Complete Genome Sequences of pLMA1 and pLMA7, Two Large Linear Plasmids of Micrococcus Strains Isolated from a High-Altitude Lake in Argentina.

The two linear plasmids pLMA1 (109,112 bp) and pLMA7 (82,075 bp) from Micrococcus strains were isolated from a high-altitude lake in the Argentinean Puna, sequenced, and annotated. These extrachromosomal elements are probably conjugative and harbor genes potentially involved in coping with the harsh conditions in such extreme environments.

SMRT sequencing of the Campylobacter coli BfR-CA-9557 genome sequence reveals unique methylation motifs.

BACKGROUND: Campylobacter species are the most prevalent bacterial pathogen causing acute enteritis worldwide. In contrast to Campylobacter jejuni, about 5 % of Campylobacter coli strains exhibit susceptibility to restriction endonuclease digestion by DpnI cutting specifically 5'-G(m)ATC-3' motifs. This indicates significant differences in DNA methylation between both microbial species. The goal of the study was to analyze the methylome of a C. coli strain susceptible to DpnI digestion, to identify its methylation motifs and restriction modification systems (RM-systems), and compare them to related organisms like C. jejuni and Helicobacter pylori. RESULTS: Using one SMRT cell and the PacBio RS sequencing technology followed by PacBio Modification and Motif Analysis the complete genome of the DpnI susceptible strain C. coli BfR-CA-9557 was sequenced to 500-fold coverage and assembled into a single contig of 1.7 Mbp. The genome contains a CJIE1-like element prophage and is phylogenetically closer to C. coli clade 1 isolates than clade 3. 45,881 6-methylated adenines (ca. 2.7 % of genome positions) that are predominantly arranged in eight different methylation motifs and 1,788 4-methylated cytosines (ca. 0.1 %) have been detected. Only two of these motifs correspond to known restriction modification motifs. Characteristic for this methylome was the very high fraction of methylation of motifs with mostly above 99 %. CONCLUSIONS: Only five dominant methylation motifs have been identified in C. jejuni, which have been associated with known RM-systems. C. coli BFR-CA-9557 shares one (RAATTY) of these, but four ORFs could be assigned to putative Type I RM-systems, seven ORFs to Type II RM-systems and three ORFs to Type IV RM-systems. In accordance with DpnI prescreening RM-system IIP, methylation of GATC motifs was detected in C. coli BfR-CA-9557. A homologous IIP RM-system has been described for H. pylori. The remaining methylation motifs are specific for C. coli BfR-CA-9557 and have been neither detected in C. jejuni nor in H. pylori. The results of this study give us new insights into epigenetics of Campylobacteraceae and provide the groundwork to resolve the function of RM-systems in C. coli.

The genome of Variovorax paradoxus strain TBEA6 provides new understandings for the catabolism of 3,3'-thiodipropionic acid and hence the production of polythioesters.

The betaproteobacterium Variovorax paradoxus strain TBEA6 is capable of using 3,3'-thiodipropionic acid (TDP) as sole carbon and energy source for growth. This thioether is employed for several industrial applications. It can be applied as precursor for the biotechnical production of polythioesters (PTE), which represent persistent bioplastics. Consequently, the genome of V. paradoxus strain TBEA6 was sequenced. The draft genome sequence comprises approximately 7.2Mbp and 6852 predicted open reading frames. Furthermore, transposon mutagenesis to unravel the catabolism of TDP in strain TBEA6 was performed. Screening of 20,000 mutants mapped the insertions of Tn5::mob in 32 mutants, which all showed no growth with TDP as sole carbon source. Based on the annotated genome sequence together with transposon-induced mutagenesis, defined gene deletions, in silico analyses and comparative genomics, a comprehensive pathway for the catabolism of TDP is proposed: TDP is imported via the tripartite tricarboxcylate transport system and/or the TRAP-type dicarboxylate transport system. The initial cleavage of TDP into 3-hydroxypropionic acid (3HP) and 3-mercaptopropionic acid (3MP), which serves as precursor substrate for PTE synthesis, is most probably performed by the FAD-dependent oxidoreductase Fox. 3HP is presumably catabolized via malonate semialdehyde, whereas 3MP is oxygenated by the 3MP-dioxygenase Mdo yielding 3-sulfinopropionic acid (3SP). Afterwards, 3SP is linked to coenzyme A. The next step is the abstraction of sulfite by a desulfinase, and the resulting propionyl-CoA enters the central metabolism. Sulfite is oxidized to sulfate by the sulfite-oxidizing enzyme SoeABC and is subsequently excreted by the cells by the sulfate exporter Pse.

Complete Genome Sequence of the Linear Plasmid pJD12 Hosted by Micrococcus sp. D12, Isolated from a High-Altitude Volcanic Lake in Argentina.

The linear plasmid pDJ12 from Micrococcus D12, isolated from the high-altitude volcanic Diamante Lake in the northwest of Argentina, was completely sequenced and annotated. It is noteworthy that the element is probably conjugative and harbors genes potentially instrumental in coping with stress conditions that prevail in such an extreme environment.

Complete Genome Sequence of Bacillus subtilis subsp. subtilis Strain 3NA.

Bacillus subtilis 3NA reaches high cell densities during fed-batch fermentation and is an interesting target for further optimization as a production strain. Here, we announce the full genome of B. subtilis 3NA. The presence of specific Bacillus subtilis 168 and W23 genetic features suggests that 3NA is a hybrid of these strains.

Engineering Bacillus subtilis for the conversion of the antimetabolite 4-hydroxy-l-threonine to pyridoxine.

Until now, pyridoxine (PN), the most commonly supplemented B6 vitamer for animals and humans, is chemically synthesized for commercial purposes. Thus, the development of a microbial fermentation process is of great interest for the biotech industry. Recently, we constructed a Bacillus subtilis strain that formed significant amounts of PN via a non-native deoxyxylulose 5'-phosphate-(DXP)-dependent vitamin B6 pathway. Here we report the optimization of the condensing reaction of this pathway that consists of the 4-hydroxy-l-threonine-phosphate dehydrogenase PdxA, the pyridoxine 5'-phosphate synthase PdxJ and the native DXP synthase, Dxs. To allow feeding of high amounts of 4-hydroxy-threonine (4-HO-Thr) that can be converted to PN by B. subtilis overexpressing PdxA and PdxJ, we first adapted the bacteria to tolerate the antimetabolite 4-HO-Thr. The adapted bacteria produced 28-34mg/l PN from 4-HO-Thr while the wild-type parent produced only 12mg/l PN. Moreover, by expressing different pdxA and pdxJ alleles in the adapted strain we identified a better combination of PdxA and PdxJ enzymes than reported previously, and the resulting strain produced 65mg/l PN. To further enhance productivity mutants were isolated that efficiently take up and convert deoxyxylulose (DX) to DXP, which is incorporated into PN. Although these mutants were very efficient to convert low amount of exogenous DX, at higher DX levels they performed only slightly better. The present study uncovered several enzymes with promiscuous activity and it revealed that host metabolic pathways compete with the heterologous pathway for 4-HO-Thr. Moreover, the study revealed that the B. subtilis genome is quite flexible with respect to adaptive mutations, a property, which is very important for strain engineering.

New mode of energy metabolism in the seventh order of methanogens as revealed by comparative genome analysis of "Candidatus methanoplasma termitum".

The recently discovered seventh order of methanogens, the Methanomassiliicoccales (previously referred to as "Methanoplasmatales"), so far consists exclusively of obligately hydrogen-dependent methylotrophs. We sequenced the complete genome of "Candidatus Methanoplasma termitum" from a highly enriched culture obtained from the intestinal tract of termites and compared it with the previously published genomes of three other strains from the human gut, including the first isolate of the order. Like all other strains, "Ca. Methanoplasma termitum" lacks the entire pathway for CO2 reduction to methyl coenzyme Mand produces methane by hydrogen-dependent reduction of methanol or methylamines, which is consistent with additional physiological data. However, the shared absence of cytochromes and an energy-converting hydrogenase for the reoxidation of the ferredoxin produced by the soluble heterodisulfide reductase indicates that Methanomassiliicoccales employ a new mode of energy metabolism, which differs from that proposed for the obligately methylotrophic Methanosphaera stadtmanae. Instead, all strains possess a novel complex that is related to the F420:methanophenazine oxidoreductase (Fpo) of Methanosarcinales butlacks an F420-oxidizing module, resembling the apparently ferredoxin-dependent Fpo-like homolog in Methanosaeta thermophila. Since all Methanomassiliicoccales also lack the subunit E of the membrane-bound heterodisulfide reductase (HdrDE), wepropose that the Fpo-like complex interacts directly with subunit D, forming an energy-converting ferredoxin: heterodisulfideoxidoreductase. The dual function of heterodisulfide in Methanomassiliicoccales, which serves both in electron bifurcation and as terminal acceptor in a membrane-associated redox process, may be a unique characteristic of the novel order.

Unravelling the complete genome sequence of Advenella mimigardefordensis strain DPN7T and novel insights in the catabolism of the xenobiotic polythioester precursor 3,3'-dithiodipropionate.

Advenella mimigardefordensis strain DPN7(T) is a remarkable betaproteobacterium because of its extraordinary ability to use the synthetic disulfide 3,3'-dithiodipropionic acid (DTDP) as the sole carbon source and electron donor for aerobic growth. One application of DTDP is as a precursor substrate for biotechnically synthesized polythioesters (PTEs), which are interesting non-degradable biopolymers applicable for plastics materials. Metabolic engineering for optimization of PTE production requires an understanding of DTDP conversion. The genome of A. mimigardefordensis strain DPN7(T) was sequenced and annotated. The circular chromosome was found to be composed of 4,740,516 bp and 4112 predicted ORFs, whereas the circular plasmid consisted of 23,610 bp and 24 predicted ORFs. The genes participating in DTDP catabolism had been characterized in detail previously, but knowing the complete genome sequence and with support of Tn5: :mob-induced mutants, putatively involved transporter proteins and a transcriptional regulator were also identified. Most probably, DTDP is transported into the cell by a specific tripartite tricarboxylate transport system and is then cleaved by the disulfide reductase LpdA, sulfoxygenated by the 3-mercaptopropionate dioxygenase Mdo, activated by the CoA ligase SucCD and desulfinated by the acyl-CoA dehydrogenase-like desulfinase AcdA. Regulation of this pathway is presumably performed by a transcriptional regulator of the xenobiotic response element family. The excessive sulfate that is inevitably produced is secreted by the cells by a unique sulfate exporter of the CPA (cation : proton antiporter) superfamily.

Genome-guided insights into the versatile metabolic capabilities of the mercaptosuccinate-utilizing ß-proteobacterium Variovorax paradoxus strain B4.

Variovorax paradoxus B4 is able to utilize 2-mercaptosuccinate (MS) as sole carbon, sulfur and energy source. The whole genome of V. paradoxus B4 was sequenced, annotated and evaluated with special focus on genomic elements related to MS metabolism. The genome encodes two chromosomes harbouring 5 795 261 and 1 353 255 bp. A total of 6753 putative protein-coding sequences were identified. Based on the genome and in combination with results from previous studies, a putative pathway for the degradation of MS could be postulated. The putative molybdopterin oxidoreductase identified during transposon mutagenesis probably catalyses the conversion of MS first into sulfinosuccinate and then into sulfosuccinate by successive transfer of oxygen atoms. Subsequently, the cleavage of sulfosuccinate yields oxaloacetate and sulfite, while the latter is oxidized to sulfate. The expression of the putative molybdopterin oxidoreductase was induced by MS, but not by gluconate, as confirmed by reverse transcriptase polymerase chain reaction. Further, in silico studies combined with experiments and comparative genomics revealed high metabolic diversity of strain B4. It bears a high potential as plant growth-promoting bacterium and as candidate for degradation and detoxification of xenobiotics and other hardly degradable substances. Additionally, the strain is of special interest for production of polythioesters with sulfur-containing precursors as MS.

Complete Genome Sequence of pAP13, a Large Linear Plasmid of a Brevibacterium Strain Isolated from a Saline Lake at 4,200 Meters above Sea Level in Argentina.

pAP13 is an 89-kb linear plasmid hosted by Brevibacterium sp. strain Ap13, an actinobacterium isolated from the feces of a flamingo from an extremely high-altitude lake in Argentina. Because of the ecological importance of the genus Brevibacterium, the absolute lack of information concerning Brevibacterium linear plasmids, and the possible ecological significance of this unusual plasmid, pAP13 was completely sequenced, including the inversely oriented termini.

First complete sequence of a giant linear plasmid from a micrococcus strain isolated from an extremely high-altitude lake.

Micrococcus sp. strain V7, an actinobacterial strain adapted to the extreme conditions of the Laguna Vilama, an extremely high-altitude (4,600 m above sea level) lake in the Argentinian Puna, was found to carry the giant linear plasmid pLMV7. We determined its sequence (92,815 bp) as a prerequisite to the investigation of its role in survival in such a harsh environment.

Evolution of enterohemorrhagic escherichia coli O26 based on single-nucleotide polymorphisms.

Enterohemorrhagic Escherichia coli (EHEC) O26:H11/H⁻ is the predominant non-O157 EHEC serotype among patients with diarrhea, bloody diarrhea, and hemolytic uremic syndrome (HUS) worldwide. To elucidate their phylogeny and association between their phylogenetic background and clinical outcome of the infection, we investigated 120 EHEC O26:H11/H⁻ strains isolated between 1965 and 2012 from asymptomatic carriers and patients with diarrhea or HUS. Whole-genome shotgun sequencing (WGS) was applied to ten representative EHEC O26 isolates to determine single nucleotide polymorphism (SNP) localizations within a predefined set of core genes. A multiplex SNP assay, comprising a randomly distributed subset of 48 SNPs, was established to detect SNPs in 110 additional EHEC O26 strains. Within approximately 1 Mb of core genes, WGS resulted in 476 high-quality bi-allelic SNP localizations. Forty-eight of these were subsequently investigated in 110 EHEC O26 and four different SNP clonal complexes (SNP-CC) were identified. SNP-CC2 was significantly associated with the development of HUS. Within the subsequently established evolutionary model of EHEC O26, we dated the emergence of human EHEC O26 to approximately 19,700 years ago and demonstrated a recent evolution within humans into the 4 SNP-CCs over the past 1,650 years. WGS and subsequent SNP typing enabled us to gain new insights into the evolution of EHEC O26 suggesting a common theme in this EHEC group with analogies to EHEC O157. In addition, the SNP-CC analysis may help to assess a risk in infected individuals for the progression to HUS and to implement more specific infection control measures.

Complete Genome Sequence of Staphylococcus aureus 6850, a Highly Cytotoxic and Clinically Virulent Methicillin-Sensitive Strain with Distant Relatedness to Prototype Strains.

Staphylococcus aureus is a frequent human commensal bacterium and pathogen. Here we report the complete genome sequence of strain 6850 (spa type t185; sequence type 50 [ST50]), a highly cytotoxic and clinically virulent methicillin-sensitive strain from a patient with complicated S. aureus bacteremia associated with osteomyelitis and septic arthritis.

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification.

in order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 α/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification / Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas

In order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.

Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST), se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un gen codificante de una hipotetica lipasa/esterasa. La secuencia deducida de amino acidos (RBest1) contiene los motivos conservados de enzimas lipoliticas y esta relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la unica enzima procariota estudiada perteneciente al subgrupo 4.4 de a/ß hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-esteres, respectivamente, revelaron que la enzima es altamente especifica para compuestos butiricos (C4), comportandose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquimicamente. La actividad optima de esterasa fue observada a pH 6,5 y las temperaturas optimas fueron entre 38 y 45 °C. Se establecio que la actividad enzimatica, determinada por hidrolisis de p-nitrofenil esteres, es afectada en presencia de diferentes solventes organicos miscibles y no miscibles, y tambien sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas ionicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptacion molecular en presencia de compuestos organicos, asi como la resistencia de las proteinas halofilas.

Complete genome sequence and metabolic potential of the quinaldine-degrading bacterium Arthrobacter sp. Rue61a.

BACKGROUND: Bacteria of the genus Arthrobacter are ubiquitous in soil environments and can be considered as true survivalists. Arthrobacter sp. strain Rue61a is an isolate from sewage sludge able to utilize quinaldine (2-methylquinoline) as sole carbon and energy source. The genome provides insight into the molecular basis of the versatility and robustness of this environmental Arthrobacter strain. RESULTS: The genome of Arthrobacter sp. Rue61a consists of a single circular chromosome of 4,736,495 bp with an average G + C content of 62.32%, the circular 231,551-bp plasmid pARUE232, and the linear 112,992-bp plasmid pARUE113 that was already published. Plasmid pARUE232 is proposed to contribute to the resistance of Arthrobacter sp. Rue61a to arsenate and Pb2+, whereas the linear plasmid confers the ability to convert quinaldine to anthranilate. Remarkably, degradation of anthranilate exclusively proceeds via a CoA-thioester pathway. Apart from quinaldine utilization, strain Rue61a has a limited set of aromatic degradation pathways, enabling the utilization of 4-hydroxy-substituted aromatic carboxylic acids, which are characteristic products of lignin depolymerization, via ortho cleavage of protocatechuate. However, 4-hydroxyphenylacetate degradation likely proceeds via meta cleavage of homoprotocatechuate. The genome of strain Rue61a contains numerous genes associated with osmoprotection, and a high number of genes coding for transporters. It encodes a broad spectrum of enzymes for the uptake and utilization of various sugars and organic nitrogen compounds. A. aurescens TC-1 is the closest sequenced relative of strain Rue61a. CONCLUSIONS: The genome of Arthrobacter sp. Rue61a reflects the saprophytic lifestyle and nutritional versatility of the organism and a strong adaptive potential to environmental stress. The circular plasmid pARUE232 and the linear plasmid pARUE113 contribute to heavy metal resistance and to the ability to degrade quinaldine, respectively.

Complete genome sequence of the broad-host-range strain Sinorhizobium fredii USDA257.

Here we announce the complete genome sequence of the symbiotic and nitrogen-fixing bacterium Sinorhizobium fredii USDA257. The genome shares a high degree of sequence similarity with the closely related broad-host-range strains S. fredii NGR234 and HH103. Most strikingly, the USDA257 genome encodes a wealth of secretory systems.

Involvement of two latex-clearing proteins during rubber degradation and insights into the subsequent degradation pathway revealed by the genome sequence of Gordonia polyisoprenivorans strain VH2.

The increasing production of synthetic and natural poly(cis-1,4-isoprene) rubber leads to huge challenges in waste management. Only a few bacteria are known to degrade rubber, and little is known about the mechanism of microbial rubber degradation. The genome of Gordonia polyisoprenivorans strain VH2, which is one of the most effective rubber-degrading bacteria, was sequenced and annotated to elucidate the degradation pathway and other features of this actinomycete. The genome consists of a circular chromosome of 5,669,805 bp and a circular plasmid of 174,494 bp with average GC contents of 67.0% and 65.7%, respectively. It contains 5,110 putative protein-coding sequences, including many candidate genes responsible for rubber degradation and other biotechnically relevant pathways. Furthermore, we detected two homologues of a latex-clearing protein, which is supposed to be a key enzyme in rubber degradation. The deletion of these two genes for the first time revealed clear evidence that latex-clearing protein is essential for the microbial utilization of rubber. Based on the genome sequence, we predict a pathway for the microbial degradation of rubber which is supported by previous and current data on transposon mutagenesis, deletion mutants, applied comparative genomics, and literature search.

Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC).

The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum ß-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic E scherichia c oli (EAHEC).