Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148.

Shewanella oneidensis MR-1 generates electricity from lactic acid, but cannot utilize starch. On the other hand, Streptococcus bovis 148 metabolizes starch and produces lactic acid. Therefore, two methods were trialed for starch-fueled microbial fuel cell (MFC) in this study. In electric generation by two-step fermentation (EGT) method, starch was first converted to lactic acid by S. bovis 148. The S. bovis 148 were then removed by centrifugation, and the fermented broth was preserved for electricity generation by S. oneidensis MR-1. Another method was electric generation by parallel fermentation (EGP) method. In this method, the cultivation and subsequent fermentation processes of S. bovis 148 and S. oneidensis MR-1 were performed simultaneously. After 1, 2, and 3 terms (5-day intervals) of S. oneidensis MR-1 in the EGT fermented broth of S. bovis 148, the maximum currents at each term were 1.8, 2.4, and 2.8 mA, and the maximum current densities at each term were 41.0, 43.6, and 49.9 mW/m2, respectively. In the EGP method, starch was also converted into lactic acid with electricity generation. The maximum current density was 140-200 mA/m2, and the maximum power density of this method was 12.1 mW/m2.

Species identification of Streptococcus bovis group isolates causing bacteremia: a comparison of two MALDI-TOF MS systems.

This study compared two MALDI-TOF MS systems (Biotyper and VITEK MS) on clinical Streptococcus bovis group isolates (n=66). The VITEK MS gave fewer misidentifications and a higher rate of correct identifications than the Biotyper. Only the identification of S. lutetiensis by the VITEK MS was reliable. Additional optimization of the available system databases is needed.

Streptococcus bovis new taxonomy: does subspecies distinction matter?

Bacteremia with Streptococcus bovis/equinus complex strains is associated with hepatobiliary disease, colorectal lesions (CL), and infective endocarditis (IE). This study addressed the clinical significance of subspecies distinction of previously designated S. bovis blood culture isolates according to the updated nomenclature. During 2002-2013, all blood culture isolates previously designated as S. bovis were recultured and identified using 16S rRNA gene sequencing and MALDI-TOF (Bruker BioTyper and Vitek MS, bioMérieux). Clinical data of patients aged ≥18 years were reviewed. A review of four recent case series was performed as well. Forty blood isolates were identified using 16S rRNA sequencing. Twenty-six bacteremic patients had S. gallolyticus ssp. pasteurianus, six had S. gallolyticus ssp. gallolyticus, two had S. gallolyticus ssp. macedonicus, and six had S. infantarius bacteremia. Species diagnosis using Vitek and bioMérieux MALDI-TOF technology was applicable in 37 and 36 samples, respectively, and was successful in all samples (100 %). Subspecies identification was confirmed in 30 (83 %) samples (as compared with 16S rRNA sequencing detection). IE was diagnosed in 22 (59 %) patients and CL in 8 (20 %) patients. Both complications were associated with all subspecies. Combining our results with those of four recent series resulted in, overall, 320 bacteremic cases, of which 88 (28 %) had CL and 66 (21 %) had IE. All 'bovis/equinus' complex subspecies were associated with CL or IE. From a clinical point of view, species diagnosis using MALDI-TOF MS should suffice to warrant consideration of transesophageal echocardiography and colonoscopy.

[Individual catalytic activity of two functional domains of bovicin HJ50 synthase BovM].

OBJECTIVE: To reconstitute the in vitro catalytic activity of the individual dehydratase or cyclase domain of bifunctional bovicin HJ50 synthase BovM, and lay a foundation for the further investigation of catalytic mechanism of class II lantibiotic synthase LanM. METHOD: The truncated proteins of BovM containing the N-terminal dehydratase domain or C-terminal cyclase domain were expressed in E. coli and purified. Substrate BovA, the precursor of bovicin HJ50, was incubated with these truncated BovM proteins in in vitro reaction system. The antimicrobial activity assay and MALDI-TOF MS analysis were used to monitor the dehydratase or cyclase activity of these truncated proteins. Meanwhile, the synergistic activities of both truncated proteins were tested in vivo and in vitro. RESULTS: The N- and C-terminal domains of BovM possessed dehydration and cyclization activity respectively. However, no synergistic activity was detected between these two functional domains. CONCLUSION: The individual functional domains of BovM could execute their corresponding functions independently, but the intactness of BovM was important for its full modification activity.

Identification of ligand specificity determinants in lantibiotic bovicin HJ50 and the receptor BovK, a multitransmembrane histidine kinase.

Lantibiotic bovicin HJ50 is produced by Streptococcus bovis HJ50 and acts as the extracellular signal to autoregulate its own biosynthesis through BovK/R two-component system. Bovicin HJ50 shows a linear N-terminal and glubolar C-terminal structure, and the sensor histidine kinase BovK contains eight transmembrane segments lacking any extensive surface-exposed sensory domain. The signal recognition mechanism between bovicin HJ50 and BovK is still unknown. We performed saturated alanine scanning mutagenesis and other amino acid substitutions on bovicin HJ50 using a semi-in vitro biosynthesis. Results of the mutants inducing activities indicated that several charged and hydrophobic amino acids in ring B of bovicin HJ50, as well as two glycines were key residues to recognize BovK. Circular dichroism analyses indicated that both glycines contributed to bovicin HJ50 structural changes in the membrane. Biotin-labeled bovicin HJ50 could interact with the N-terminal sensor of BovK, and several charged residues and a conserved hydrophobic region in the N-terminal portion of BovK sensor domain were important for interacting with the signal bovicin HJ50. By combining the results, we suggested a mechanism of bovicin HJ50 recognizing and activating BovK mainly through electrostatic and hydrophobic interactions.

[Effects of secondary structure of the leader peptide on modification and processing of bovicin HJ50].

OBJECTIVE: Elucidating the correlation between the secondary structure of the leader peptide of lantibiotic bovicin HJ50 and its modification and processing. METHODS: The variants with mutated leader peptide were synthesized by semi-in vitro biosynthesis, and their modification pattern were then analyzed by MALDI-TOF MS. At the same time, the effect of leader peptide mutants on processing the modified propeptide was examined by HPLC and antimicrobial activity. RESULTS: We constructed 6 mutants (F-16A, V-15E, E-14L, E-8P, L-7D, L-4K) involved in forming secondary structure of the bovicin HJ50 leader peptide. F-16A, V-15E, L-4K showed very little effect on modification and processing whereas E-14L and E-8P caused changes in modification. In addition, we found that L-7D strongly affected the processing. CONCLUSION: The conserved helix structure in the leader peptide of bovicin HJ50 was closely related to the activity of BovM and BovT150, and the presence of secondary structure was very important to modification and processing of bovicin HJ50.

Interaction with lipid II induces conformational changes in bovicin HC5 structure.

Bovicin HC5 is a lantibiotic produced by Streptococcus bovis HC5 that targets the cell wall precursor lipid II. An understanding of the modes of action against target bacteria can help broadening the clinical applicability of lantibiotics in human and veterinary medicine. In this study, the interaction of bovicin HC5 with lipid II was examined using tryptophan fluorescence and circular dichroism spectroscopy with model membrane systems that do or do not allow pore formation by bovicin HC5. In the presence of lipid II, a blue-shift of 12 nm could be observed for the fluorescence emission maximum of the tryptophan residue for all of the membrane systems tested. This change in fluorescence emission was paralleled by a decrease in accessibility toward acrylamide and phospholipids carrying a spin-label at the acyl chain; the tryptophan residue of bovicin HC5 was located near the twelfth position of the membrane phospholipid acyl chains. Moreover, the binding of lipid II by bovicin HC5 induced remarkable conformational changes in the bovicin HC5 structure. The interaction of bovicin HC5 with lipid II was highly stable even at pH 2.0. These results indicate that bovicin HC5 interacts directly with lipid II and that the topology of this interaction changes under different conditions, which is relevant for the biological activity of the peptide. To our knowledge, bovicin HC5 is the only bacteriocin described thus far that is able to interact with its target in extreme pH values, and this fact might be related to its unique structure and stability.

Differentiation of species of the Streptococcus bovis/equinus-complex by MALDI-TOF Mass Spectrometry in comparison to sodA sequence analyses.

The Streptococcus bovis/equinus complex is a heterogeneous group within the group D streptococci with important clinical relevance regarding infective endocarditis, sepsis and colon carcinoma. The taxonomic identification of species and sub-species of this complex, by the standard methods remains difficult. In the present study, we compared the cluster analysis of 88 strains of species of the S. bovis/equinus complex by sequence analysis of the manganese-dependent superoxide dismutase gene (sodA) and by Matrix Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS). We observed a high congruence of strain grouping by MALDI-TOF MS in comparison with sodA sequence analyses, demonstrating the accuracy and reliability of MALDI-TOF MS in comparison to DNA sequence-based method. By generating mass spectra for each species and sub-species, we were able to discriminate all members of the S. bovis/equinus complex. Furthermore, we demonstrated reliable identifications to the species level by MALDI-TOF MS, independently of cultivation conditions.

Effect of pH on the activity of bovicin HC5, a bacteriocin from Streptococcus bovis HC5.

The bacteriocin, bovicin HC5, catalyzed potassium efflux from Streptococcus bovis JB1, and this activity was highly pH dependent. When the pH was near neutral, glucose-energized cells were not affected by bovicin HC5, but the intracellular steady-state concentration of potassium decreased at acidic pH values. The idea that pH was affecting bovicin HC5 binding was supported by the observation that acidic pH also enhanced the efflux of potassium from non-energized cells that had been loaded with potassium. The relationship between bovicin HC5 concentration and potassium depletion was a saturation function, but cooperativity plots indicated that the binding of one bovicin molecule to the cell membrane facilitated the binding of another.

Factors affecting the antibacterial activity of the ruminal bacterium, Streptococcus bovis HC5.

Streptococcus bovis HC5 inhibits a variety of S. bovis strains and other Gram-positive bacteria, but factors affecting this activity had not been defined. Batch culture studies indicated that S. bovis HC5 did not inhibit S. bovis JB1 (a non-bacteriocin-producing strain) until glucose was depleted and cells were entering stationary phase, but slow-dilution-rate, continuous cultures (0.2 h(-1)) had as much antibacterial activity as stationary-phase batch cultures. Because the activity of continuous cultures (0.2-1.2 h(-1)) was inversely related to the glucose consumption rate, it appeared that the antibacterial activity was being catabolite repressed by glucose. When the pH of continuous cultures (0.2 h(-1)) was decreased from 6.7 to 5.4, antibacterial activity doubled, but this activity declined at pH values less than 5.0. Continuous cultures (0.2 h(-1)) that had only ammonia as a nitrogen source had antibacterial activity, and large amounts of Trypticase (10 mg ml(-1)) caused only a 2.0-fold decline in the amount of HC5 cell-associated protein that was needed to prevent S. bovis JB1 growth. Because S. bovis HC5 was able to produce antibacterial activity over a wide range of culture conditions, there is an increased likelihood that this activity could have commercial application.

Bovicin HC5, a bacteriocin from Streptococcus bovis HC5.

Previous work indicated that Streptococcus bovis HC5 had significant antibacterial activity, and even nisin-resistant S. bovis JB1 cells could be strongly inhibited. S. bovis HC5 inhibited a variety of Gram-positive bacteria and the spectrum of activity was similar to monensin, a commonly used feed additive. The crude extracts (ammonium sulfate precipitation) were inactivated by Pronase E and trypsin, but the activity was resistant to heat, proteinase K and alpha-chymotrypsin. Most of the antibacterial activity was cell associated, but it could be liberated by acidic NaCl (100 mM, pH 2.0) without significant cell lysis. When glycolysing S. bovis JB1 cells were treated with either crude or acidic NaCl extracts, intracellular potassium declined and this result indicated the antibacterial activity was mediated by a pore-forming peptide. The peptide could be purified by HPLC and matrix-assisted laser desorption ionization time-of-flight analysis indicated that it had a molecular mass of approximately 2440 Da. The terminal amino acid sequence was VGXRYASXPGXSWKYVXF. The unnamed amino acid residues (designated by X) had approximately the same position as dehydroalanines found in some lantibiotics, but samples that were reduced and alkylated prior to Edman degradation did not have cysteine residues. The only other bacteriocin that had significant similarity was the lantibiotic precursor of Streptococcus pyogenes SF370, but the identity was only 55%. Based on these results, the bacteriocin of S. bovis HC5 appears to be novel and the authors now designate it as bovicin HC5.

The replicon of the cryptic Plasmid pSBO1 isolated from Streptococcus bovis JB1.

The cryptic plasmid pSBO1 (3904 bp) was isolated from Streptococcus bovis JB1. pSBO1 contained an open reading frame (ORF) that is homologous to sequences encoding the replication protein (Rep) in pEFC1 (isolated from Enterococcus faecalis), pSK639 (Staphylococcus epidermidis), pLA103 (Lactobacillus acidophilus), and pUCL287 (Tetragenococcus halophila). In addition, four 22-bp direct repeats (DRs) were located upstream of the putative replication gene (rep) of pSBO1. Recombinant plasmids (pSBE10 and pSBE11) containing the DRs and putative rep of pSBO1 replicated in S. bovis 12-U-1 and no8 strains. This result indicates that the putative rep encoded Rep and that the replicon of pSBO1 contained the DRs and the rep. Gel shift assays showed that the Rep of pSBO1 bound the 22-bp DRs.

Identification of virulence associated markers in the cell wall of pigeon Streptococcus gallolyticus strains.

The cell wall protein profiles of 56 isolates of Streptococcus gallolyticus of differing virulence for pigeons were compared by SDS-PAGE. Additionally, Western blot analysis was performed on the cell wall proteins of 14 strains using sera of pigeons, experimentally infected with A(+)T1 or A(-)T2 strains of S. gallolyticus. The profile of silver stained gels exhibited a complex array of 20-50 bands ranging from less than 6.5-210kDa. A band with molecular mass of 114kDa was only observed in isolates that belonged to the highly virulent A(+)T1, A(+)T2, A(+)T3 and A(-)T1 culture supernatant groups. A band with a slightly higher molecular mass (115kDa) as well as a 207kDa band were only detected in isolates that belonged to the moderately A(-)T3 or low A(-)T2 virulent culture supernatant groups. The 114 and 115kDa band were recognised by all homologous and heterologous pigeon sera used whereas the 207kDa band was only recognised by sera of pigeons infected with a A(-)T2 strain. These findings may indicate that the 114, 115 and 207kDa bands are useful as additional virulence associated markers for pigeon S. gallolyticus strains.