Genome-based metabolic and phylogenomic analysis of three Terrisporobacter species.

Acetogenic bacteria are of high interest for biotechnological applications as industrial platform organisms, however, acetogenic strains from the genus Terrisporobacter have hitherto been neglected. To date, three published type strains of the genus Terrisporobacter are only covered by draft genome sequences, and the genes and pathway responsible for acetogenesis have not been analyzed. Here, we report complete genome sequences of the bacterial type strains Terrisporobacter petrolearius JCM 19845T, Terrisporobacter mayombei DSM 6539T and Terrisporobacter glycolicus DSM 1288T. Functional annotation, KEGG pathway module reconstructions and screening for virulence factors were performed. Various species-specific vitamin, cofactor and amino acid auxotrophies were identified and a model for acetogenesis of Terrisporobacter was constructed. The complete genomes harbored a gene cluster for the reductive proline-dependent branch of the Stickland reaction located on an approximately 21 kb plasmid, which is exclusively found in the Terrisporobacter genus. Phylogenomic analysis of available Terrisporobacter genomes suggested a reclassification of most isolates as T. glycolicus into T. petrolearius.

Brevundimonas and Serratia as host systems for assessing associated environmental viromes and phage diversity by complementary approaches.

Focusing on visible plaques for phage isolation leaves the question if we miss the diversity of non-plaque forming phages. We addressed this question through direct plaque-based isolation by employing the new hosts Brevundimonas pondensis LVF1 and Serratia marcescens LVF3 dsDNA, ssDNA, dsRNA, and ssRNA host-associated metavirome analysis. Of the 25 distinctive dsDNA phage isolates, 14 were associated with Brevundimonas and 11 with Serratia. TEM analysis revealed that 6 were myoviruses, 18 siphoviruses and 1 podovirus, while phages infecting Brevundimonas belonged all to siphoviruses. The associated viromes suggested a higher phage diversity in summer than in winter, and dsDNA phages were the dominant group. Isolation of vB_SmaP-Kaonashi was possible after investigating the viromes associated with Serratia, demonstrating the great potential of accompanying host-associated metavirome analysis. The ssDNA virome analysis showed that the B. pondensis LVF1 host is associated with Microviridae and Inoviridae phages, although none of them were isolated. The results demonstrated that the classical isolation technique is not exhausted, leading to the isolation of new dsDNA phages. It can be further improved by combination with metavirome techniques, which revealed further diversity.

Interference and co-existence of staphylococci and Cutibacterium acnes within the healthy human skin microbiome.

Human skin is populated by trillions of microbes collectively called the skin microbiome. Staphylococcus epidermidis and Cutibacterium acnes are among the most abundant members of this ecosystem, with described roles in skin health and disease. However, knowledge regarding the health beneficial effects of these ubiquitous skin residents is still limited. Here, we profiled the staphylococcal and C. acnes landscape across four different skin sites of 30 individuals (120 skin samples) using amplicon-based next-generation sequencing. Relative abundance profiles obtained indicated the existence of phylotype-specific co-existence and exclusion scenarios. Co-culture experiments with 557 staphylococcal strains identified 30 strains exhibiting anti-C. acnes activities. Notably, staphylococcal strains were found to selectively exclude acne-associated C. acnes and co-exist with healthy skin-associated phylotypes, through regulation of the antimicrobial activity. Overall, these findings highlight the importance of skin-resident staphylococci and suggest that selective microbial interference is a contributor to healthy skin homeostasis.

Complete Genome Sequence of Bacillus frigoritolerans JHS1.

Bacillus frigoritolerans JHS1 was isolated from the soil of a tomato plant (Solanum lycopersicum). The genome consists of one circular chromosome (5,552,463 bp) and a plasmid (16,118 bp) with an overall GC content of 40.57%. Using TYGS for taxonomic classification, strain JHS1 was assigned to the species Bacillus frigoritolerans.

Phenotypic and Transcriptomic Analyses of Seven Clinical Stenotrophomonas maltophilia Isolates Identify a Small Set of Shared and Commonly Regulated Genes Involved in the Biofilm Lifestyle.

Stenotrophomonas maltophilia is one of the most frequently isolated multidrug-resistant nosocomial opportunistic pathogens. It contributes to disease progression in cystic fibrosis (CF) patients and is frequently isolated from wounds, infected tissues, and catheter surfaces. On these diverse surfaces S. maltophilia lives in single-species or multispecies biofilms. Since very little is known about common processes in biofilms of different S. maltophilia isolates, we analyzed the biofilm profiles of 300 clinical and environmental isolates from Europe of the recently identified main lineages Sgn3, Sgn4, and Sm2 to Sm18. The analysis of the biofilm architecture of 40 clinical isolates revealed the presence of multicellular structures and high phenotypic variability at a strain-specific level. Further, transcriptome analyses of biofilm cells of seven clinical isolates identified a set of 106 shared strongly expressed genes and 33 strain-specifically expressed genes. Surprisingly, the transcriptome profiles of biofilm versus planktonic cells revealed that just 9.43% ± 1.36% of all genes were differentially regulated. This implies that just a small set of shared and commonly regulated genes is involved in the biofilm lifestyle. Strikingly, iron uptake appears to be a key factor involved in this metabolic shift. Further, metabolic analyses implied that S. maltophilia employs a mostly fermentative growth mode under biofilm conditions. The transcriptome data of this study together with the phenotypic and metabolic analyses represent so far the largest data set on S. maltophilia biofilm versus planktonic cells. This study will lay the foundation for the identification of strategies for fighting S. maltophilia biofilms in clinical and industrial settings.IMPORTANCE Microorganisms living in a biofilm are much more tolerant to antibiotics and antimicrobial substances than planktonic cells are. Thus, the treatment of infections caused by microorganisms living in biofilms is extremely difficult. Nosocomial infections (among others) caused by S. maltophilia, particularly lung infection among CF patients, have increased in prevalence in recent years. The intrinsic multidrug resistance of S. maltophilia and the increased tolerance to antimicrobial agents of its biofilm cells make the treatment of S. maltophilia infection difficult. The significance of our research is based on understanding the common mechanisms involved in biofilm formation of different S. maltophilia isolates, understanding the diversity of biofilm architectures among strains of this species, and identifying the differently regulated processes in biofilm versus planktonic cells. These results will lay the foundation for the treatment of S. maltophilia biofilms.

Phage vB_BveM-Goe7 represents a new genus in the subfamily Bastillevirinae.

Bacillus velezensis FZB42 is a Gram-positive, endospore-forming rhizobacterium that is associated with plant roots and promotes plant growth. It was used as host to isolate phage vB_BveM-Goe7 (Goe7). Goe7 exhibits a Myoviridae morphology with a contractile tail and an icosahedral head. Its genome is 158,674 bp in size and contains 5137-bp-long terminal repeats (LTRs). It also contains five tRNA-encoding genes and 251 coding DNA sequences (CDS), of which 65 were annotated. The adsorption constant of Goe7 is 6.1 ± 0.24 × 10-8 ml/min, with a latency period of 75 min and a burst size of 114 particles per burst. A BLASTn sequence comparison against the non-redundant nucleotide database of NCBI revealed that Goe7 is most similar to Bacillus subtilis phage vB_BsuM-Goe3.

Proteomic and transcriptomic elucidation of the mutant ralstonia eutropha G+1 with regard to glucose utilization.

By taking advantage of the available genome sequence of Ralstonia eutropha H16, glucose uptake in the UV-generated glucose-utilizing mutant R. eutropha G(+)1 was investigated by transcriptomic and proteomic analyses. Data revealed clear evidence that glucose is transported by a usually N-acetylglucosamine-specific phosphotransferase system (PTS)-type transport system, which in this mutant is probably overexpressed due to a derepression of the encoding nag operon by an identified insertion mutation in gene H16_A0310 (nagR). Furthermore, a missense mutation in nagE (membrane component EIICB), which yields a substitution of an alanine by threonine in NagE and may additionally increase glucose uptake, was identified. Phosphorylation of glucose is subsequently mediated by NagF (cytosolic PTS component EIIA-HPr-EI) or glucokinase (GlK), respectively. The inability of the defined deletion mutant R. eutropha G(+)1 ΔnagFEC to utilize glucose strongly confirms this finding. In addition, secondary effects of glucose, which is now intracellularly available as a carbon source, on the metabolism of the mutant cells in the stationary growth phase occurred: intracellular glucose degradation is stimulated by the stronger expression of enzymes involved in the 2-keto-3-deoxygluconate 6-phosphate (KDPG) pathway and in subsequent reactions yielding pyruvate. The intermediate phosphoenolpyruvate (PEP) in turn supports further glucose uptake by the Nag PTS. Pyruvate is then decarboxylated by the pyruvate dehydrogenase multienzyme complex to acetyl coenzyme A (acetyl-CoA), which is directed to poly(3-hydroxybutyrate). The polyester is then synthesized to a greater extent, as also indicated by the upregulation of various enzymes of poly-ß-hydroxybutyrate (PHB) metabolism. The larger amounts of NADPH required for PHB synthesis are delivered by significantly increased quantities of proton-translocating NAD(P) transhydrogenases. The current study successfully combined transcriptomic and proteomic investigations to unravel the phenotype of this hitherto-undefined glucose-utilizing mutant.

Genome sequence of the polysaccharide-degrading, thermophilic anaerobe Spirochaeta thermophila DSM 6192.

Spirochaeta thermophila is a thermophilic, free-living anaerobe that is able to degrade various α- and ß-linked sugar polymers, including cellulose. We report here the complete genome sequence of S. thermophila DSM 6192, which is the first genome sequence of a thermophilic, free-living member of the Spirochaetes phylum. The genome data reveal a high density of genes encoding enzymes from more than 30 glycoside hydrolase families, a noncellulosomal enzyme system for (hemi)cellulose degradation, and indicate the presence of a novel carbohydrate-binding module.

Investigations on the microbial catabolism of the organic sulfur compounds TDP and DTDP in Ralstonia eutropha H16 employing DNA microarrays.

In this study, we have investigated the transcriptome of Ralstonia eutropha H16 during cultivation with gluconate in presence of 3,3'-thiodipropionic acid (TDP) or 3,3'-dithiodipropionic acid (DTDP) during biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptopropionate). Genome-wide transcriptome analyses revealed several genes which were upregulated during cultivation in presence of the above-mentioned compounds. Obtained data strongly suggest that two ABC-type transport system and three probable extracytoplasmic solute receptors mediate the uptake of TDP and DTDP, respectively. In addition, genes encoding the hydrolase S-adenosylhomocysteinase AhcY and the thiol-disulfide interchange proteins DsbA, DsbD, and FrnE were upregulated during cultivation on DTDP and, in case of AhcY and FrnE, on TDP as well. It is assumed that the corresponding enzymes are involved in the cleavage of TDP and DTDP. Several genes of the fatty acid metabolism exhibited increased expression levels: genes encoding two acetyltransferases, a predicted acyltransferase, the acetoacetyl-CoA reductase phaB3, an enoyl-CoA hydratase as well as an acyl dehydratase, an acetyl-CoA synthetase, two acyl-CoA dehydrogenases, the methylmalonyl-CoA mutase encoded by sbm1 and sbm2 and phaY1 were detected. Furthermore, ORF H16_A0217 encoding a hypothetical protein and exhibiting 54% amino acids identical to an acyl-CoA thioesterase from Saccharomonospora viridis was found to be highly upregulated. As the 2-methylcitrate synthase PrpC exhibited a three- to fourfold increased activity in cells grown in presence of TDP or DTDP as compared to gluconate, metabolization of the cleavage products 3MP and 3-hydroxypropionate to propionyl-CoA is proposed.

Genome-wide transcriptome analyses of the 'Knallgas' bacterium Ralstonia eutropha H16 with regard to polyhydroxyalkanoate metabolism.

Ralstonia eutropha H16 is probably the best-studied 'Knallgas' bacterium and producer of poly(3-hydroxybutyrate) (PHB). Genome-wide transcriptome analyses were employed to detect genes that are differentially transcribed during PHB biosynthesis. For this purpose, four transcriptomes from different growth phases of the wild-type H16 and of the two PHB-negative mutants PHB(-)4 and Delta phaC1 were compared: (i) cells from the exponential growth phase with cells that were in transition to stationary growth phase, and (ii) cells from the transition phase with cells from the stationary growth phase of R. eutropha H16, as well as (iii) cells from the transition phase of R. eutropha H16 with those from the transition phase of R. eutropha PHB(-)4 and (iv) cells from the transition phase of R. eutropha Delta phaC1 with those from the transition phase of R. eutropha PHB(-)4. Among a large number of genes exhibiting significant changes in transcription level, several genes within the functional class of lipid metabolism were detected. In strain H16, phaP3, accC2, fabZ, fabG and H16_A3307 exhibited a decreased transcription level in the stationary growth phase compared with the transition phase, whereas phaP1, H16_A3311, phaZ2 and phaZ6 were found to be induced in the stationary growth phase. Compared with PHB(-)4, we found that phaA, phaB1, paaH1, H16_A3307, phaP3, accC2 and fabG were induced in the wild-type, and phaP1, phaP4, phaZ2 and phaZ6 exhibited an elevated transcription level in PHB(-)4. In strain Delta phaC1, phaA and phaB1 were highly induced compared with PHB(-)4. Additionally, the results of this study suggest that mutant strain PHB(-)4 is defective in PHB biosynthesis and fatty acid metabolism. A significant downregulation of the two cbb operons in mutant strain PHB(-)4 was observed. The putative polyhydroxyalkanoate (PHA) synthase phaC2 identified in strain H16 was further investigated by several functional analyses. Mutant PHB(-)4 could be phenotypically complemented by expression of phaC2 from a plasmid; on the other hand, in the mutant H16Delta phaC1, no PHA production was observed. PhaC2 activity could not be detected in any experiment.

The genome of Methanosarcina mazei: evidence for lateral gene transfer between bacteria and archaea.

The Archaeon Methanosarcina mazei and related species are of great ecological importance as they are the only organisms fermenting acetate, methylamines and methanol to methane, carbon dioxide and ammonia (in case of methylamines). Since acetate is the precursor of 60% of the methane produced on earth these organisms contribute significantly to the production of this greenhouse gas, e.g. in rice paddies. The 4,096,345 base pairs circular chromosome of M. mazei is more than twice as large as the genomes of the methanogenic Archaea currently completely sequenced (Bult et al., 1996; Smith et al., 1997). 3,371 open reading frames (ORFs) were identified. Based on currently available sequence data 376 of these ORFs are Methanosarcina-specific and 1,043 ORFs find their closest homologue in the bacterial domain. 544 of these ORFs reach significant similarity values only in the bacterial domain. They include 56 of the 102 transposases, and proteins involved in gluconeogenesis, proline biosynthesis, transport processes, DNA-repair, environmental sensing, gene regulation, and stress response. Striking examples are the occurrence of the bacterial GroEL/GroES chaperone system and the presence of tetrahydrofolate-dependent enzymes. These findings might indicate that lateral gene transfer has played an important evolutionary role in forging the physiology of this metabolically versatile methanogen.