OpenUCT :: Browsing by Author "Coyne, Vernon"

Browsing by Author "Coyne, Vernon"

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Open Access
A biological study of the cellular response to heat stress in the South African alga Gracilaria gracilis
(2012) Boom, Taryn; Coyne, Vernon; Rafudeen, Suhail
Gracilaria gracilis is a commercially important alga, previously harvested from the wild South African population in Saldanha Bay as a feed for marine organisms and as a source of commercially important agar. Since 1974 however, a number of sporadic population collapses has lead to the destruction of this once flourishing resource. After numerous failed attempts at re-establishing this industry, the need to develop an alternative farming strategy became evident. In order to devise such a solution, a better understanding of the tolerances and responses of this alga to the environmental parameters responsible for the downfall of the population is required. Although the exact reasons remain unclear, Jaffray et al., 1997 have reported that increased water temperature in Saldanha Bay may be a contributing factor as the population collapses have repeatedly occurred during summer months. Thus the effect of heat stress on G. gracilis has been selected for this study.
Open Access
Characterisation of nitrogen stress response genes of the marine Alga Gracilaria Gracilis
(2003) Gebrekiros, Simon T; Coyne, Vernon
Low environmental nutrient concentration is the main factor limiting natural production and success of Gracilaria gracilis cultivation in Saldanha Bay, South Africa and nitrogen is the single element of all the nutrients required by seaweeds that is most frequently limiting to growth. Biomass, relative growth rate, the concentration of nitrogen in the growth medium, the mean nitrogen uptake rate of the plant and the amount of nitrogen in the thallus were determined for nitrogen enriched and nitrogen deprived G. gracilis cultures grown in the laboratory.
Open Access
Characterisation of the effect of stress on nitrogen metabolism in the commercially important agarophyte, Gracilaria gracilis
(2012) Reddy, Amelia Francine; Coyne, Vernon
Gracilaria gracilis occurs naturally in Saldanha Bay, and was important to the South African export industry as it is a source of two important types of agar, namely food grade and sugar reactive agar. However, a number of abiotic and biotic factors, such as nutrient limitation, has virtually destroyed the G. gracilis resource. An understanding of the physiological response of the alga to nutrient limitation will aid in re-establishing and sustaining G. gracilis populations. However, modelling algal physiology and growth in response to abiotic stresses such as nutrient limitation requires an understanding of the underlying metabolic processes. The present study aimed to address this by investigating nitrogen metabolism and the mechanisms regulating nitrogen metabolism in G. gracilis. This was achieved by profiling changes in gene and protein expression, and activity of two major nitrogen metabolic enzymes, nitrate reductase and glutamine synthetase. Long term culture of G. gracilis in nitrogen replete and lacking conditions indicated that nutrient limitation causes a reduction in intracellular nitrogen and nitrogen protein stores such as phycoerythrin. When various sources were introduced to the culture medium to replenish nitrogen starved G. gracilis, changes in nitrate reductase and glutamine synthetase mRNA, protein and activity seemed to be dependent on the nutrient history of the cells, intracellular and extracellular nitrogen concentrations, metabolites of nitrogen assimilation and other metabolic processes such as carbon metabolism and photosynthesis. Nutrient studies suggested that multiple G. gracilis nitrate reductase and glutamine synthetase isoforms are present and differentially regulated via transcriptional, post-trancriptional, translational and posttranslational mechanisms. Furthermore, the insensitivity of these nitrogen metabolic enzymes to ammonium inhibition and the ability to alter the GS1:GS2 activity ratio possibly represents adaptive strategies developed by G. gracilis to survive nitrogen limitation. Immunocytochemical investigations confirmed the presence of multiple nitrate reductase and glutamine synthetase isoforms. The enzymes were successfully localised to the cell wall, chloroplast and cytosol of G. gracilis. A novel finding was the immuno-localisation of glutamine synthetase to intracellular starch granules. Overall, findings in the current study have suggested multiple roles for these metabolic enzymes that include nitrogen assimilation/transport, cell wall biosynthesis and senescence. This study led to the development of a model of the metabolic changes that occur in nitrogen replete and deplete G. gracilis and provides a firm foundation for future studies of the nitrogen stress response in G. gracilis. Characterisation of the G. gracilis nitrogen stress response may ultimately revive mariculture of this commercially important alga in South Africa.
Open Access
Characterisation of the population genetics of farm-bred Haliotis midae using microsatellite DNA markers
(2006) Naidoo, René Kathleen; Coyne, Vernon
The abalone Haliotis midae is a gastropod mollusc which is of commercial importance in South Africa due to its high export value. During this study the genetic structure of farmed Haliotis midae was investigated in order to determine whether microsatellite DNA analysis could be used to separate farmed abalone into phenotypically different groups with respect to growth rate. Microsatellites display high levels of variability and this makes them suitable for a wide variety of applications in aquaculture, particularly where genetic differentiation between population groups may be limited. This study investigates the genetic composition of 120 individuals from 2 spawning events that had been classed as either fast or slow growing by the farm managers. Three highly polymorphic microsatellite loci were selected and used to determine the extent of the genetic diversity which exists amongst the 120 individuals tested from the lacobsbaai abalone farm. Additionally, these microsatellite loci were used to determine whether abalone classed as either fast or slow growing could be differentiated into specific genetic population groups. Neighbour-joining trees constructed using genetic distance data obtained for all three loci demonstrated that a distinct separation between the fast and slow growing abalone was evident. It was also found that there has been a loss of genetic diversity on the lacobsbaai abalone farm in terms of heterozygosity. This has been attributed to the high levels of inbreeding as evidenced by the high Fis values. All population groups were found to deviate significantly from Hardy-Weinberg equilibrium and this is most likely due to the occurrence of non-random mating on the lacobsbaai abalone farm. This study has successfully demonstrated that a combination of micro satellite loci with high allelic diversity can potentially be employed as a tool for distinguishing between fast and slow growing farmed abalone. This study needs to be validated by testing larger sample sizes and the use of additional farms.
Open Access
Cloning and molecular characterisation of four alginate lyase genes from Vibrio midae SY9 : an enteric bacterium from the abalone Haliotis midae
(2010) Cross, Bronwen; Coyne, Vernon
Alginate is a linear, un-branched polysaccharide of (1-4)-linked -D-mannuronate (M) and its C-5 epimer, -L-guluronate acid (G). These uronic acids are arranged in three different block types in the alginate polymer; poly-M, poly-G or poly-MG. Alginate lyases are enzymes that utilize a -elimination reaction to depolymerise the alginate polymer resulting in cleavage of the (1-4)-O-glycosidic linkage between monomers and the formation of an unsaturated uronic acid at the new non-reducing terminus. Alginate lyases have been isolated from a wide range of sources including marine invertebrates and marine bacteria, which often produce more than one alginate lyase enzyme. Haliotis midae is the commercially important abalone species found along the South African coast. Over-fishing and poaching of this species has led to a depletion of the naturally occurring populations and closure of the recreational and commercial fisheries. Abalone farming was initiated and has rapidly increased in response to the increasing demand for this delicacy. However, there are many problems associated with abalone aquaculture, the most significant being disease and the slow growth rates of the animals. The use of probiotics in abalone aquaculture is a potential solution to both of these problems.
Open Access
Development of a Dusky kob scFv gene phage display library for the discovery of antibodies to Brome mosaic virus - a proxy for a novel, emerging fish pathogen
(2021) Naylor, Kyle Andrew; Coyne, Vernon
Fish farming is rapidly becoming the world's fastest growing production sector, achieving an annual growth rate of approximately 8.9% since the early 1970s. However, high stocking densities result in elevated stress levels in farmed fish, leading to increased susceptibility to infection by opportunistic pathogens and parasites. Antibody phage display is a method that allows foreign peptides or proteins to be expressed on the phage surface through translational fusion with phage coat proteins. Consequently, antibodies expressed by a diverse repertoire of genes coding for the single chain variable fragment (scFv) of immunoglobulin M can be isolated and screened for affinity to a specific infectious agent or parasite. In this study, a phage display library displaying scFvs derived from combination pairings of Dusky kob (Argyrosomus japonicas) variable heavy and light chain fragments, sourced from the splenic B cells of healthy Dusky kob, was constructed. The library was subjected to two rounds of biopanning against brome mosaic virus (BMV), a grass virus to which Dusky kob would have no prior exposure that served as a proxy for an emerging fish pathogen. Five clones were identified as having high affinity and specificity to BMV, as determined by phage enzymelinked immunosorbent assay (ELISA) and phage western blot analysis, respectively. To validate the diagnostic and therapeutic potential of antibody fragments isolated from this phage display library, the gene encoding the antibody fragment of the clone displaying the highest affinity to BMV was selected and expressed using a yeast surface display system. ELISA analysis of serum sampled from Dusky kob exposed to BMV by injection demonstrated that the yeast displayed anti-BMV antibody could successfully detect BMV in the blood serum of BMV-infected Dusky kob with similar sensitivity to a commercially available counterpart. Similarly, this study demonstrated the neutralising effect of yeast displayed anti-BMV antibodies which were found to successfully reduce BMV infection in barley. Overall, these findings demonstrate the feasibility of a Dusky kob phage display library as a source of diagnostically and therapeutically important antibodies against emerging fish pathogens or parasites that threaten the fish farming industry of South Africa.
Open Access
Development of a transformation protocol and cell culture system for the commercially important species of red macroalga, Gracilaria gracilis
(2011) Huddy, Suzanne Margaret; Coyne, Vernon; Meyers, Ann
A better understanding of how this commercially important seaweed responds at a genetic level to stresses faced in the aquaculture environment would not only be advantageous to the South African industry, but this knowledge is essential for selecting and/or engineering macroalgal strains that are either more tolerant or resistant to these stresses. This requires in vivo analysis of G. gracilis gene function and regulation in order to introduce new or improved genes into G. gracilis, and for this to be possible, a protocol for transformation of recombinant DNA into G. gracilis is required. In this study a transformation and tissue culture system for G. gracilis was developed. These tools provide the necessary groundwork for future genetic manipulation studies that are essential for improving our understanding of the role that various genes play in stress response and tolerance in G. gracilis.
Open Access
Identification and partial characterisation of the HSP70 gene of the South African abalone Haliotis midae
(2011) Lyons, Jarrod; Coyne, Vernon
Identifying genes which are up-regulated in response to stress has become important in understanding invertebrate immunity. The HSP70 gene from Haliotis midae was successfuly cloned and sequenced, and was found to have highest similarity (99%) with Haliotis diversicolor.
Open Access
Identification of a potential antimicrobial peptide derived from Haliotis midae haemocyanin
(2019) North, Jarid; Coyne, Vernon
Haliotis midae aquaculture within South Africa remains afflicted by infectious diseases. Understanding of how the abalone’s innate immune response functions is one of the greatest hindrances to assisting with the defence or detection of pathogenic attacks on farms. The multifunctional oxygen transporter haemocyanin was previously found to be upregulated in response to bacterial infection (Beltran 2015), indicating it may play a role in the defence response of Haliotis midae. The current knowledge regarding haemocyanin’s role in the abalone innate immune response is incomplete. A number of studies have been published that investigated haemocyanin’s potential as a broad spectrum antimicrobial peptide in many arthropod species. There has been only one study conducted in molluscs, which utilised synthetic peptides derived from a haemocyanin consensus sequence. In most organisms the haemocyanin protein is comprised of a string of eight roughly 50 kDa functional units (FU) annotated a-h. The current study determined the nucleotide sequence of the final four functional units on the C-terminal end of H. midae haemocyanin and examined the potential antimicrobial activity of the peptide product. The haliotisin coding sequence identified by Zhuang et al. (2015) was detected in FU-e of the H. midae haemocyanin and subcloned into an Escherichia coli expression vector for recombinant production of the peptide. This peptide showed some activity against Staphylococcus aureus and E. coli, suggesting it may function as an antimicrobial peptide. This study provides the first evidence that an antimicrobial peptide derived from the H. midae haemocyanin could be functioning as a component of the abalone innate immune response.
Open Access
Identification of differentially expressed genes in the commercially important agarophyte, Gracilaria gracilis, following nitrogen deprivation
(2006) Lebi, Tanya; Coyne, Vernon
The red agarophytic alga, Gracilaria gracilis, occurs naturally within Saldahna Bay, South Africa. Gracilaria species are commercially exploited for their hydrocolloid agar, valued at US$132 million per annum (FAO, 2002). G. gracilis is thus a valuable resource for South Africa in terms of generating foreign currency through export. Nitrogen limitation occurring within Saldahna Bay during the summer to autumn months, however, is considered to be the major factor preventing commercial cultivation of G. gracilis. Algae possess various survival mechanisms that generally involve the activation or repression of various gene regulatory systems. Such regulatory systems aid in preserving energetic homeostasis at a cellular level, thereby allowing maximal algal survival during adverse environmental conditions. Microarray technology is a highthroughput global gene analysis tool, allowing for the simultaneous identification of thousands of differentially expressed genes in a single assay. Combined with genomic technologies such as genetic engineering, microarray technology has the potential to identify genes that confer enhanced in vivo tolerance to a specific stress. In the current investigation, microarray technology was utilized to identify genes differentially expressed in G. gracilis in response to nitrogen limitation. A total of 39 differentially expressed genes were identified. Ofthe 35 genes that were sequenced, 13 were assigned a putative function based on sequence similarity to genes deposited in various databases. The remaining sequences corresponded to either unknown hypothetical proteins or else had no significant homology to any of the sequences in the databases. Overall, the current investigation has provided a foundation for future research into the biological role(s) of each of the identified differentially expressed genes within their true isogenic background. Future studies may thus allow for improved crop tolerance and stability, enhancing aquaculture of this valuable resource in Southern Africa.
Open Access
The investigation of bacterial pathogens of the red macroalga gracilaria gracilis and its response to bacterial infection
(1998) Jaffray, Ann Elizabeth; Coyne, Vernon
The red agar-containing macroalga G. gracilis (Stackhouse) Steentoft, Irvine et, Farnham has occurred in Saldanha Bay, South Africa for many years. However, in recent years a number of collapses in this G. gracilis population were recorded, in some instances almost erradicating the entire population. One of the causes of these collapses is thought to be bacterial disease about which there is very little known. The bacterial pathogens of this macroalga were thus investigated to determine the nature of disease occurrence and how G. gracilis responds to such infections. A large number of culturable bacterial epiphytes isolated from G. gracilis from Saldanha Bay, South Africa, and Luderitz, Namibia were characterised and compared. The number of culturable bacteria isolated from the seawater surrounding the macroalgae was significantly lower than that which occurred epiphytically on the macro algal thalli. Most of the bacteria isolated were Gram-negative, motile rods, and many were classified to genus level. Scanning electron microscopy confirmed that a large epiphytic population of coccoid and rod-shaped bacteria occurs primarily on the main thalli of the rnacroalga and that significantly fewer (and often none) reside on the thallus growth tips.
Open Access
Investigation of the effect of a probiotic-supplemented diet on the haemocyte proteome of the abalone Haliotis midae
(2016) Dias, Valera Lucena; Coyne, Vernon
Haliotis midae is an economically important South African abalone species which has been cultured since the 1990s. H. midae farming constitutes approximately 93% of the country's total marine aquaculture industry. However, the slow growth rate of this animal and the potential for disease outbreaks remain a concern for farmers. Therefore, government and private institutions have joined efforts to investigate ways of enhancing abalone production, to increase abalone growth rates and improve disease resistance. Several studies have shown that probiotic microorganisms can significantly increase the growth rate and disease resistance of H. midae. To date, no comprehensive studies have been conducted to characterise these physiological improvements at the molecular level. Thus, the aim of this study was to evaluate the effect of a probiotic-supplemented diet on the haemocyte proteome in H. midae. Two probiotic-strains, Vibrio midae SY9 and Debaryomuces hansenii AY1, were introduced into H. midae via a kelp-based feed. Changes in the haemocyte proteome were analysed using isobaric tag for relative and absolute quantification (iTRAQ) coupled with LC-MS/MS. A total of 128 haemocyte proteins were identified. Proteins that were found to vary significantly in their expression levels in haemocytes sampled from abalone fed the probiotic-supplemented diet were identified as COP9 signalosome subunit 4, phosphorylase, T-complex protein 1 subunit gamma, V-type proton ATPase subunit B, Rab 1 and Ra-related protein Rab 1A. Differential expression of COP9 signalosome subunit 4 (up-regulated) and Ras-related protein Rab 1A (down-regulated) was confirmed by western blot analysis. Bioinformatics analysis revealed proteins with immune class GO terms that functioned in metabolism, apoptosis, cell adhesion, immune response, stress response, and response to endogenous and external stimulus. Hierarchical clustering analyses showed that proteins with similar expression patterns mostly belonged to the same immune classes. Analysis of protein interaction networks indicated that all the differentially expressed proteins may indirectly interact with each other. It was also found that the neurotrophic tyrosine kinase receptor was the central molecule within the interaction network, suggesting that this protein may play a crucial role within the protein interaction network that contains all the differentially expressed proteins. Biochemical pathway analysis indicated that phagosomal maturation was the most significant canonical pathway identified, in which V-type proton ATPase and Ras-related protein Rab have fundamental importance. Changes in Ras-related protein Rab 1A expression were further investigated in the cytosolic and membrane fractions of haemocyte cells using western blot analysis and cellular immunochemistry. The expression of this protein was found to be down-regulated both in cytosolic and membrane fractions from haemocytes sampled from H. midae fed a probioticsupplemented diet. Although an association between Ras-related protein Rab 1A and F-actin (cell cytoskeleton) was not detected, confocal microscopy confirmed Ras-related protein Rab 1A down-regulation. Thus, results from this study suggest that Ras-related protein Rab 1A may play a key role in H. midae immune response, when this species of abalone is fed with a probiotic-supplemented diet. This is the first time that a large-scale proteomics approach has been used to investigate proteome changes in haemocytes sampled from H. midae fed a probiotic-supplemented diet. The findings of this study, regarding the protein profile, interaction networks, molecular pathways and a putative molecular indicator of H. midae immune response, provide a foundation from which future studies can be conducted in order to increase our understanding of how probiotics affect the abalone immune system.
Open Access
Investigation of the effect of ocean acidification on the haemocyte proteome of the South African abalone, Haliotis midae
(2020) Carroll,SarahLeigh; Coyne, Vernon
Haliotis midae is an economically important marine invertebrate that is farmed in South Africa, contributing more than half of the revenue generated by the aquaculture industry. However, the future sustainability of abalone farming in South Africa is threatened by the ongoing climate crisis. The effect of climate change is unrelenting for organisms such as abalone, which rely on a succinct balance of physico-chemical environmental properties. Indeed, the ocean environment is susceptible to these imbalances and has already witnessed changes in seawater temperatures and pH. Over the last century, global ocean surface temperatures have increased by 0.74°C and seawater pH has declined by 0.1 units, while global predictions for 2100 suggest oceans will experience a decline in pH by 0.3-0.5 units. Thus, ocean acidification (OA) is a growing cause for concern since it adversely affects marine organisms such as corals and calcareous marine invertebrates. Research focusing on the effect of climate change on marine life has grown tremendously over the last two decades, with an emphasis on molluscs such as mussels and oysters which are considered ideal proxies for measuring environmental change and the underlying molecular effects thereof. However, research on abalone in this arena has primarily focused on larvae and the physiological effects of OA on development and shell growth. The underlying molecular mechanisms involved in the Haliotis midae stress response to ocean acidification have largely remained unexplored. Thus, this study sought to elucidate the effect of OA on the haemocyte proteome of H. midae, as well as to gain insight into the underlying molecular mechanisms that characterize the stress response of this abalone species. This study employed a comparative shotgun proteomics approach using isobaric tagging for relative and absolute quantification (iTRAQ) coupled with LC-MS/MS to investigate the proteomic response of H. midae haemocytes to reduced pH conditions representative of future predictions of ocean acidification. Four independent iTRAQ experiments were conducted where haemocytes were sampled after 12, 72 and 168 hours of exposure of H. midae to OA conditions (pH 7.5, represents OA predicted by 2100). Following quantitative analysis, 227 non-redundant and differentially expressed proteins were detected across the four independent experiments. Proteins of statistical significance (p ≤ 0.05) and biological relevance (foldchange ≥ 1.2) were identified. The 227 proteins were grouped according to their expression profile using Weighted Gene Cluster Network Analysis to gain an insight into the biological processes underlying the stress response of H. midae to OA conditions. Sequence similarity, Gene Ontology, data mining and network modelling based on other molluscs and well-characterised genomes were utilized for assigning putative functions to the grouped proteins. This revealed a multifaceted interplay of various biological processes and signaling pathways in H. midae, such as the induction of anaerobic metabolism, cytoskeletal stabilization and the induction of the ERK/MAPK signaling cascade. Notably, this analysis demonstrated a possible link between the stress and immune responses which has only been observed in other molluscs. Their complex association suggests an overlap of pathways, with putative dual functionality of proteins such as MAPK, CAMK, Serpin B2 and haemocyanin, all of which have been implicated in the stress and innate immune responses of other organisms. The combined data from the quantitative (Chapter 2) and functional analyses (Chapter 3) resulted in the compilation of a group of 33 candidate protein biomarkers of OA stress. A holistic and complete picture of the potential regulatory mechanisms involved in the stress response of H. midae was generated through protein-protein interaction network modelling and data mining. Aquarium-based experiments were conducted to validate the candidate OA biomarker proteins, as well as a set of previously identified biomarker candidates of acute temperature stress in H. midae (Calder and Coyne, unpublished), using label-free protein quantification (LFQ) coupled to LC-MS/MS analysis. Furthermore, the effect of a combination of reduced pH and elevated temperature on the candidate biomarker proteins was investigated. Five candidate biomarker proteins of OA stress were validated, while 10 candidates of acute temperature stress were detected and validated. The combined stress condition identified 7 potential biomarkers. Candidate biomarkers of OA stress were predominantly associated with the innate immune system, while those responding to temperature stress were largely associated with energetics and oxidative stress. Potential biomarkers of the combined stressors were found to be associated with signal transduction and intracellular trafficking. This component of the research project not only highlighted the importance of validation in biomarker discovery, but demonstrated the usefulness of an LFQ-proteomics approach for biomarker validation. This is the first time high-throughput shotgun proteomics has been employed to investigate the H. midae haemocyte stress response. This study provides a solid foundation for elucidating the putative functional stress response of a non-model organism, and highlights the complex dynamics and interplay between the stress and immune responses of H. midae. On-farm experimentation will be conducted to test whether the candidate biomarkers identified in this project can reliably detect abiotically stressed H. midae and whether they can be integrated into a “suite” of biomarkers for a health monitoring program for farmed H. midae. Successful implementation of such a health monitoring program will ensure the future sustainability of the South African abalone aquaculture industry despite the severity of climate change.
Open Access
Investigation of the Gracilaria gracilis proteome response to nitrogen limitation
(2012) Naidoo, René Kathleen; Coyne, Vernon; Rafudeen, Suhail
In the past, commercial quantities of G. gracilis were harvested from Saldanha Bay until population collapses of the natural resource necessitated the need for research into alternative aquaculture programs for G. gracilis cultivation. One of the reasons for the G. gracilis population collapse was attributed to adverse conditions during summer which led to thermal stratification of the water column and subsequent nutrient limiting conditions. Inorganic nitrogen has been identified as the major nutrient factor limiting growth and production of G. gracilis populations in South Africa. Although the physiological mechanisms implemented by G. gracilis which allow adaption to low nitrogen environments have been investigated, not much is known about the molecular mechanisms which underlie these adaptions. Thus, it is necessary to elucidate the molecular basis of these adaptions in G. gracilis to complement the existing physiological data.
Open Access
Investigation of the role of the extracellular β-agarase, produced by the bacterial epiphyte Pseudoalteromonas sp. LS2i, in the virulence response towards the agarophyte Gracilaria gracilis
(2014-07-30) Gildenhuys, Carin; Coyne, Vernon
Gracilaria gracilis that grows naturally at Saldanha Bay, South Africa is economically important as a source of agar. The Gracilaria yields from natural beds at Saldanha Bay are however unreliable, and consequently the South African Gracilaria industry has experienced a number of setbacks over the years. The only way a consistent supply can be assured is by mariculture to supplement the natural harvests. In 1993 the Seaweed Research Institute (SRU) found that mariculture of G. gracilis in Saldanha Bay is feasible but that there is potential to improve yields by technical research and development (Anderson et al.1996a). Jaffray and Coyne (1996) developed a pathogenicity assay that demonstrated that agarolytic bacteria isolated from Saldanha Bay Gracilaria induced disease symptoms such as thallus bleaching, while non-agarolytic isolates did not. It is thought that unfavorable environmental conditions such as elevated water temperature and nutrient depletion, which occur during the summer months in the surface layers of the water column in Saldanha Bay, induce the onset of agarase production or result in changes in the bacterial community structure in which agarase-producers become more dominant. By using the pathogenicity assay, Jaffray and Coyne (1996) identified the highly agarolytic Gracilaria gracilis pathogen, Pseudoalteromonas sp. LS2i. The aim of this study was to characterize the bacterial pathogen, Pseudoalteromonas sp. LS2i to further our understanding of virulence regulation and specifically, the role of the agarase enzymes in the process of seaweed-pathogen interaction. Two agarolytic clones, pEB1 and pJB1, were obtained after constructing and screening a Pseudoalteromonas sp. LS2i genomic library in Esherichia coli. Restriction enzyme mapping suggested that both clones contain the same agarase gene. Southern hybridization studies confirmed the origin of the cloned DNA and sequencing studies revealed the 1062 bp ORF, putative promoter region, putative ribosome binding site and putative transcriptional start point of the cloned agarase gene. The ORF showed sequence identity to several other β-agarases and was identified as a member of the GH-16 family of glycoside hydrolases. The agarase was purified from the E. coli JM109 (pEB3) transformant. The molecular weight was estimated to be 39 kDa by SDS-PAGE. Zymogram analysis confirmed that the purified protein is agarolytic and TLC analysis revealed that the predominant end-products of agar hydrolysis are neoagarohexaose and neoagarobiose, which indicates the same mode of action as that observed for the agarase produced extracellularly by Pseudoalteromonas sp. LS2i.
Open Access
Isolation and characterization of a β(1-4) agarase of an epiphytic bacterial pathogen, Pseudoalteromonas gracilis B9, of the red alga, Gracilaria gracilis.
(2001) Schroeder, Declan Cosmo; Coyne, Vernon
Bibliography: p. 201-216.
Open Access
Midaesin - a histone H2A antimicrobial peptide from the South African abalone, Haliotis midae
(2021) French, Lee; Coyne, Vernon
The South African abalone, Haliotis midae, is a commercially important shellfish that is farmed in the Western Cape. Histone H2A-derived peptides investigated from various organisms have been shown to act as antimicrobial peptides (AMPs) against a range of microbes. Therefore, the aim of the study was to determine whether the H. midae histone H2A acts as an AMP and thus plays a role in the innate immune system of this abalone. A peptide fragment corresponding to the first 42 amino acids of the N-terminus of histone H2A was examined in silico. This 4.63 kDa peptide, referred to as Midaesin, was found to possess a high proportion of hydrophobic and basic residues. Secondary structure prediction of Midaesin revealed the presence of an amphipathic α-helix. Alignment of the Midaesin peptide to other known histone H2A-derived AMPs revealed significant sequence similarity. Additionally, RT-qPCR of total cDNA isolated from cultured H. midae haemocytes exposed to heat-killed V. anguillarum for 1h showed that the histone H2A transcript was upregulated, implying a role in the immune response. A PCR-amplified DNA fragment coding for the Midaesin peptide was cloned into a bacterial expression vector and purified. Midaesin-containing peptides inhibited the growth of Staphylococcus aureus, Escherichia coli and Vibrio anguillarum at 10 µM in liquid growth inhibition assays. Scanning electron microscopy and fluorescent microscopy, employing the membrane impermeable dye propidium iodide, revealed membrane disruption of V. anguillarum cells exposed to 13 µM or 30 µM of the Midaesin-containing peptides for 30 minutes, respectively. Secondary structure analysis by circular dichroism indicated a shift in the secondary structure of the Midaesin-containing peptide upon incubation with V. anguillarum cells for 30 minutes with a trend towards more α-helical content. Taken together, the above indicates that histone H2A may be involved in the immune response of H. midae and that Midaesin has the potential to act as an AMP.
Open Access
Molecular and functional characterization of the melanin biosynthetic genes from Vibrio cholerae 569B
(1998) Schroeder, Irmagard Cherè; Coyne, Vernon
V. cholerae 569B is a bacterium infamous for its role as the causative agent of the diarrhoeal disease cholera. Although the bacterium occurs naturally in brackish waters and estuaries, cholera outbreaks are closely linked to specific environmental conditions. For example, most outbreaks occur during the summer months when the bacterium experiences an increase in water temperature, play a role in activating virulence in V. cholerae 569B, the exact mechanism remains to be elucidated. Previously it was observed that when V. cholerae is exposed to elevated temperature and salinity, the bacterium initiates the synthesis of a brown-black pigment known as melanin. The function of the pigment and the genes involved in its synthesis was unknown. We therefore set out to determine the function of pigmentation in V. cholerae 569B, since pigmentation could significantly enhance the survival of the bacterium during adverse conditions and therefore aid in the persistence of the organism in the environment.
Open Access
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
(2010) Huddy, Robert John; Coyne, Vernon
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 (P<0.05) enhanced growth parameters compared to abalone fed either the basal diet or the V. midae SY9 supplemented diet. However, there was no significant difference (P>0.05) 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 SY9
Open Access
Optimising the growth of Cryptococcus species SS1, a potential probiotic for farmed abalone
(2004) Van Wyk, Jennifer Caroline; Coyne, Vernon; Harrison, Sue
Farmed abalone is a reliable and good quality source of abalone. Cryptococcus species SS1 was isolated from the gut of the South African abalone, Haliotis midae, and has been identified as a potential probiotic for farmed abalone. The implementation of strain SS1 as a probiotic for aquacultured abalone required the design of a fermentation system to produce hjgh concentrations of the yeast strain in order to supply the probiotic to commercial abalone producers. The aim of this project was to assist in the recommendation of a commercial fermentation process that is economically feasible for the production of strain SS1. This involved evaluation of all the main factors that will contribute to the cost of the fermentation; i.e. cultivation medium, fermentation space and time, and productivity.