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See detailThe extracellular RNA complement of Escherichia coli
Ghosal, Anubrata UL; Upadhyaya, Bimal Babu UL; Fritz, Joëlle UL et al

in MicrobiologyOpen (2015)

he secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but ... [more ▼]

he secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacte- ria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA comple- ment. Our results demonstrate that a large part of the extracellular RNA com- plement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV- free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA- fimL and ves-spy intergenic regions. Our study provides the first detailed char- acterization of the extracellular RNA complement of the enteric model bacte- rium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication. [less ▲]

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See detailParacoccus denitrificans possesses two BioR homologs having a role in regulation of biotin metabolism
Feng, Youjun; Kumar, Ritesh; Ravcheev, Dmitry UL et al

in MicrobiologyOpen (2015), 4

Recently, we determined that BioR, the GntR family of transcription factor, acts as a repressor for biotin metabolism exclusively distributed in certain species of a-proteobacteria, including the zoonotic ... [more ▼]

Recently, we determined that BioR, the GntR family of transcription factor, acts as a repressor for biotin metabolism exclusively distributed in certain species of a-proteobacteria, including the zoonotic agent Brucella melitensis and the plant pathogen Agrobacterium tumefaciens. However, the scenario is unusual in Paracoccus denitrificans, another closely related member of the same phylum a-proteobacteria featuring with denitrification. Not only does it encode two BioR homologs Pden_1431 and Pden_2922 (designated as BioR1 and BioR2, respectively), but also has six predictive BioR-recognizable sites (the two bioR homolog each has one site, whereas the two bio operons (bioBFDAGC and bioYB) each contains two tandem BioR boxes). It raised the possibility that unexpected complexity is present in BioR-mediated biotin regulation. Here we report that this is the case. The identity of the purified BioR proteins (BioR1 and BioR2) was confirmed with LC-QToF-MS. Phylogenetic analyses combined with GC percentage raised a possibility that the bioR2 gene might be acquired by horizontal gene transfer. Gel shift assays revealed that the predicted BioR-binding sites are functional for the two BioR homologs, in much similarity to the scenario seen with the BioR site of A. tumefaciens bioBFDAZ. Using the A. tumefaciens reporter system carrying a plasmid-borne LacZ fusion, we revealed that the two homologs of P. denitrificans BioR are functional repressors for biotin metabolism. As anticipated, not only does the addition of exogenous biotin stimulate efficiently the expression of bioYB operon encoding biotin transport/uptake system BioY, but also inhibits the transcription of the bioBFDAGC operon resembling the de novo biotin synthetic pathway. EMSA-based screening failed to demonstrate that the biotin-related metabolite is involved in BioR-DNA interplay, which is consistent with our former observation with Brucella BioR. Our finding defined a complex regulatory network for biotin metabolism in P. denitrificans by two BioR proteins. [less ▲]

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See detailTwo novel regulators of N-acetyl-galactosamine utilization pathway and distinct roles in bacterial infections
Zhang, Huimin; Ravcheev, Dmitry UL; Hu, Dan et al

in MicrobiologyOpen (2015), 4(6), 983-1000

Bacterial pathogens can exploit metabolic pathways to facilitate their successful infection cycles, but little is known about roles of d-galactosamine (GalN)/ N-acetyl-d- galactosamine (GalNAc) catabolism ... [more ▼]

Bacterial pathogens can exploit metabolic pathways to facilitate their successful infection cycles, but little is known about roles of d-galactosamine (GalN)/ N-acetyl-d- galactosamine (GalNAc) catabolism pathway in bacterial pathogenesis. Here, we report the genomic reconstruction of GalN/GalNAc utilization pathway in Streptococci and the diversified aga regulons. We delineated two new paralogous AgaR regulators for the GalN/GalNAc catabolism pathway. The electrophoretic mobility shift assays experiment demonstrated that AgaR2 (AgaR1) binds the predicted palindromes, and the combined in vivo data from reverse transcription quantitative polymerase chain reaction and RNA-seq suggested that AgaR2 (not AgaR1) can effectively repress the transcription of the target genes. Removal of agaR2 (not agaR1) from Streptococcus suis 05ZYH33 augments significantly the abilities of both adherence to Hep-2 cells and anti-phagocytosis against RAW264.7 macrophage. As anticipated, the dysfunction in AgaR2-mediated regulation of S. suis impairs its pathogenicity in experimental models of both mice and piglets. Our finding discovered two novel regulators specific for GalN/GalNAc catabolism and assigned them distinct roles into bacterial infections. To the best of our knowledge, it might represent a first paradigm that links the GalN/GalNAc catabolism pathway to bacterial pathogenesis. [less ▲]

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