The effect of carbon and nitrogen sources on bovicin HC5 production by Streptococcus bovis HC5

Aims:  To investigate the effect of media composition and agroindustrial residues on bovicin HC5 production by Streptococcus bovis HC5.
Methods and Results:  Batch cultures of S. bovis HC5 were grown in basal medium containing different carbon and nitrogen sources. The activity of cell-free and cell-associated bovicin HC5 was determined in culture supernatants and acidic extracts obtained from cell pellets, respectively. Streptococcus bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l⁻¹ of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when yeast extract and trypticase (0·5 and 1·0 g l⁻¹, respectively) were added together to the basal medium. Streptococcus bovis HC5 cultures produced bovicin HC5 in cheese whey and sugar cane juice and maximal volumetric productivity was obtained after 12 h of incubation.
Conclusions:  Streptococcus bovis HC5 is a versatile lactic acid bacterium that can utilize several carbon and nitrogen sources for bovicin HC5 production. This bacterium could be a useful model to study bacteriocin production in the rumen ecosystem.
Significance and Impact of the Study:  The use of agroindustrial residues as carbon sources could have an economical impact on bovicin HC5 production. To our knowledge, this is the first report to show the use of sugar cane juice for bacteriocin production by lactic acid bacteria.     

Bovicin HC5 inhibits wasteful amino acid degradation by mixed ruminal bacteria in vitro

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5) that inhibits pure cultures of hyper ammonia-producing bacteria (HAB). Experiments were preformed to see if: (1) S. bovis HC5 cells could inhibit the deamination of amino acids by mixed ruminal bacteria taken directly from a cow, (2) semi-purified bovicin was as effective as S. bovis HC5 cells, and 3) semi-purified and the feed additive monensin were affecting the same types of ammonia-producing ruminal bacteria. Because purified and semi-purified bovicin HC5 was as effective as S. bovis HC5 cells, it appeared that bovicin HC5 was penetrating the cell membranes of HAB before it could be degraded by peptidases and proteinases. Mixed ruminal bacteria that were successively transferred and enriched nine times with trypticase did not become significantly more resistant to either bovicin HC5 (50 AU mL⁻¹) or monensin (5 μM), and amplified rDNA restriction analysis indicated that bovicin HC5 and monensin appeared to be selecting against the same types of bacteria.     

Bacterial competition between a bacteriocin-producing and a bacteriocin-negative strain of Streptococcus bovis in batch and continuous culture

A bacteriocin-producing Streptococcus bovis strain (HC5) outcompeted a sensitive strain (JB1) before it reached stationary phase (pH 6.4), even though it grew 10% slower and cell-free bovicin HC5 could not yet be detected. The success of bacteriocin-negative S. bovis isolates was enhanced by the presence of another sensitive bacterium (Clostridium sticklandii SR). PCR based on repetitive DNA sequences indicated that S. bovis HC5 was not simply transferring bacteriocin genes to S. bovis JB1. When the two S. bovis strains were coinoculated into minimal medium, bacteriocin-negative isolates predominated, and this effect could be explained by the longer lag time (0.5 vs. 1.5 h) of S. bovis HC5. If the glucose concentration of the minimal medium was increased from 2 to 7 mg mL(-1), the effect of lag time was diminished and bacteriocin-producing isolates once again dominated the coculture. When the competition was examined in continuous culture, it became apparent that batch culture inocula were never able to displace a strain that had already reached steady state, even if the inoculum was large. This result indicated that bacterial selection for substrate affinity was even more important than bacteriocin production.     

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.     

Production of bacteriocins by Streptococcus bovis strains from Australian ruminants

Aims: To examine the prevalence of bacteriocin production in Streptococcus bovis isolates from Australian ruminants and the feasibility of industrial production of bacteriocin. Methods and Results: Streptococcus bovis strains were tested for production of bacteriocin‐like inhibitory substances (BLIS) by antagonism assay against Lactococcus lactis. BLIS production was associated with source animal location (i.e. proximity of other bacteriocin‐positive source animals) rather than ruminant species/breed or diet. One bacteriocin showing strong inhibitory activity (Sb15) was isolated and examined. Protein sequence, stability and activity spectrum of this bovicin were very similar to bovicin HC5. Production could be increased through serial culturing, and increased productivity could be partially maintained during cold storage of cultures. Conclusions: BLIS production is geographically widely distributed in Eastern Australia, and it appears that the bacteriocin⁺ trait is maintained in animals at the same location. The HC5‐like bacteriocin, originally identified in North America, is also found in Australia. Production of bacteriocin can be increased through serial culturing. Significance and Impact of the Study: The HC5‐like bacteriocins appear to have a broad global distribution. Serial culturing may provide a route towards commercial manufacturing for use in industrial applications, and purified bacteriocin from S. bovis Sb15 could potentially be used to prevent food spoilage or as a feed additive to promote growth in ruminant species.     

The effect of bovicin HC5, a bacteriocin from Streptococcus bovis HC5, on ruminal methane production in vitro

A Pseudomonas monteilli strain (designated C11) that uses the phosphotriester coroxon as its sole phosphorus source has been isolated. Native PAGE and activity staining identified a single isozyme with significant phosphotriesterase activity in the soluble fraction of the cell. This phosphotriesterase could hydrolyse both coumaphos and coroxon. The hydrolysis product of coroxon, diethylphosphate, and the thion analogue, coumaphos, could not serve as phosphorus sources when added to the growth medium. The majority of the phosphotriesterase and phosphatase activity was contained in the soluble fraction of the cell. Phosphatase activity was inhibited by vanadate as well as by dialysis against the metal chelator, EDTA. Phosphotriesterase activity was not affected by either vanadate or dialysis with EDTA or 1,10-phenanthroline. Phosphotriesterase activity was regulated by the amounts of both phosphate and coroxon in the medium, whereas total phosphatase activity was regulated by phosphate but not coroxon. A lack of hybridisation using a probe against the opd (organophosphate degradation) gene encoding a phosphotriesterase from Flavobacterium sp. ATCC27551 against bulk DNA from P. monteilli C11 suggested that this strain does not contain opd. The work presented here indicates the presence of a novel phosphotriesterase in P. monteilli C11.     

The ability of non-bacteriocin producing Streptococcus bovis strains to bind and transfer bovicin HC5 to other sensitive bacteria

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5), but S. bovis JB1 does not have antimicrobial activity. Preliminary experiments revealed an anomaly. When S. bovis JB1 cells were washed in stationary phase S. bovis HC5 cell-free culture supernatant, the S. bovis JB1 cells were subsequently able to inhibit hyper-ammonia producing ruminal bacteria (Clostridium sticklandii, Clostridium aminophilum and Peptostreptococcus anaerobius). Other non-bacteriocin producing S. bovis strains also had the ability to bind and transfer semi-purified bovicin HC5. Bovicin HC5 that was bound to S. bovis JB1 was much more resistant to Pronase E than cell-free bovicin HC5, but it could be inactivated if the incubation period was 24 h. Acidic NaCl treatment (100 mM, pH 2.0) liberates half of the bovicin HC5 from S. bovis HC5, but it did not prevent bovicin HC5 from binding to S. bovis JB1. Acidic NaCl liberated some bovicin HC5 from S. bovis JB1, but the decrease in activity was only 2-fold. Bovicin HC5 is a positively charged peptide, and the ability of S. bovis JB1 to bind bovicin HC5 could be inhibited by either calcium or magnesium (100 mM). Acidic NaCl-treated S. bovis JB1 cells were unable to accumulate potassium, but they were still able to bind bovicin HC5 and prevent potassium accumulation by untreated S. bovis JB1 cells. Based on these results, bovicin HC5 bound to S. bovis JB1 cells still acts as a pore-forming lantibiotic.     

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.     

奶牛瘤胃中牛链球菌的分离鉴定

采用厌氧分离技术从奶牛瘤胃中分离出1株细菌,通过对其形态、培养特性、生理生化特性、16S rRNA基因序列测定与同源性分析等研究,确定分离菌株为牛链球菌（Streptococcus bovis）,为进一步研究其对瘤胃发酵的影响奠定了基础。     

Heterologous expression of an endoglucanase gene (endA) from the ruminai anaerobe Ruminococcus flavefaciens 17 in Streptococcus bovis and Streptococcus sanguis

Abstract The heterologous expression of a cloned endoglucanase gene ( endA ) from the ruminai bacterium Ruminococcus flavefaciens 17 was demonstrated in the Streptococcus species S. bovis JB1 and S. sanguis DLL The endA gene was introduced into S. bovis and S. sanguis using the Escherichia coli/Streptococcus shuttle vector pVA838. Expression of the gene was detected by clearing zones around the recombinant colonies on agar plates containing carboxymethylcellulose stained with Congo red. S. bovis JB1 containing the endA gene was capable of utilizing cellotetraose at a faster rate than the parent strain. This is the first demonstration that Streptococcus species can express a gene from a Ruminococcus flavefaciens strain.     

Alternative strategies of 2-deoxyglucose resistance and low affinity glucose transport in the ruminal bacteria, Streptococcus bovis and Selenomonas ruminantium

Abstract Streptococcus bovis and Selenomonas ruminantium grew in the presence of the glucose analog, 2-deoxyglucose (2-DG), but the cells no longer had high affinity glucose transport. In S. bovis , 2-DG resistance was correlated with a decrease in phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase (PTS) activity. The 2-DG-selected S. bovis cells relied solely upon a low affinity, facilitated diffusion mechanism of glucose transport and a 2-DG-resistant glucokinase (ATP-dependent). The glucokinase activity of S. ruminantium was competitively inhibited by 2-DG, and the 2-DG selected cells continued to use PEP-dependent PTS as a mechanism of glucose transport. In this latter case, the 2-DG selected cells switched from a mannosephosphotransferase (enzyme II) that phosphorylated glucose, mannose, and 2-DG, but not α-methylglucoside to a glucosephosphotransferase (enzyme II) that phosphorylated glucose and α-methylglucoside but not 2-DG or mannose. The glucosephosphotransferase (enzyme II) had a very low affinity for glucose and the transport kinetics were similar to the facilitated diffusion system of S. bovis .     

Characterization of Streptococcus bovis from the rumen of the dromedary camel and Rusa deer

AIMS: Isolation and characterization of Streptococcus bovis from the dromedary camel and Rusa deer. METHODS AND RESULTS: Bacteria were isolated from the rumen contents of four camels and two deer fed lucerne hay by culturing on the semi-selective medium MRS agar. Based on Gram morphology and RFLP analysis seven isolates, MPR1, MPR2, MPR3, MPR4, MPR5, RD09 and RD11 were selected and putatively identified as Streptococcus. The identity of these isolates was later confirmed by comparative DNA sequence analysis of the 16S rRNA gene with the homologous sequence from S. bovis strains, JB1, C14b1, NCFB2476, SbR1, SbR7 and Sb5, from cattle and sheep, and the Streptococcus equinus strain NCD01037T. The percentage similarity amongst all strains was >99%, confirming the identification of the camel isolates as S. bovis. The strains were further characterized by their ability to utilize a range of carbohydrates, the production of volatile fatty acids (VFA) and lactate and the determination of the doubling time in basal medium 10 supplemented with glucose. All the isolates produced l-lactate as a major fermentation end product, while four of five camel isolates produced VFA. The range of carbohydrates utilized by all the strains tested, including those from cattle and sheep were identical, except that all camel isolates and the deer isolate RD11 were additionally able to utilize arabinose. CONCLUSIONS: Streptococcus bovis was successfully isolated from the rumen of camels and deer, and shown by molecular and biochemical characterization to be almost identical to S. bovis isolates from cattle and sheep. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus bovis is considered a key lactic acid producing bacterium from the gastrointestinal tract of ruminants, and has been implicated as a causative agent of lactic acidosis. This study is the first report of the isolation and characterization of S. bovis from the dromedary camel and Rusa deer, and suggests a major contributive role of this bacterium to fermentative acidosis.     

Modelling and predicting the effect of temperature,water activity and pH on growth of Streptococcus iniae in Tilapia

Aims: To evaluate and model the growth of Streptococcus iniae affect by temperatures (10–45°C), water activity (A_w; 0·995–0·957), and pH (5–8). Methods and Results: Temperatures, A_w, and pH were adjusted. The behaviour of S. iniae was studied and modelled. Growth curves were fitted by using logistic, Gompertz, and Baranyi models. The maximum growth rates obtained from the primary model were then modelled as a function of temperature, A_w, and pH using the Belehradek‐type models for secondary model. The optimum values for growth were found to be in the range of 35–40°C, A_w 0.995–1, and pH 6–7. The statistical characteristics of the models were validated by r², mean square error, bias, and accuracy factors. The results of validation indicated that Baranyi model performed the best. Conclusions: The effect of temperature, A_w/NaCl, pH control of S. iniae in tilapia could be satisfactorily predicted under current experimental conditions, and the proposed models could serve as a tool for this purpose. Significance and Impact of the Study: The suggested predictive model can be used for risk assessment concerning S. iniae in tilapia.     

Significant decrease of ADP release rate underlies the potent activity of dimethylenastron to inhibit mitotic kinesin Eg5 and cancer cell proliferation

Eg5 is a mitotic kinesin that plays a crucial role in the formation of bipolar mitotic spindles, by hydrolyzing ATP to push apart anti-parallel microtubules. Dimethylenastron is potent specific small molecule inhibitor of Eg5. The mechanism by which dimethylenastron inhibits Eg5 function remains unclear. By comparing with enastron, here we report that dimethylenastron prevents the growth of pancreatic and lung cancer cells more effectively, by halting mitotic progression and triggering apoptosis. We analyze their interactions with ADP-bound Eg5 crystal structure, and find that dimethylenastron binds Eg5 motor domain with higher affinity. In addition, dimethylenastron allosterically blocks the conformational change of the “sandwich”-like ADP-binding pocket more effectively. We subsequently use biochemical approach to reveal that dimethylenastron slows ADP release more significantly than enastron. These data thus provide biological, structural and mechanistic insights into the potent inhibitory activity of dimethylenastron.     

Factors affecting the use of 13C(alpha) chemical shifts to determine, refine, and validate protein structures

Interest centers here on the analysis of two different, but related, phenomena that affect side-chain conformations and consequently 13C(alpha) chemical shifts and their applications to determine, refine, and validate protein structures. The first is whether 13C(alpha) chemical shifts, computed at the DFT level of approximation with charged residues is a better approximation of observed 13C(alpha) chemical shifts than those computed with neutral residues for proteins in solution. Accurate computation of 13C(alpha) chemical shifts requires a proper representation of the charges, which might not take on integral values. For this analysis, the charges for 139 conformations of the protein ubiquitin were determined by explicit consideration of protein binding equilibria, at a given pH, that is, by exploring the 2(xi) possible ionization states of the whole molecule, with xi being the number of ionizable groups. The results of this analysis, as revealed by the shielding/deshielding of the 13C(alpha) nucleus, indicated that: (i) there is a significant difference in the computed 13C(alpha) chemical shifts, between basic and acidic groups, as a function of the degree of charge of the side chain; (ii) this difference is attributed to the distance between the ionizable groups and the 13C(alpha) nucleus, which is shorter for the acidic Asp and Glu groups as compared with that for the basic Lys and Arg groups; and (iii) the use of neutral, rather than charged, basic and acidic groups is a better approximation of the observed 13C(alpha) chemical shifts of a protein in solution. The second is how side-chain flexibility influences computed 13C(alpha) chemical shifts in an additional set of ubiquitin conformations, in which the side chains are generated from an NMR-derived structure with the backbone conformation assumed to be fixed. The 13C(alpha) chemical shift of a given amino acid residue in a protein is determined, mainly, by its own backbone and side-chain torsional angles, independent of the neighboring residues; the conformation of a given residue itself, however, depends on the environment of this residue and, hence, on the whole protein structure. As a consequence, this analysis reveals the role and impact of an accurate side-chain computation in the determination and refinement of protein conformation. The results of this analysis are: (i) a lower error between computed and observed 13C(alpha) chemical shifts (by up to 3.7 ppm), was found for approximately 68% and approximately 63% of all ionizable residues and all non-Ala/Pro/Gly residues, respectively, in the additional set of conformations, compared with results for the model from which the set was derived; and (ii) all the additional conformations exhibit a lower root-mean-square-deviation (1.97 ppm < or = rmsd < or = 2.13 ppm), between computed and observed 13C(alpha) chemical shifts, than the rmsd (2.32 ppm) computed for the starting conformation from which this additional set was derived. As a validation test, an analysis of the additional set of ubiquitin conformations, comparing computed and observed values of both 13C(alpha) chemical shifts and chi(1) torsional angles (given by the vicinal coupling constants, 3J(N-Cgamma) and 3J(C'-Cgamma), is discussed.     

The effect of net charge on the solubility, activity, and stability of ribonuclease Sa

The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.