Fumarate and nitrite reduction by Prevotella nigrescens and Prevotella buccae isolated from Chronic Periodontitis patients.

OBJECTIVE: This study is an investigation of anaerobic nitrite and fumarate reduction/respiration abilities of two characterised Prevotella species namely Prevotella nigrescens (SS6B) and Prevotella buccae (GS6B) isolated from the periodontal pockets of chronic periodontitis (ChP) patients. METHODS: Isolation and identification of the periodontal bacteria from 20 patients showing clinical symptoms of ChP. Characterisation of anaerobic nitrite and fumarate reduction was done in P. nigrescens (SS6B) and P. buccae (GS6B) using reduction assays, inhibition assays with use of specific inhibitors, growth assays and enzyme activity assays. Degenerate PCR was used to detect and amplify nitrite reductase (nrfA) and fumarate reductase (frdA) gene sequences in these Prevotella isolates. In addition, molecular and in silico analysis of the amplified anaerobic reductase gene sequences was performed using NCBI conserved domain analysis, Interpro database and MegaX. RESULTS: We provided experimental evidence for presence of active nitrite and fumarate reductase activities through enzyme activity, reduction, inhibitor and growth assays. Moreover, we were able to detect presence of 505 bps nrfA gene fragment and 400 bps frdA gene fragment in these Prevotella spp. These fragments show similarity to multiheme ammonia forming cytochrome c nitrite reductases and fumarate reductases flavoprotein subunit, respectively. CONCLUSION: Anaerobic nitrite and fumarate respiration abilities in P. nigrescens and P. buccae isolates appear to be important for detoxification process and growth, respectively.

Prevotella: An insight into its characteristics and associated virulence factors.

Prevotella species, a gram-negative obligate anaerobe, is commonly associated with human infections such as dental caries and periodontitis, as well as other conditions such as chronic osteomyelitis, bite-related infections, rheumatoid arthritis and intestinal diseases like ulcerative colitis. This generally harmless commensal possesses virulence factors such as adhesins, hemolysins, secretion systems exopolysaccharide, LPS, proteases, quorum sensing molecules and antibiotic resistance to evolve into a well-adapted pathogen capable of causing successful infection and proliferation in the host tissue. This review describes several of these virulence factors and their advantage to Prevotella spp. in causing inflammatory diseases like periodontitis. In addition, using genome analysis of Prevotella reference strains, we examined other putative virulence determinants which can provide insights as biomarkers and be the targets for effective interventions in Prevotella related diseases like periodontitis.

Synthesis of Alginate Nanogels with Polyvalent 3D Transition Metal Cations: Applications in Urease Immobilization.

Biocompatible nanogels are highly in demand and have the potential to be used in various applications, e.g., for the encapsulation of sensitive biomacromolecules. In the present study, we have developed water-in-oil microemulsions of sodium alginate sol/hexane/Span 20 as a template for controlled synthesis of alginate nanogels, cross-linked with 3d transition metal cations (Mn2+, Fe3+, and Co2+). The results suggest that the stable template of 110 nm dimensions can be obtained by microemulsion technique using Span 20 at concentrations of 10mM and above, showing a zeta potential of -57.3 mV. A comparison of the effects of the cross-links on the morphology, surface charge, protein (urease enzyme) encapsulation properties, and stability of the resulting nanogels were studied. Alginate nanogels, cross-linked with Mn2+, Fe3+, or Co2+ did not show any gradation in the hydrodynamic diameter. The shape of alginate nanogels, cross-linked with Mn2+ or Co2+, were spherical; whereas, nanogels cross-linked with Fe3+ (Fe-alginate) were non-spherical and rice-shaped. The zeta potential, enzyme loading efficiency, and enzyme activity of Fe-alginate was the highest among all the nanogels studied. It was found that the morphology of particles influenced the percent immobilization, loading capacity, and loading efficiency of encapsulated enzymes. These particles are promising candidates for biosensing and efficient drug delivery due to their relatively high loading capacity, biocompatibility, easy fabrication, and easy handling.

The octahaem SirA catalyses dissimilatory sulfite reduction in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a metal reducer that uses a large number of electron acceptors including thiosulfate, polysulfide and sulfite. The enzyme required for thiosulfate and polysulfide respiration has been recently identified, but the mechanisms of sulfite reduction remained unexplored. Analysis of MR-1 cultures grown anaerobically with sulfite suggested that the dissimilatory sulfite reductase catalyses six-electron reduction of sulfite to sulfide. Reduction of sulfite required menaquinones but was independent of the intermediate electron carrier CymA. Furthermore, the terminal sulfite reductase, SirA, was identified as an octahaem c cytochrome with an atypical haem binding site. The sulfite reductase of S. oneidensis MR-1 does not appear to be a sirohaem enzyme, but represents a new class of sulfite reductases. The gene that encodes SirA is located within a 10-gene locus that is predicted to encode a component of a specialized haem lyase, a menaquinone oxidase and copper transport proteins. This locus was identified in the genomes of several Shewanella species and appears to be linked to the ability of these organisms to reduce sulfite under anaerobic conditions.

Regulation and Maturation of the Shewanella oneidensis Sulfite Reductase SirA.

Shewanella oneidensis, a metal reducer and facultative anaerobe, expresses a large number of c-type cytochromes, many of which function as anaerobic reductases. All of these proteins contain the typical heme-binding motif CXXCH and require the Ccm proteins for maturation. Two c-type cytochrome reductases also possess atypical heme-binding sites, the NrfA nitrite reductase (CXXCK) and the SirA sulfite reductase (CX12NKGCH). S. oneidensis MR-1 encodes two cytochrome c synthetases (CcmF and SirE) and two apocytochrome c chaperones (CcmI and SirG). SirE located in the sir gene cluster is required for the maturation of SirA, but not NrfA. Here we show that maturation of SirA requires the combined function of the two apocytochrome c chaperones CcmI and SirG. Loss of either protein resulted in decreased sulfite reductase. Furthermore, SirA was not detected in a mutant that lacked both chaperones, perhaps due to misfolding or instability. These results suggest that CcmI interacts with SirEFG during SirA maturation, and with CcmF during maturation of NrfA. Additionally, we show that CRP regulates expression of sirA via the newly identified transcriptional regulatory protein, SirR.