stability. In the presence of the sixth plasmid, copy number changes of other ECRs, in particular, a decrease of the 86-kb plasmid, were observed. The most conspicuous finding was the strong influence of plasmids on chromosomal gene expression, especially the repression of the CtrA regulon and the activation of the denitrification gene cluster. Expression is inversely controlled by either the presence of the 102-kb plasmid or the absence of the 86-kb plasmid. We identified regulatory genes on both plasmids, i.e., a sigma 70 factor and a quorum sensing synthase, that might be responsible for these major changes. The tremendous effects that were probably even underestimated challenge the current understanding of the relevance of volatile plasmids not only for the original host but also for new recipients after conjugation. IMPORTANCE Plasmids are small DNA molecules that replicate independently of the bacterial chromosome. The common view of the role of plasmids is dominated by the accumulation of resistance genes, which is responsible for the antibiotic crisis in health care and livestock breeding. Beyond rapid adaptations to a changing environment, no general relevance for the host cell's regulome was attributed to these volatile ECRs. The current study shows for the model organism D. shibae that its chromosomal gene expression is strongly influenced by two plasmids. We provide evidence that the gain or loss of plasmids not only results in minor alterations of the genetic repertoire but also can have tremendous effects on bacterial physiology. The central role of some plasmids in the regulatory network of the host could also explain their persistence despite fitness costs, which has been described as the "plasmid paradox."

Extremophile Metal Resistance: Plasmid-Encoded Functions in Streptomyces mirabilis.

The extreme metal tolerance of up to 130 mM NiSO4 in Streptomyces mirabilis P16B-1 was investigated. Genome sequencing revealed the presence of a large linear plasmid, pI. To identify plasmid-encoded determinants of metal resistance, a newly established transformation system was used to characterize the predicted plasmid-encoded loci nreB, hoxN, and copYZ. Reintroduction into the plasmid-cured S. mirabilis ΔpI confirmed that the predicted metal transporter gene nreB constitutes a nickel resistance factor, which was further supported by its heterologous expression in Escherichia coli. In contrast, the predicted nickel exporter gene hoxN decreased nickel tolerance, while copper tolerance was enhanced. The predicted copper-dependent transcriptional regulator gene copY did not induce tolerance toward either metal. Since genes for transfer were identified on the plasmid, its conjugational transfer to the metal-sensitive Streptomyces lividans TK24 was checked. This resulted in acquired tolerance toward 30 mM nickel and additionally increased the tolerance toward copper and cobalt, while oxidative stress tolerance remained unchanged. Intracellular nickel concentrations decreased in the transconjugant strain. The high extracellular nickel concentrations allowed for biomineralization. Plasmid transfer could also be confirmed into the co-occurring actinomycete Kribbella spp. in soil microcosms. IMPORTANCE Living in extremely metal-rich environments requires specific adaptations, and often, specific metal tolerance genes are encoded on a transferable plasmid. Here, Streptomyces mirabilis P16B-1, isolated from a former mining area and able to grow with up to 130 mM NiSO4, was investigated. The bacterial chromosome, as well as a giant plasmid, was sequenced. The plasmid-borne gene nreB was confirmed to confer metal resistance. A newly established transformation system allowed us to construct a plasmid-cured S. mirabilis as well as an nreB-rescued strain in addition to confirming nreB encoding nickel resistance if heterologously expressed in E. coli. The potential of intra- and interspecific plasmid transfer, together with the presence of metal resistance factors on that plasmid, underlines the importance of plasmids for transfer of resistance factors within a bacterial soil community.