pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

Background The ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications. Results A quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli - Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified. Conclusions We show that a shuttle vector-based protein affinity 'pull-down' approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.published_or_final_versio

Multilocus sequence analysis of phylogroup 1 and 2 oral treponeme strains

More than 75 ‘species-level' phylotypes of spirochete bacteria belonging to the genus Treponema reside within the human oral cavity. The majority of these oral treponeme phylotypes correspond to as-yet uncultivated taxa, or strains of uncertain standing in taxonomy. Here, we analyze phylogenetic and taxonomic relationships between oral treponeme strains using a multilocus sequence analysis (MLSA) scheme based on the highly-conserved 16S rRNA, pyrH, recA and flaA genes. We utilize this MLSA scheme to analyze genetic data from a curated collection of oral treponeme strains (n=71) of diverse geographical origins. This comprises phylogroup 1 (n=23) and phylogroup 2 (n=48) treponeme strains; including all relevant ATCC reference strains. The taxonomy of all strains was confirmed or inferred via the analysis of ca. 1,450 bp 16S rRNA gene sequences using a combination of bioinformatic and phylogenetic approaches. Taxonomic and phylogenetic relationships between the respective treponeme strains were further investigated by analyzing individual and concatenated flaA (1,074 nt), recA (1,377 nt) and pyrH (696 nt) gene sequence datasets. Our data confirmed the species differentiation between Treponema denticola (n=41) and Treponema putidum (n=7) strains. Notably, our results clearly supported the differentiation of the 23 phylogroup 1 treponeme strains into 5 distinct ‘species-level' phylotypes. These respectively corresponded to ‘Treponema vincentii' (n=11), Treponema medium (n=1); ‘Treponema sinensis' (T. sp. IA; n=4); Treponema sp. IB (n=3); and Treponema sp. IC (n=4). In conclusion, our MLSA-based approach can be used to effectively discriminate oral treponeme taxa, confirm taxonomic assignment, and enable the delineation of species boundaries with high confidence.published_or_final_versio

Complete Genome Sequence of the Oral Spirochete Bacterium Treponema putidum Strain OMZ 758T (ATCC 700334T)

The oral spirochete bacterium Treponema putidum inhabits human periodontal niches. The complete genome sequence of the OMZ 758(T) (ATCC 700334(T)) strain of this species was determined, revealing a 2,796,913-bp chromosome, with a G+C content of 37.30% and a single plasmid (pTPu1; 3,649 bp) identical to pTS1 from Treponema denticola

Complete Genome Sequence for Treponema sp. OMZ 838 (ATCC 700772, DSM 16789), Isolated from a Necrotizing Ulcerative Gingivitis Lesion

The oral treponeme bacterium Treponema sp. OMZ 838 was originally isolated from a human necrotizing ulcerative gingivitis (NUG) lesion. Its taxonomic status remains uncertain. The complete genome sequence length was determined to be 2,708,067 bp, with a G+C content of 44.58%, and 2,236 predicted coding DNA sequences (CDS)

Gene expression of bacterial collagenolytic proteases in root caries

Objective: It is unknown whether bacteria play a role in the collagen matrix degradation that occurs during caries progression. Our aim was to characterize the expression level of genes involved in bacterial collagenolytic proteases in root biofilms with and without caries. Method: we collected samples from active cavitated root caries lesions (RC, n = 30) and from sound root surfaces (SRS, n = 10). Total microbial RNA was isolated and cDNA sequenced on the Illumina Hi-Seq2500. Reads were mapped to 162 oral bacterial reference genomes. Genes encoding putative bacterial collagenolytic proteases were identified. Normalization and differential expression analysis was performed on all metatranscriptomes (FDR8) but none in SRS were Pseudoramibacter alactolyticus [HMPREF0721_RS02020; HMPREF0721_RS04640], Scardovia inopinata [SCIP_RS02440] and Olsenella uli DSM7084 [OLSU_RS02990]. Conclusion: Our findings suggest that the U32 proteases may be related to carious dentine. The contribution of a small number of species to dentine degradation should be further investigated. These proteases may have potential in future biotechnological and medical applications, serving as targets for the development of therapeutic agents

Snapshot of the three domains of life in the oral niche

Trade Mixer & Poster Session B: abstract no. 329The human oral microbiome is a complex microbial community constantly influenced by several external factors such as diet, oral hygiene, lifestyle and antibiotic exposures, to name a few. Hence it may be expected that subjects with different geographical, socioeconomic or ethnic backgrounds will have different oral microbial community structures. Recent studies using next generation sequencing (NGS) have almost exclusively focused on the oral microbiota of individuals from North America or Europe, who have ‘good’ oral health, or common oral diseases such as periodontitis or caries. Very few studies have been done on subjects of Asian lineage. Among Chinese communities, oral diseases such as periodontitis are relatively prevalent; hence it is important to elucidate their oral microbial communities, to identify putative differences. To address this, we analyzed the subgingival plaque of subjects with periodontitis versus periodontitis-free controls using 454 pyrosequencing; targeting hypervariable regions of the 16S rRNA for Eubacteria and Archaea, and the 18S rRNA for the Eukarya. Analysis of the pyrosequencing data revealed that Actinobacteria, Bacteriodetes, Firmicutes, Fusobacteria, Proterobacteria and Spirochaetes were the common phyla recovered from both the periodontitis and periodontitis-free subjects, although in different proportions. Firmicutes was the most abundant phylum recovered from periodontitis-free subjects followed by Actinobacteria. As for the periodontitis subjects, Bacteriodetes was predominant, with the majority of sequences recovered corresponding to Porphyromonas. Furthermore, levels of the genus Treponema were also significantly higher in periodontitis subjects. Only sparse fungi sequences were recovered and Saccharomycetes was more in number among the periodontitis-free subjects. Lastly, Archaea was only evident in the periodontitis subjects and 100% of the sequences recovered were highly similar to Methanobrevibacter oralis. This study gives an overview of the diversity of the three domains of life in the oral micro-niche.link_to_OA_fulltex

PZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

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