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Chitin and its derivative chitosan are abundant natural polysaccharides with many potential industrial applications. Metagenomic analysis of chitin-enriched soil samples using the Roche Genome Sequencer FLX platform led to the identification of several novel genes for chitin and chitosan modifying enzymes (CCMEs) which may be used to produce novel chitosans. The sequencing approach yielded 2,281,090 reads with an average length of 378 bp amounting to a total sequence information of approximately 851Mb. Assembly of the obtained sequences comprised 699,710 reads representing 30.68% of all reads. A total of 6625 contigs larger than 500 bp containing 16,289 predicted genes are included in the assembly. Taxonomic profiling of the indigenous microbial community by applying the software CARMA revealed that 96.1% of the reads were of bacterial origin including 17% assigned to the family Xanthomonadaceae. Several putative genes encoding CCMEs were identified by comparison against the GenBank database, inclusive a full-length chitinase gene which was codon optimized for Escherichia coli and heterologously synthesized as a Strep-tagged protein in E. coli Rosetta 2 using the pET vector system. Approximately 5 mg of the novel active chitinase was purified as demonstrated by dot assay analysis using glycol chitin as a substrate. Next generation metagenomic sequencing, thus, emerges as a new and powerful tool for the identification of potentially novel biocatalysts of biotechnological value. (C) 2014 Elsevier B.V. All rights reserved.

Industrial units, manufacturing dyes, chemicals, solvents, and xenobiotic compounds, produce liquid and solid wastes, which upon conventional treatment are released in the nearby environment and thus are the major cause of pollution. Soil collected from contaminated Kharicut Canal bank (N 22°57.878'; E 072°38.478'), Ahmedabad, Gujarat, India was used for metagenomic DNA preparation to study the capabilities of intrinsic microbial community in dealing with xenobiotics. Sequencing of metagenomic DNA on the Genome Sequencer FLX System using titanium chemistry resulted in 409,782 reads accounting for 133,529,997 bases of sequence information. Taxonomic analyses and gene annotations were carried out using the bioinformatics platform Sequence Analysis and Management System for Metagenomic Datasets. Taxonomic profiling was carried out by three different complementary approaches: (a) 16S rDNA, (b) environmental gene tags, and (c) lowest common ancestor. The most abundant phylum and genus were found to be "Proteobacteria" and "Pseudomonas," respectively. Metagenome reads were mapped on sequenced microbial genomes and the highest numbers of reads were allocated to Pseudomonas stutzeri A1501. Assignment of obtained metagenome reads to Gene Ontology terms, Clusters of Orthologous Groups of protein categories, protein family numbers, and Kyoto Encyclopedia of Genes and Genomes hits revealed genomic potential of indigenous microbial community. In total, 157,024 reads corresponded to 37,028 different KEGG hits, and amongst them, 11,574 reads corresponded to 131 different enzymes potentially involved in xenobiotic biodegradation. These enzymes were mapped on biodegradation pathways of xenobiotics to elucidate their roles in possible catalytic reactions. Consequently, information obtained from the present study will act as a baseline which, subsequently along with other "-omic" studies, will help in designing future bioremediation strategies in effluent treatment plants and environmental clean-up projects.

The IncF antibiotic resistance and virulence plasmid pRSB225, isolated from an unknown bacterium released with the purified wastewater from a municipal sewage treatment plant into the environment has been analysed at the genomic level by pyrosequencing. The 164,550 bp plasmid comprises 210 coding sequences (cds). It is composed of three replicons (RepFIA, RepFIB, and RepFII) and encodes further plasmid-specific functions for stable maintenance and inheritance and conjugative plasmid transfer. The plasmid is self-transmissible and shows a narrow host range limited to the family Enterobacteriaceae . The accessory modules of the plasmid mainly comprise genes conferring resistance to ampicillin ( bla TEM-1b ), chloramphenicol ( catA1 ), erythromycin ( mphA ), kanamycin and neomycin ( aphA1 ), streptomycin ( strAB ), sulphonamides ( sul2 ), tetracycline ( tetA (B)) and trimethoprim ( dfrA14 ), as well as mercuric ions ( mer genes). In addition, putative virulence-associated genes coding for iron uptake ( iutA / iucABCD , sitABCD , and a putative high-affinity Fe 2+ uptake system) and for a toxin/antitoxin system ( vagCD ) were identified on the plasmid. All antibiotic and heavy metal resistance genes are located either on class 1 (Tn 10 -remnant, Tn 4352B ) and class 2 transposons (Tn 2 -remnant, Tn 21 , Tn 402 -remnant) or a class 1 integron, whereas almost all putative virulence genes are associated with IS elements (IS 1 , IS 26 ), indicating that transposition and/or recombination events were responsible for acquisition of the accessory pRSB225 modules. Particular modules of plasmid pRSB225 are related to corresponding segments of different virulence plasmids harboured by pathogenic Escherichia coli strains. Moreover, pRSB225 modules were also detected in entero-aggregative-haemorrhagic E. coli (EAHEC) draft genome sequences suggesting that IncF plasmids related to pRSB225 mediated gene transfer into pathogenic E. coli derivatives.

Lactobacillus buchneri belongs to the group of heterofermentative lactic acid bacteria and is a common member of the silage microbiome. Here we report the completely annotated genomic sequence of L. buchneri CD034, a strain isolated from stable grass silage. The whole genome of L. buchneri CD034 was sequenced on the Roche Genome Sequencer FLX platform. It was found to consist of four replicons, a circular chromosome, and three plasmids. The circular chromosome was predicted to encode 2319 proteins and contains a genomic island and two prophages which significantly differ in G+C-content from the remaining chromosome. It possesses all genes for enzymes of a complete phosphoketolase pathway, whereas two enzymes necessary for glycolysis are lacking. This confirms the classification of L. buchneri CD034 as an obligate heterofermentative lactic acid bacterium. A set of genes considered to be involved in the lactate degradation pathway and genes putatively involved in the breakdown of plant cell wall polymers were identified. Moreover, several genes encoding putative S-layer proteins and two CRISPR systems, belonging to the subclasses I-E and II-A, are located on the chromosome. The largest plasmid pCD034-3 was predicted to encode 57 genes, including a putative polysaccharide synthesis gene cluster, whereas the functions of the two smaller plasmids, pCD034-1 and pCD034-2, remain cryptic. Phylogenetic analysis based on sequence comparison of the conserved marker gene rpoA reveals that L. buchneri CD034 is more closely related to Lactobacillus hilgardii strains than to Lactobacillus brevis and Lactobacillus plantarum strains. Comparison of the L. buchneri CD034 core genome to other fully sequenced and closely related members of the genus Lactobacillus disclosed a high degree of conservation between L. buchneri CD034 and the recently sequenced L. buchneri strain NRRL B-30929 and a more distant relationship to L. buchneri ATCC 11577 and L. brevis ssp. gravesensis ATCC 27305, which cluster together with L. hilgardii type strain ATCC 8290. L. buchneri CD034 genome information will certainly provide the basis for further postgenome studies with the objective to optimize application of the strain in silage production.

Structural composition and gene content of a biogas-producing microbial community from a production-scale biogas plant fed with renewable primary products was recently analyzed by means of a metagenome sequencing approach. To determine the transcriptionally active part of the same biogas community and to identify key transcripts for the biogas production process, the metatranscriptome of the microorganisms was sequenced for the first time. The metatranscriptome sequence dataset generated on the Genome Sequencer FLX platform is represented by 484,920 sequence reads. Taxonomic profiling of the active part of the community by classification of 16S ribosomal sequence tags revealed that members of the Euryarchaeota and Firmicutes account for the dominant phyla. Only smaller fractions of the 16S ribosomal sequence tags were assigned to the phyla Bacteroidetes, Actinobacteria and Synergistetes. Among the mRNA-derived sequence tags from the metatranscriptome dataset, transcripts encoding enzymes involved in substrate hydrolysis, acidogenesis, acetate formation and methanogenesis could be identified. Transcripts for enzymes functioning in methanogenesis are among the most abundant mRNA tags indicating that the corresponding pathway is very active in the methanogenic sub-community. As a frame of reference for evaluation of metatranscriptome sequence data, the 16S rDNA-based taxonomic profile of the community was analyzed by means of high-throughput 16S rDNA amplicon sequencing. Processing of the obtained amplicon reads resulted in 18,598 high-quality 16S rDNA sequences covering the V3-V4 hypervariable region of the 16S rRNA gene. Comparison of the taxonomic profiles deduced from 16S rDNA amplicon sequences and the metatranscriptome dataset indicates a high transcriptional activity of archaeal species. Overall, it was shown that the most abundant species dominating the community also contributed the majority of the transcripts. In the future, key transcripts for the biogas production process will provide valuable markers for evaluation of the performance of biogas-producing microbial communities with the objective to optimize the biotechnology of this process.

The final step of the biogas production process, the methanogenesis, is frequently dominated by members of the genus Methanoculleus. In particular, the species Methanoculleus bourgensis was identified to play a role in different biogas reactor systems. The genome of the type strain M. bourgensis MS2(T), originally isolated from a sewage sludge digestor, was completely sequenced to analyze putative adaptive genome features conferring competitiveness within biogas reactor environments to the strain. Sequencing and assembly of the M. bourgensis MS2(T) genome yielded a chromosome with a size of 2,789,773 bp. Comparative analysis of M. bourgensis MS2(T) and Methanoculleus marisnigri JR1 revealed significant similarities. The absence of genes for a putative ammonium uptake system may indicate that M. bourgensis MS2(T) is adapted to environments rich in ammonium/ammonia. Specific genes featuring predicted functions in the context of osmolyte production were detected in the genome of M. bourgensis MS2(T). Mapping of metagenome sequences derived from a production-scale biogas plant revealed that M. bourgensis MS2(T) almost completely comprises the genetic information of dominant methanogens present in the biogas reactor analyzed. Hence, availability of the M. bourgensis MS2(T) genome sequence may be valuable regarding further research addressing the performance of Methanoculleus species in agricultural biogas plants. (C) 2014 Elsevier B.V. All rights reserved.

The whole sequence of plasmid pENVA carrying the extended-spectrum β-lactamase gene bla CTX-M-15 was determined. It was identified from a series of clonally related Klebsiella pneumoniae sequence type 274 strains recovered from companion animals. This plasmid was 253,984 bp in size and harbored, in addition to bla CTX-M-15 , a large array of genes encoding resistance to many antibiotic molecules, including β-lactams ( bla TEM-1 , bla DHA-1 ), aminoglycosides ( aacA2 , aadA1 ), tetracycline ( tetA ), quinolones ( qnrB4 ), trimethoprim ( dfrA15 ), and sulfonamides (two copies of sul1 ). In addition, genes encoding resistance to mercury, tellurium, nickel, and quaternary compounds were identified. It also carried genes encoding DNA damage protection and mutagenesis repair and a locus for a CRISPR system, which corresponds to an immune system involved in protection against bacteriophages and plasmids. Comparative analysis of the plasmid scaffold showed that it possessed a structure similar to that of only a single plasmid, which was pNDM-MAR encoding the carbapenemase NDM-1 and identified from human K. pneumoniae isolates. Both plasmids possessed two replicons, namely, those of IncFIB-like and IncHIB-like plasmids, which were significantly different from those previously characterized. The bla CTX-M-15 gene, together with the other antibiotic resistance genes, was part of a large module likely acquired through a transposition process. We characterized here a new plasmid type carrying the bla CTX-M-15 gene identified in a K. pneumoniae isolate of animal origin. The extent to which this plasmid type may spread efficiently and possibly further enhance the dissemination of bla CTX-M-15 among animal and human isolates remains to be determined.

Methanobacterium sp. Mb1, a hydrogenotrophic methanogenic Archaeon, was isolated from a rural biogas plant producing methane-rich biogas from maize silage and cattle manure in Germany. Here we report the complete genome sequence of the novel methanogenic isolate Methanobacterium sp. Mb1 harboring a 2,029,766 bp circular chromosome featuring a GC content of 39.74%. The genome encodes two rRNA operons, 41 tRNA genes and 2021 coding sequences and represents the smallest genome currently known within the genus Methanobacterium. (C) 2013 Elsevier B.V. All rights reserved.

Silage is green fodder conserved by lactic acid fermentation performed by epiphytic lactic acid bacteria under anaerobic conditions. To improve the ensiling process and the quality of the resulting silage, starter cultures are added to the fresh forage. A detailed analysis of the microbial community playing a role in grass ensiling has been carried out by high throughput sequencing technologies. Moreover, the influence of the inoculant Lactobacillus buchneri CD034 on the microbial community composition was studied. For this purpose, grass was ensiled untreated or inoculated with L. buchneri CD034. The fresh forage as well as silages after 14 and 58 days of fermentation were characterized physico-chemically. Characteristic silage conditions such as increased titers of lactic acid bacteria and higher concentrations of acetic acid were observed in the inoculated silage in comparison to the untreated samples. Taxonomic community profiles deduced from 16S rDNA amplicon sequences indicated that the relative abundance of Lactococci diminished in the course of fermentations and that the proportion of bacteria belonging to the phyla Proteobacteria and Bacteroidetes increased during the fermentation of untreated silage. In the inoculated silage, members of these phyla were repressed due to an increased abundance of Lactobacilli. In addition, metagenome analyses of silage samples confirmed taxonomic profiles based on 16S rDNA amplicons. Moreover, Lactobacillus plantarum, Lactobacillus brevis and Lactococcus lactis were found to be dominant species within silages as analyzed by means of fragment recruitments of metagenomic sequence reads on complete reference genome sequences. Fragment recruitments also provided clear evidence for the competitiveness of the inoculant strain L. buchneri CD034 during the fermentation of the inoculated silage. The inoculation strain was able to outcompete other community members and also affected physico-chemical characteristics of the silage.

Twenty-six isolates of Clostridium perfringens of different MLST types from chickens with necrotic enteritis (NE) (15 netB-positive) or from healthy chickens (6 netB-positive, 5 netB-negative) were found to contain 1–4 large plasmids, with most netB-positive isolates containing 3 large and variably sized plasmids which were more numerous and larger than plasmids in netB-negative isolates. NetB and cpb2 were found on different plasmids consistent with previous studies. The pathogenicity locus NELoc1, which includes netB, was largely conserved in these plasmids whereas NeLoc3, present in the cpb2 containing plasmids, was less well conserved. A netB-positive and a cpb2-positive plasmid were likely to be conjugative, and the plasmids were completely sequenced. Both plasmids possessed the intact tcp conjugative region characteristic of C. perfringens conjugative plasmids. Comparative genomic analysis of nine CpCPs, including the two plasmids described here, showed extensive gene rearrangements including pathogenicity locus and accessory gene insertions around rather than within the backbone region. The pattern that emerges from this analysis is that the major toxin-containing regions of the variety of virulence-associated CpCPs are organized as complex pathogenicity loci. How these different but related CpCPs can co-exist in the same host has been an unanswered question. Analysis of the replication-partition region of these plasmids suggests that this region controls plasmid incompatibility, and that CpCPs can be grouped into at least four incompatibility groups.

Methanoculleus bourgensis , of the order Methanomicrobiales , is a dominant methanogenic archaeon in many biogas-producing reactor systems fed with renewable primary products. It is capable of synthesizing methane via the hydrogenotrophic pathway utilizing hydrogen and carbon dioxide or formate as the substrates. Here we report the complete and finished genome sequence of M. bourgensis strain MS2 T , isolated from a sewage sludge digester.

The dissemination of antibiotic resistance genes among bacteria often occurs by means of plasmids. Wastewater treatment plants (WWTP) were previously recognized as hot spots for the horizontal transfer of genetic material. One of the plasmid groups that is often associated with drug resistance is the incompatibility group IncN. The aim of this study was to gain insights into the diversity and evolutionary history of IncN plasmids by determining and comparing the complete genome sequences of the four novel multi-drug resistance plasmids pRSB201, pRSB203, pRSB205 and pRSB206 that were exogenously isolated from the final effluent of a municipal WWTP. Their sizes range between 42,875 bp and 56,488 bp and they share a common set of backbone modules that encode plasmid replication initiation, conjugative transfer, and plasmid maintenance and control. All plasmids are transferable at high rates between Escherichia coli strains, but did not show a broad host range. Different genes conferring resistances to ampicillin, streptomycin, spectinomycin, sulfonamides, tetracycline and trimethoprim were identified in accessory modules inserted in these plasmids. Comparative analysis of the four WWTP IncN plasmids and IncN plasmids deposited in the NCBI database enabled the definition of a core set of backbone genes for this group. Moreover, this approach revealed a close phylogenetic relationship between the IncN plasmids isolated from environmental and clinical samples. Phylogenetic analysis also suggests the existence of host-specific IncN plasmid subgroups. In conclusion, IncN plasmids likely contribute to the dissemination of resistance determinants between environmental bacteria and clinical strains. This is of particular importance since multi-drug resistance IncN plasmids have been previously identified in members of the Enterobacteriaceae that cause severe infections in humans.

Although IncP-1 plasmids are important for horizontal gene transfer among bacteria, in particular antibiotic resistance spread, so far only three plasmids from the subgroup IncP-1 alpha have been completely sequenced. In this study we doubled this number. The three IncP-1 alpha plasmids pB5, pB11 and pSP21 were isolated from bacteria of two different sewage treatment plants and sequenced by a combination of next-generation and capillary sequencing technologies. A comparative analysis including the previously analysed IncP-1 alpha plasmids RK2, pTB11 and pBS228 revealed a highly conserved plasmid backbone (at least 99.9% DNA sequence identity) comprising 54 core genes. The accessory elements of the plasmid pB5 constitute a class 1 integron interrupting the parC gene and an IS6100 copy inserted into the integron. In addition, the tetracycline resistance genes tetAR and the ISTB11-like element are located between the klc operon and the trfA-ssb operon. Plasmid pB11 is loaded with a Tn5053-like mercury resistance transposon between the parCBA and parDE operons and contains tetAR that are identical to those identified in plasmid pB5 and the insertion sequence ISSP21. Plasmid pSP21 harbours an ISPa7 element in a Tn402 transposon including a class 1 integron between the partitioning genes parCBA and parDE. The IS-element ISSP21 (99.89% DNA sequence identity to ISSP21 from pB11), inserted downstream of the tetR gene and a copy of ISTB11 (identical to ISTB11 on pTB11) inserted between the genes pncA and pinR. On all three plasmids the accessory genes are almost always located between the backbone modules confirming the importance of the backbone functions for plasmid maintenance. The striking backbone conservation among the six completely sequenced IncP-1 alpha plasmids is in contrast to the much higher diversity within the IncP-1 alpha subgroup. (C) 2010 Elsevier B. V. All rights reserved.