Arabinan saccharification by biogas reactor metagenome-derived arabinosyl hydrolases.

BACKGROUND: Plant cell walls represent the most plentiful renewable organic resource on earth, but due to their heterogeneity, complex structure and partial recalcitrance, their use as biotechnological feedstock is still limited. RESULTS: In order to identify efficient enzymes for polysaccharide breakdown, we have carried out functional screening of metagenomic fosmid libraries from biogas fermenter microbial communities grown on sugar beet pulp, an arabinan-rich agricultural residue, or other sources containing microbes that efficiently depolymerize polysaccharides, using CPH (chromogenic polysaccharide hydrogel) or ICB (insoluble chromogenic biomass) labeled polysaccharide substrates. Seventy-one depolymerase-encoding genes were identified from 55 active fosmid clones by using Illumina and Sanger sequencing and dbCAN CAZyme (carbohydrate-active enzyme) annotation. An around 56 kb assembled DNA fragment putatively originating from Xylanivirga thermophila strain or a close relative was analyzed in detail. It contained 48 ORFs (open reading frames), of which 31 were assigned to sugar metabolism. Interestingly, a large number of genes for enzymes putatively involved in degradation and utilization of arabinose-containing carbohydrates were found. Seven putative arabinosyl hydrolases from this DNA fragment belonging to glycoside hydrolase (GH) families GH51 and GH43 were biochemically characterized, revealing two with endo-arabinanase activity and four with exo-α-L-arabinofuranosidase activity but with complementary cleavage properties. These enzymes were found to act synergistically and can completely hydrolyze SBA (sugar beet arabinan) and DA (debranched arabinan). CONCLUSIONS: We screened 32,776 fosmid clones from several metagenomic libraries with chromogenic lignocellulosic substrates for functional enzymes to advance the understanding about the saccharification of recalcitrant lignocellulose. Seven putative X. thermophila arabinosyl hydrolases were characterized for pectic substrate degradation. The arabinosyl hydrolases displayed maximum activity and significant long-term stability around 50 °C. The enzyme cocktails composed in this study fully degraded the arabinan substrates and thus could serve for arabinose production in food and biofuel industries.

Identification of New Chromosomal Loci Involved in com Genes Expression and Natural Transformation in the Actinobacterial Model Organism Micrococcus luteus.

Historically, Micrococcus luteus was one of the first organisms used to study natural transformation, one of the main routes of horizontal gene transfer among prokaryotes. However, little is known about the molecular basis of competence development in M. luteus or any other representative of the phylum of high-GC Gram-positive bacteria (Actinobacteria), while this means of genetic exchange has been studied in great detail in Gram-negative and low-GC Gram-positive bacteria (Firmicutes). In order to identify new genetic elements involved in regulation of the comEA-comEC competence operon in M. luteus, we conducted random chemical mutagenesis of a reporter strain expressing lacZ under the control of the comEA-comEC promoter, followed by the screening of dysregulated mutants. Mutants with (i) upregulated com promoter under competence-repressing conditions and (ii) mutants with a repressed com promoter under competence-inducing conditions were isolated. After genotype and phenotype screening, the genomes of several mutant strains were sequenced. A selection of putative com-influencing mutations was reinserted into the genome of the M. luteus reporter strain as markerless single-nucleotide mutations to confirm their effect on com gene expression. This strategy revealed mutations affecting com gene expression at genetic loci different from previously known genes involved in natural transformation. Several of these mutations decreased transformation frequencies by several orders of magnitude, thus indicating significant roles in competence development or DNA acquisition in M. luteus. Among the identified loci, there was a new locus containing genes with similarity to genes of the tad clusters of M. luteus and other bacteria.

Characterization of Two α-l-Arabinofuranosidases from Acetivibrio mesophilus and Their Synergistic Effect in Degradation of Arabinose-Containing Substrates.

Arabinofuranosidases are important accessory enzymes involved in the degradation of arabinose-containing poly- and oligosaccharides. Two arabinofuranosidases from the recently described novel anaerobic cellulolytic bacterium Acetivibrio mesophilus, designated AmAraf51 and AmAraf43, were heterologously expressed in Escherichia coli and biochemically characterized. AmAraf51 not only removed arabinose moieties at O-3, O-2 and terminal O-5 positions of arabinose-containing oligosaccharides, but also exhibited exo-ß-xylosidase side activity. In comparison, AmAraf43 preferably cleaved 1,3-linkages from arabinosyl disubstitutions. AmAraf51 and AmAraf43 demonstrated maximum activity at 70 °C and 57 °C, respectively. Judging from the genetic context and substrate specificity, AmAraf51 may decompose internalized arabino/xylo-oligosaccharides. The embedding of the AmAraf43 gene between genes for several putative xylanolytic enzymes, along with its enzymatic properties suggests that AmAraf43 cleaves arabinose decorations from heteroxylans extracellularly. The enzymes revealed completely converse activity profiles towards arabinan/arabinoxylan: AmAraf51 displayed strong activity on arabinan, while AmAraf43 prefers arabinoxylan. AmAraf51 dramatically stimulated the saccharification level of wheat arabinoxylan (WAX-RS) and sugar beet arabinan when administered along with xylanase M_Xyn10 or arabinanase PpAbn43, respectively. For WAX-RS degradation, the yield of arabinose and xylose was boosted 13.77-fold and 4.96-fold, respectively. The bifunctional activity, thermostability and high catalytic efficiency make AmAraf51 an interesting candidate for industrial applications.

The novel EHEC gene asa overlaps the TEGT transporter gene in antisense and is regulated by NaCl and growth phase.

Only a few overlapping gene pairs are known in the best-analyzed bacterial model organism Escherichia coli. Automatic annotation programs usually annotate only one out of six reading frames at a locus, allowing only small overlaps between protein-coding sequences. However, both RNAseq and RIBOseq show signals corresponding to non-trivially overlapping reading frames in antisense to annotated genes, which may constitute protein-coding genes. The transcription and translation of the novel 264 nt gene asa, which overlaps in antisense to a putative TEGT (Testis-Enhanced Gene Transfer) transporter gene is detected in pathogenic E. coli, but not in two apathogenic E. coli strains. The gene in E. coli O157:H7 (EHEC) was further analyzed. An overexpression phenotype was identified in two stress conditions, i.e. excess in salt or arginine. For this, EHEC overexpressing asa was grown competitively against EHEC with a translationally arrested asa mutant gene. RT-qPCR revealed conditional expression dependent on growth phase, sodium chloride, and arginine. Two potential promoters were computationally identified and experimentally verified by reporter gene expression and determination of the transcription start site. The protein Asa was verified by Western blot. Close homologues of asa have not been found in protein databases, but bioinformatic analyses showed that it may be membrane associated, having a largely disordered structure.

The Novel Anaerobiosis-Responsive Overlapping Gene ano Is Overlapping Antisense to the Annotated Gene ECs2385 of Escherichia coli O157:H7 Sakai.

Current notion presumes that only one protein is encoded at a given bacterial genetic locus. However, transcription and translation of an overlapping open reading frame (ORF) of 186 bp length were discovered by RNAseq and RIBOseq experiments. This ORF is almost completely embedded in the annotated L,D-transpeptidase gene ECs2385 of Escherichia coli O157:H7 Sakai in the antisense reading frame -3. The ORF is transcribed as part of a bicistronic mRNA, which includes the annotated upstream gene ECs2384, encoding a murein lipoprotein. The transcriptional start site of the operon resides 38 bp upstream of the ECs2384 start codon and is driven by a predicted σ70 promoter, which is constitutively active under different growth conditions. The bicistronic operon contains a ρ-independent terminator just upstream of the novel gene, significantly decreasing its transcription. The novel gene can be stably expressed as an EGFP-fusion protein and a translationally arrested mutant of ano, unable to produce the protein, shows a growth advantage in competitive growth experiments compared to the wild type under anaerobiosis. Therefore, the novel antisense overlapping gene is named ano (anaerobiosis responsive overlapping gene). A phylostratigraphic analysis indicates that ano originated very recently de novo by overprinting after the Escherichia/Shigella clade separated from other enterobacteria. Therefore, ano is one of the very rare cases of overlapping genes known in the genus Escherichia.

A novel short L-arginine responsive protein-coding gene (laoB) antiparallel overlapping to a CadC-like transcriptional regulator in Escherichia coli O157:H7 Sakai originated by overprinting.

BACKGROUND: Due to the DNA triplet code, it is possible that the sequences of two or more protein-coding genes overlap to a large degree. However, such non-trivial overlaps are usually excluded by genome annotation pipelines and, thus, only a few overlapping gene pairs have been described in bacteria. In contrast, transcriptome and translatome sequencing reveals many signals originated from the antisense strand of annotated genes, of which we analyzed an example gene pair in more detail. RESULTS: A small open reading frame of Escherichia coli O157:H7 strain Sakai (EHEC), designated laoB (L-arginine responsive overlapping gene), is embedded in reading frame -2 in the antisense strand of ECs5115, encoding a CadC-like transcriptional regulator. This overlapping gene shows evidence of transcription and translation in Luria-Bertani (LB) and brain-heart infusion (BHI) medium based on RNA sequencing (RNAseq) and ribosomal-footprint sequencing (RIBOseq). The transcriptional start site is 289 base pairs (bp) upstream of the start codon and transcription termination is 155 bp downstream of the stop codon. Overexpression of LaoB fused to an enhanced green fluorescent protein (EGFP) reporter was possible. The sequence upstream of the transcriptional start site displayed strong promoter activity under different conditions, whereas promoter activity was significantly decreased in the presence of L-arginine. A strand-specific translationally arrested mutant of laoB provided a significant growth advantage in competitive growth experiments in the presence of L-arginine compared to the wild type, which returned to wild type level after complementation of laoB in trans. A phylostratigraphic analysis indicated that the novel gene is restricted to the Escherichia/Shigella clade and might have originated recently by overprinting leading to the expression of part of the antisense strand of ECs5115. CONCLUSIONS: Here, we present evidence of a novel small protein-coding gene laoB encoded in the antisense frame -2 of the annotated gene ECs5115. Clearly, laoB is evolutionarily young and it originated in the Escherichia/Shigella clade by overprinting, a process which may cause the de novo evolution of bacterial genes like laoB.

Methanogen communities in stools of humans of different age and health status and co-occurrence with bacteria.

Hydrogenotrophic methanogens live in a synthrophic relationship with the human gut microbiota as the terminal part of the anaerobic food chain. Methanobrevibacter smithii of the Methanobacteriales is the prevailing archaeal species. Recently, methylotrophic archaea of the novel order Methanomassiliicoccales were isolated from human stools. Few data exist on the prevalence, abundance, persistence and ecology of these methanogens in humans. This study investigated methanogen communities in 26 healthy and obese children (8-14 years) and 18 adults (28-78 years) using quantitative PCR. Samples were obtained from nine females before and after giving birth. Bacterial groups linked to the abundance of methanogens in adult females were identified using a 16S rRNA gene amplicon data set. A total of 89% and 65% of adults and children, respectively, carried Methanobacteriales. Methanomassiliicoccales were recovered from 50% of the adults and one child. Mean relative abundance of Methanomassiliicoccales in adults was lower than that of Methanobacteriales (0.10% versus 0.52%). Both Methanobacteriales and Methanomassiliicoccales formed stable communities in females before and after giving birth. Methanobacteriales co-occurred with bacterial taxonomic groups associated with the trophic chain from carbohydrate degradation to hydrogen and formate formation. Relative abundance was inversely correlated to Blautia. Negative correlation with little characterized groups within the Clostridiales indicated possible interactions of Methanomassiliicoccales with the bacterial community.