Molecular characterisation of the vibrio midae sy9 extra cellular alkaline serine protease and its role in the previously observed probiotic effect on the growth of Haliotis midae

Abalone are marine gastropods that command a very high market price, particularly in the Far East where they are a highly sought after sea food delicacy. South Africa has a rapidly developing abalone aquaculture industry, based on the cultivation of Haliotis midae in landbased race-way systems. The relatively slow growth rates of abalone represent a major constraint on the abalone aquaculture industry. However, there is mounting experimental evidence showing that the health and physiology of aquacultured species can be improved through the prophylactic use of probiotic bacteria. Previous research by Macey and Coyne (2005) showed that H. midae fed a high protein artificial diet, ABFEED(R) S34, supplemented with the bacterium Vibrio midae SY9 have enhanced digestion, growth and immune responses. Probiotic microorganisms are thought to function in a variety of ways, which include the secretion of extracellular enzymes that may enhance digestion in the host organism. However, most of the investigations conducted on probiotic microorganisms for aquacultured species have failed to elucidate the exact mode of action. In this study, the predominant V. midae SY9 extracellular alkaline protease, VmproA, was investigated in an attempt to determine the role of VmproA in the growth enhancing probiotic effect observed by Macey and Coyne (2005) for abalone fed V. midae SY9 supplemented feed. The V. midae SY9 gene, vmproA, encoding the protease was cloned from a previously constructed genomic library and characterised. Nucleotide sequencing and analysis indicated that vmproA encodes a protein, VmproA, which has high similarity to a Vibrio alginolyticus extracellular detergent resistant alkaline serine protease. Furthermore, during the course of this investigation it became apparent that VmproA may represent an extracellular alkaline detergent-stable, member of the proteinase K-like subfamily of the subtilase superfamily of serine proteases. The detergent-stable protease gene, vmproA, was targeted for gene mutagenesis through vmproA gene duplication and disruption, resulting in the construction of the mutant strains V. midae SY9Pro2 and V. midae SY9Mut2, respectively. VmproA gene duplication and disruption did not significantly influence the growth of the mutant strains in batch culture in comparison to V. midae SY9. V. midae SY9Pro2 produced and secreted VmproA and had equivalent levels of extracellular protease activity to V. midae SY9 when cultivated in a high protein medium. However, insertional inactivation of vmproA resulted in a loss of VmproA production and secretion. This also resulted in a significant reduction in the extracellular protease levels produced by V. midae SY9Mut2 in comparison with that of V. midae SY9. The effect of dietary supplementation with either V. midae SY9, V. midae SY9Pro2 or V. midae SY9Mut2 on H. midae growth performance was investigated in a growth trial. The basal diet of ABFEED(R) S34 weaning chips was separately supplemented with the V. midae SY9 strains by vacuum impregnation so as to achieve a viable cell concentration of greater than 108 CFU g-1 ABFEED(R). After 180 days, H. midae receiving either the V. midae SY9Pro2 or V. midae SY9Mut2 supplemented diets displayed significantly (P0.05) between animals fed the V. midae SY9 supplemented diet or the control diet. In situ alkaline protease levels within the crop/stomach and intestinal digestive tract regions were significantly enhanced (P<0.05) in H. midae fed V. midae SY9 supplemented ABFEED(R) S34 compared to animals fed the basal diet or the basal diet supplemented with either V. midae SY9Pro2 or V. midae SY9Mut2. In situ hybridization and immunohistochemistry were employed to examine the in vivo localisation of dietary supplemented V. midae SY9 cells and VmproA within the H. midae digestive tract. V. midae SY9 was chromosomally tagged with the mini-Tn10-gfp-kan transposon and the resulting strain, V. midae SY

Analysis of the microbial community associated with a bioprocess system for bioremediation of thiocyanate- and cyanide-laden mine water effluents

Gold extraction by cyanidation from refractory gold ores results in the formation of thiocyanate- and cyanide-contaminated wastewater effluents that must be treated before recycle or discard. Activated sludge processes, such as ASTER™, can be used for biodegradation of these effluent streams. The destruction of these compounds is catalyzed by a mixed microbial culture, however, very little is known about the community composition and metabolic potential of the thiocyanate- and cyanide-degrading microorganisms within the community. Here we describe our on-going attempts to better understand the key microorganisms, within the ASTER™ bioprocess, that contribute to the destruction of thiocyanate and cyanide, and how this knowledge relates to further process optimisation

Evaluation of the ASTERTM process in the presence of suspended solids

The ability to recycle and reuse process water is a major contributing factor toward increased sustainability in the mining industry. However, the presence of toxic compounds has prevented this in most bioleaching operations. The ASTERTM process has been used for the bioremediation of cyanide (CN) and thiocyanate (SCN−) containing effluents at demonstration and commercial scale, increasing the potential for recycling of the treated effluent. The process relies on a complex consortium of microorganisms and laboratory tests have shown that the biomass retention, in suspended flocs or attached biofilm, significantly improved SCN− degradation rates. The current research evaluated the process performance in the presence of suspended solids (up to 5.5% m/v) ahead of implementation at a site where complete tailings removal is not possible. Experiments were performed in four 1 l CSTRs (with three primary reactors in parallel at an 8 h residence time, feeding one secondary reactor at a 2.7 h residence time). Stable operation at the design specifications (5.5% solids, 100 mg/l SCN− feed, effluent SCN− 57 mg/l/h was achieved, despite no obvious floc formation. Microbial ecology studies (16S rRNA clone library) revealed reduced diversity relative to reactors operated without suspended solids

Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome

Background: The importance of the accessory enzymes such as α-L-arabinofuranosidases (AFases) in synergistic interactions within cellulolytic mixtures has introduced a paradigm shift in the search for hydrolytic enzymes. The aim of this study was to characterize novel AFase genes encoding enzymes with differing temperature optima and thermostabilities for use in hydrolytic cocktails. Results Three fosmids, pFos-H4, E3 and D3 were selected from the cloned metagenome of high temperature compost, expressed in Escherichia coli and subsequently purified to homogeneity from cell lysate. All the AFases were clustered within the GH51 AFase family and shared a homo-hexameric structure. Both AFase-E3 and H4 showed optimal activity at 60 °C while AFase-D3 had unique properties as it showed optimal activity at 25 °C as well as the ability to maintain substantial activity at temperatures as high as 90 °C. However, AFase-E3 was the most thermostable amongst the three AFases showing full activity even at 70 °C. The maximum activity was observed at a pH profile between pH 4.0–6.0 for all three AFases with optimal activity for AFase H4, D3 and E3 at pH 5.0, 4.5 and 4.0, respectively. All the AFases showed KM range between 0.31 mM and 0.43 mM, Kcat range between 131 s− 1 and 219 s− 1 and the specific activity for AFase-H4, AFases-E3 and was 143, 228 and 175 U/mg, respectively. AFases-E3 and D3 displayed activities against pNP-β-L-arabinopyranoside and pNP-β-L-mannopyranoside respectively, and both hydrolysed pNP-β-D-glucopyranoside. Conclusion All three AFases displayed different biochemical characteristics despite all showing conserved overall structural similarity with typical domains of AFases belonging to GH51 family. The hydrolysis of cellobiose by a GH51 family AFase is demonstrated for the first time in this study

An unusual feruloyl esterase belonging to family VIII esterases and displaying a broad substrate range

A thermophilic compost metagenomic library constructed in Escherichia coli was functionally screenedfor novel esterases. Of the 110,592 fosmid clones screened, 25 clones demonstrated degradative activ-ity on glyceryl tributyrate (a hit rate of 1:4,423). Four clones displayed ferulic acid esterase activityand were sequenced using 454 Titanium sequencing technology. EstG34, a 410 amino acid protein, wasidentified as having high sequence identity with a number of bacterial -lactamases. EstG34 has theS-X-X-K motif which is conserved in class C -lactamases and family VIII carboxylesterases. Purifiedrecombinant EstG34 had a molecular mass of 42 kDa and displayed hydrolytic activity towards a vari-ety of p-nitrophenyl esters, hydroxycinnamic acid esters and -naphthol acetate. EstG34 represents thefirst family VIII carboxylesterase and -lactamase fold enzyme, able to hydrolyse ferulate and a numberof other hydroxycinnamic acid esters. In addition, EstG34 is the first reported FAE to not adopt the / hydrolase conformation. The sequence similarity and wide substrate utilization capability of this esterasecomplicates its placement within current classification systems, but also draws attention to the enzyme’spotential versatility.The National Research Foundation (NRF) and the Technology Innovation Agency (TIA), South Africa(Project number PB99/08).http://www.elsevier.com/locate/molcatb2016-08-31hb201

Erratum to: Methods for evaluating medical tests and biomarkers

[This corrects the article DOI: 10.1186/s41512-016-0001-y.]

Evidence synthesis to inform model-based cost-effectiveness evaluations of diagnostic tests: a methodological systematic review of health technology assessments

Background: Evaluations of diagnostic tests are challenging because of the indirect nature of their impact on patient outcomes. Model-based health economic evaluations of tests allow different types of evidence from various sources to be incorporated and enable cost-effectiveness estimates to be made beyond the duration of available study data. To parameterize a health-economic model fully, all the ways a test impacts on patient health must be quantified, including but not limited to diagnostic test accuracy. Methods: We assessed all UK NIHR HTA reports published May 2009-July 2015. Reports were included if they evaluated a diagnostic test, included a model-based health economic evaluation and included a systematic review and meta-analysis of test accuracy. From each eligible report we extracted information on the following topics: 1) what evidence aside from test accuracy was searched for and synthesised, 2) which methods were used to synthesise test accuracy evidence and how did the results inform the economic model, 3) how/whether threshold effects were explored, 4) how the potential dependency between multiple tests in a pathway was accounted for, and 5) for evaluations of tests targeted at the primary care setting, how evidence from differing healthcare settings was incorporated. Results: The bivariate or HSROC model was implemented in 20/22 reports that met all inclusion criteria. Test accuracy data for health economic modelling was obtained from meta-analyses completely in four reports, partially in fourteen reports and not at all in four reports. Only 2/7 reports that used a quantitative test gave clear threshold recommendations. All 22 reports explored the effect of uncertainty in accuracy parameters but most of those that used multiple tests did not allow for dependence between test results. 7/22 tests were potentially suitable for primary care but the majority found limited evidence on test accuracy in primary care settings. Conclusions: The uptake of appropriate meta-analysis methods for synthesising evidence on diagnostic test accuracy in UK NIHR HTAs has improved in recent years. Future research should focus on other evidence requirements for cost-effectiveness assessment, threshold effects for quantitative tests and the impact of multiple diagnostic tests

Erratum to: Methods for evaluating medical tests and biomarkers

[This corrects the article DOI: 10.1186/s41512-016-0001-y.]

Detection and localisation of the abalone probiotic Vibrio midae SY9 and its extracellular protease, VmproA, within the digestive tract of the South African abalone, Haliotis midae

Probiotics have been widely reported to increase the growth rate of commercially important fish and shellfish by enhancing the digestion of ingested feed through the production of extracellular enzymes such as proteases and alginases. In order to investigate this further, the objective of this study was to localise the bacterial probiont Vibrio midae SY9 and one of the extracellular proteases it produces in the digestive tract of the South African abalone Haliotis midae. This was accomplished by inserting a promotorless gfp gene into the chromosome of the bacterium which was incorporated in an artificial, fishmeal-based abalone feed. In situ histological comparison of abalone fed either a basal diet or the basal diet supplemented with V. midae SY9::Tn10.52 using a cocktail of DNA probes to the gfp gene localised the probiont to the crop/stomach and intestinal regions of the H. midae digestive tract. Generally, the ingested probiotic bacterium occurred in association with feed and particulate matter within the crop/stomach and intestinal regions, as well as adhered to the wall of the crop/stomach. Histological immunohistochemical examination using polyclonal anti-VmproA antibodies localised an extracellular protease produced by V. midae SY9 to the H. midae crop/stomach and intestine where it appeared to be associated with feed and/or other particulate matter in the abalone gut. Thus the data suggests that V. midae SY9 colonises and/or adheres to the mucous lining of the abalone gut. Furthermore, the close association observed between the bacterium, its extracellular protease and ingested feed particles supports the theory that V. midae SY9 elevates in situ digestive enzyme levels and thus enhances feed digestion in farmed abalone