Browsing by Author "Deane, Shelly M."

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    The chromosomal arsenic resistance genes of Thiobacillus ferrooxidans have an unusual arrangement and confer increased arsenic and antimony resistance to Escherichia coli
    (American Society for Microbiology, 2000-05) Butcher, Bronwyn G.; Deane, Shelly M.; Rawlings, Douglas E.
    The chromosomal arsenic resistance genes of the acidophilic, chemolithoautotrophic, biomining bacterium Thiobacillus ferrooxidans were cloned and sequenced. Homologues of four arsenic resistance genes, arsB, arsC, arsH, and a putative arsR gene, were identified. The T. ferrooxidans arsB (arsenite export) and arsC (arsenate reductase) gene products were functional when they were cloned in an Escherichia coli ars deletion mutant and conferred increased resistance to arsenite, arsenate, and antimony. Therefore, despite the fact that the ars genes originated from an obligately acidophilic bacterium, they were functional in E. coli. Although T. ferrooxidans is gram negative, its ArsC was more closely related to the ArsC molecules of gram-positive bacteria. Furthermore, a functional trxA (thioredoxin) gene was required for ArsC-mediated arsenate resistance in E. coli; this finding confirmed the gram-positive ArsC-like status of this resistance and indicated that the division of ArsC molecules based on Gram staining results is artificial. Although arsH was expressed in an E. coli- derived in vitro transcription-translation system, ArsH was not required for and did not enhance arsenic resistance in E. coli. The T. ferrooxidans ars genes were arranged in an unusual manner, and the putative arsR and arsC genes and the arsBH genes were translated in opposite directions. This divergent orientation was conserved in the four T. ferrooxidans strains investigated.
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    Development of a novel selection / counter‑selection system for chromosomal gene integrations and deletions in lactic acid bacteria
    (BMC (part of Springer Nature), 2019-03-29) Van Zyl, Winschau F.; Dicks, Leon M. T.; Deane, Shelly M.
    Background: The underlying mechanisms by which probiotic lactic acid bacteria (LAB) enhance the health of the consumer have not been fully elucidated. Verification of probiotic modes of action can be achieved by using singleor multiple-gene knockout analyses of bacterial mutants in in vitro or in vivo models. We developed a novel system based on an inducible toxin counter-selection system, allowing for rapid and efficient isolation of LAB integration or deletion mutants. The Lactococcus lactis nisin A inducible promoter was used for expression of the Escherichia coli mazF toxin gene as counter-selectable marker. Results: The flippase (FLP)/flippase recognition target (FRT) recombination system and an antisense RNA transcript were used to create markerless chromosomal gene integrations/deletions in LAB. Expression of NisR and NisK signalling proteins generated stable DNA integrations and deletions. Large sequences could be inserted or deleted in a series of steps, as demonstrated by insertion of the firefly bioluminescence gene and erythromycin resistance marker into the bacteriocin operons or adhesion genes of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA. Conclusions: The system was useful in the construction of L. plantarum 423 and E. mundtii ST4SA bacteriocin and adhesion gene mutants. This provides the unique opportunity to study the role of specific probiotic LAB genes in complex environments using reverse genetics analysis. Although this work focuses on two probiotic LAB strains, L. plantarum 423 and E. mundtii ST4SA, the system developed could be adapted to most, if not all, LAB species.
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    Diguanylate cyclase null mutant reveals that C-Di-GMP pathway regulates the motility and adherence of the extremophile bacterium acidithiobacillus caldus
    (Public Library of Science, 2015) Castro, Matias; Deane, Shelly M.; Ruiz, Lina; Rawlings, Douglas E.; Guiliani, Nicolas
    An understanding of biofilm formation is relevant to the design of biological strategies to improve the efficiency of the bioleaching process and to prevent environmental damages caused by acid mine/rock drainage. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in different biomining bacteria. In many bacteria, the intracellular levels of c-di-GMP molecules regulate the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development, through different kinds of effectors. Thus, we recently started a study of the c-di-GMP pathway in several biomining bacteria including Acidithiobacillus caldus. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). We previously reported the existence of intermediates involved in c-di-GMP pathway from different Acidithiobacillus species. Here, we report our work related to At. caldus ATCC 51756. We identified several putative-ORFs encoding DGC and PDE and effector proteins. By using total RNA extracted from At. caldus cells and RT-PCR, we demonstrated that these genes are expressed. We also demonstrated the presence of c-di-GMP by mass spectrometry and showed that genes for several of the DGC enzymes were functional by heterologous genetic complementation in Salmonella enterica serovar Typhimurium mutants. Moreover, we developed a DGC defective mutant strain (Δc1319) that strongly indicated that the c-di-GMP pathway regulates the swarming motility and adherence to sulfur surfaces by At. caldus. Together, our results revealed that At. caldus possesses a functional c-di-GMP pathway which could be significant for ores colonization during the bioleaching process.
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    In vivo bioluminescence imaging of the spatial and temporal colonization of lactobacillus plantarum 423 and enterococcus mundtii ST4SA in the intestinal tract of mice
    (BMC (part of Springer Nature), 2018-10-30) Van Zyl, Winschau F.; Deane, Shelly M.; Dicks, Leon Milner Theodore, 1961-
    Background: Lactic acid bacteria (LAB) are major inhabitants and part of the normal microflora of the gastrointestinal tract (GIT) of humans and animals. Despite substantial evidence supporting the beneficial properties of LAB, only a few studies have addressed the migration and colonization of probiotic bacteria in the GIT. The reason for this is mostly due to the limitations, or lack of, efficient reporter systems. Here we describe the development and application of a non-invasive in vivo bioluminescence reporter system to study, in real-time, the spatial and temporal persistence of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA in the intestinal tract of mice. Results: This study reports on the application of the firefly luciferase gene (ffluc) fromPhotinus pyralis to develop luciferase-expressing L. plantarum 423 and E. mundtii ST4SA, using a Lactococcus lactis NICE system on a high copy number plasmid (pNZ8048) and strong constitutive lactate dehydrogenase gene promoters (Pldh and STldh). The reporter system was used for in vivo and ex vivo monitoring of both probiotic LAB strains in the GIT of mice after single and multiple oral administrations. Enterococcus mundtii ST4SA reached the large intestine 45 min after gavage, while L. plantarum 423 reached the cecum/colon after 90 min. Both strains predominantly colonized the cecum and colon after five consecutive daily administrations. Enterococcus mundtii ST4SA persisted in faeces at higher numbers and for more days compared to L. plantarum 423. Conclusions: Our findings demonstrate the efficiency of a high-copy number vector, constitutive promoters and bioluminescence imaging to study the colonization and persistence of L. plantarum 423 and E. mundtii ST4SA in the murine GIT. The system allowed us to differentiate between intestinal transit times of the two strains in the digestive tract. This is the first report of bioluminescence imaging of a luciferase-expressing E. mundtii strain to study colonization dynamics in the murine model. The bioluminescence system developed in this study may be used to study the in vivo colonization dynamics of other probiotic LAB.
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    Migration of bacteriocins across gastrointestinal epithelial and vascular endothelial cells, as determined using in vitro simulations
    (Nature Research (part of Springer Nature), 2019-08-07) Dreyer, Leane; Smith, Carine; Deane, Shelly M.; Dicks, Leon Milner Theodore, 1961-; Van Staden, Anton D.
    Little is known about the migration of bacteriocins across human cells. In this study, we report on migration of three bacteriocins nisin, plantaricin 423 and bacST4SA across colonic adenocarcinoma (Caco-2) cells and human umbilical vein endothelial cells (HUVECs). Bacteriocins were fluorescently labelled while still maintaining antimicrobial activity. Migration of fluorescently labelled bacteriocins across monolayers was assessed in vitro using transmigration well inserts. After 3 h, 75% of nisin, 85% of plantaricin 423 and 82% of bacST4SA migrated across the Caco-2 cell monolayer. Over the same time span, 88% nisin, 93% plantaricin 423 and 91% bacST4SA migrated across the HUVEC monolayer. The viability of both cell types remained unchanged when exposed to 50 µM of nisin, plantaricin 423 or bacST4SA. The effect of human plasma on bacteriocin activity was also assessed. Activity loss was dependent on bacteriocin type and concentration, with the class-IIa bacteriocins retaining more activity compared to nisin. This is the first report of bacteriocins migrating across simulated gastrointestinal- and vascular-barriers. This study provides some of the first evidence that bacteriocins are capable of crossing the gut-blood-barrier. However, in vivo studies need to be performed to confirm these findings and expand on the role of bacteriocin migration across cell barriers
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    Molecular insights into probiotic mechanisms of action employed against intestinal pathogenic bacteria
    (Taylor & Francis, 2020-10) van Zyl, Winschau F.; Deane, Shelly M.; Dicks, Leon M. T.
    Gastrointestinal (GI) diseases, and in particular those caused by bacterial infections, are a major cause of morbidity and mortality worldwide. Treatment is becoming increasingly difficult due to the increase in number of species that have developed resistance to antibiotics. Probiotic lactic acid bacteria (LAB) have considerable potential as alternatives to antibiotics, both in prophylactic and therapeutic applications. Several studies have documented a reduction, or prevention, of GI diseases by probiotic bacteria. Since the activities of probiotic bacteria are closely linked with conditions in the host’s GI-tract (GIT) and changes in the population of enteric microorganisms, a deeper understanding of gut-microbial interactions is required in the selection of the most suitable probiotic. This necessitates a deeper understanding of the molecular capabilities of probiotic bacteria. In this review, we explore how probiotic microorganisms interact with enteric pathogens in the GIT. The significance of probiotic colonization and persistence in the GIT is also addressed.
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    Molecular insights into probiotic mechanisms of action employed against intestinal pathogenic bacteria
    (Taylor and Francis, 2020) Van Zyl, Winschau F.; Deane, Shelly M.; Dicks, Leon Milner Theodore, 1961-
    Gastrointestinal (GI) diseases, and in particular those caused by bacterial infections, are a major cause of morbidity and mortality worldwide. Treatment is becoming increasingly difficult due to the increase in number of species that have developed resistance to antibiotics. Probiotic lactic acid bacteria (LAB) have considerable potential as alternatives to antibiotics, both in prophylactic and therapeutic applications. Several studies have documented a reduction, or prevention, of GI diseases by probiotic bacteria. Since the activities of probiotic bacteria are closely linked with conditions in the host’s GI-tract (GIT) and changes in the population of enteric microorganisms, a deeper understanding of gut-microbial interactions is required in the selection of the most suitable probiotic. This necessitates a deeper understanding of the molecular capabilities of probiotic bacteria. In this review, we explore how probiotic microorganisms interact with enteric pathogens in the GIT. The significance of probiotic colonization and persistence in the GIT is also addressed.