No description available.

Avocados are an economically important crop in South Africa and are mainly exported to Europe. As with any other tropical and subtropical crop, avocados are prone to pre- and postharvest diseases. Until recently, chemical control was the only effective measure to control fungal avocado pathogens In 1987, a Bacillus subtilis isolate was found that showed promise as a biocontrol agent in both pre- and postharvest applications to control postharvest diseases. However, over time variable results has been obtained in semi commercial trials. From the original B. subtilis isolate several subcultures have been made and used over a 15 year period in various experimental trials. The dual culture technique was used to compare the biocontrol activity of the subcultures against postharvest pathogens (Colletotrichum gloeosporioides, Phomopsis perseae, Dothiorella aromatica and Lasiodiplodia theobromae). The subcultures differed significantly in their effectiveness and genetic stability. No difference between the subcultures could be found when DNA fingerprinting using RISA PCR was used. The most effective subculture, MI-14, was used in further studies. The mode of action employed by a biocontrol agent is of utmost importance and can be used to enhance its efficacy. In a previous study it was hypothesized that antibiosis as well as competition for nutrients and space is the modes of action involved in biocontrol of B. subtilis against postharvest pathogens of avocado. The direct interaction between B. subtilis and C. gloeosporioides on avocado fruit were observed using scanning electron microscopy. Cells of B. subtilis were observed to colonize the hyphae of C gloeosporioides. In some instances, hyphal walls were lysed in the presence of B. subtilis and may be due to the presence of enzymes or antibiotic substances. Conidia of C. gloeosporioides did not germinate in the presence of B. subtilis. Diffusible inhibitory metabolites active against C. gloeosporioides were produced in vitro by B. subtilis. Inhibitory volatile substances were also produced by B. subtilis and were found to be active against P. perseae, D. aromatica and L. theobromae but not C. gloeosporioides. Siderophores production as well as chitinase, amylase, lipase and proteinase activity were also observed and may play a role in antagonism. Antibiotic production by B. subtilis is a well-known phenomenon. Most antibiotics are polypeptides and lipopeptides. The involvement of phenolic metabilites in biocontrol by B subtilis is less known. A seven-day-old culture of B. subtilis in a minimal medium was analyzed for the presence of free acid phenolic compounds active against fungi. Free acid phenolic metabolites were found and separated using layer chromatography. TLC plates containing the separated spots were sprayed with Clasdosporium cladosporioides and plates were observed for inhibition zones. The phenolic substances were present at 7.06 ± 0.95 mg gallic acid ml-1. The phenolic substances fall in the hydroxycinamic acid group due to their fluorescent coloring under UV at 350 nm. The mode of action involved is also influenced by environmental factors. The effect of temperature and carbon- and nitrogen sources of the in vitro inhibitory activity of B. subtilis against C. gloeosporioides, P. perseae, D. aromatica and L. theobromae were investigated using the dual culture technique. The most effective temperature range for B. subtilis was found to be between 20 and 37°C. At temperatures lower than 15°C, B. subtilis was found to be not very effective, suggesting why postharvest applications followed directly by cold storage do not always work effectively. D-arabinose and D-(+)-mannitol evaluated as carbon source as well as L-glutamic acid, L-glutamine and L-(+)-asparagine used as nitrogen sources support in vitro antagonism against the pathogens most effectively. They also do not support the growth of C. gloeosporioides, P. perseae, D. aromatica and L. theobromae. These nutrients can potentially be the most effective ones to incorporate in commercial B. subtilis formulations. The study showed the potential role of antagonistic free acid phenolic substances, volatiles and siderophores on inhibition of fungal avocado pathogens. Further studies to confirm their in situ activity are required. In conclusion, various factors affect the efficacy of B. subtilis against postharvest pathogens of avocado. These factors should be kept in mind when applying the commercial product in order to achieve the best results. / Dissertation (MSc (Microbiology))--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

The work presented explored the in vivo and in vitro synthesis of ribosomal RNA in the Gram positive, spore-forming bacterium Bacillus subtilis. The investigation began with a study of rRNA synthesis in B. subtilis during steady state growth and under nutritional shift-up conditions. The percent of transcription which is ribosomal RNA was measured by hybridization of pulse labeled RNA to a specific DNA probe carrying the 3' end of the 23S RNA gene. The fractional rate of ribosomal RNA synthesis increased with cellular growth rate, and showed a rapid increase after a nutritional shift up. RNA synthesis during infection with an amber mutant of bacteriophage SP01 was also examined. Infected cells continued to synthesize rRNA at the preinfection rate, but could not respond to media enrichment by increasing the percent rRNA-synthesis. The latter study suggested the existence of a specific RNA polymerase that transcribed ribosomal RNA genes. The conclusions from the in vivo study led to an analysis of rRNA transcription in vitro. The isolation of the putative ribosomal RNA specific RNA polymerase was attempted by affinity chromatography on cellulose complexed with plasmid DNA containing the promoter region of the B. subtilis rrnB rRNA operon, and by sedimentation through a glycerol gradient. No difference in activity profile was observed when transcription activity at the rRNA tandem promoters was compared to activity at a non-ribosomal promoter. Since in vivo analysis of the control of rRNA synthesis in Escherichia coli suggested that regulation occurs at the level of transcription initiation, in vitro transcription initiation at the B. subtilis rRNA promoters was investigated using the single round transcription assay. Initial rates of transcription were different at each of the two tandem promoters of the B. subtilis rrnB operon: the upstream promoter, PI, initiated slowly, while the downstream promoter, P2, initiated faster. In addition, transcription initiation at the two promoters appeared to be linked. The formation of a heparin resistant complex at the PI promoter affected the stability of the heparin resistant complex formed at the P2 promoter. The kinetics of transcription initiation at the tandem rRNA promoters were examined using the tau plot analysis. RNA polymerase had a high affinity for both rRNA promoters, but the rate of initiation at these promoters was relatively slow when compared to non-ribosomal promoters. Finally, transcription initiation on two artificial tandem promoter constructs was compared with initiation on the native tandem promoter construct. In general, PI was shown to have a positive effect on transcription from downstream promoters, but had specific effects on different promoters. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Developing cultivation methods that yield chemically and isotopically defined fatty acid (FA) compositions within bacterial cytoplasmic membranes establishes an in vivo experimental platform to study membrane biophysics and cell membrane regulation using novel approaches. Yet before fully realizing the potential of this method, it is prudent to understand the systemic changes in cells induced by the labeling procedure itself. In this work, analysis of cellular membrane compositions was paired with proteomics to assess how the proteome changes in response to the directed incorporation of exogenous FAs into the membrane of Bacillus subtilis. Key findings from this analysis include an alteration in lipid headgroup distribution, with an increase in phosphatidylglycerol lipids and decrease in phosphatidylethanolamine lipids, possibly providing a fluidizing effect on the cell membrane in response to the induced change in membrane composition. Changes in the abundance of enzymes involved in FA biosynthesis and degradation are observed; along with changes in abundance of cell wall enzymes and isoprenoid lipid production. The observed changes may influence membrane organization, and indeed the well-known lipid raft-associated protein flotillin was found to be substantially down-regulated in the labeled cells – as was the actin-like protein MreB. Taken as a whole, this study provides a greater depth of understanding for this important cell membrane experimental platform and presents a number of new connections to be explored in regard to modulating cell membrane FA composition and its effects on lipid headgroup and raft/cytoskeletal associated proteins.

The effects of dietary antimicrobial removal and Bacillus subtilis supplementation on the growth and intestinal health of male broilers were investigated. Birds were fed either a control, antimicrobial, or a B. subtilis probiotic diet at different feeding phases. Birds were challenged with a 10 × dose of a coccidiosis vaccine. Supplementation of B. subtilis in for antimicrobials in the late grower and early finisher phases improved growth similar to birds fed antimicrobials until withdrawal, while antimicrobial removal without B. subtilis supplementation in those periods hindered growth. The improved growth suggests that the probiotic was able to alleviate the stress of the challenge compared to antimicrobial removal. Processing yields were improved with antimicrobial removal and B. subtilis supplementation in late grower and early finisher phase. Intestinal health was improved with lower intestinal lesions when antimicrobial were removed and B. subtilis supplemented suggesting the reduction of Eimeria species from colonizing the intestine.

The electrospray survivability of B. subtilis spores and E. coli was tested in atmospheric mobility experiments. E. coli did not survive electrospray charging and desolvation, but B. subtilis did. Experimental conditions ensured that any surviving bacteria were charged, desolvated, and de-agglomerated. B. subtilis was also found to survive both positive and negative electrospray and subsequent introduction into vacuum conditions. Attempts were made to measure the charge distribution of viable B. subtilis spores using electrostatic deflection. From those experiments, it was found that either the spores do not become highly charged under the electrospray conditions used or only spores in a low positive or negative charge state survive.

No description available.

No description available.

Formation of spores by Bacillus subtilis is a primitive system of differentiation that has become a paradigm for studying cell differentiation in prokaryotes. Differential gene expression commences soon after the single, asymmetric sporulation division through the activation of different RNA polymerase sigma factors, sigma F in the smaller prespore and sigma E in the larger mother cell. sigma E activation relies on an inter-cellular signaling emanating from sigma F-directed gene expression. Formation of the asymmetric division septum and compartmentalized activity of both sigma factors occur prior to chromosome partitioning. At the time of septation, only 30% of the chromosome destined to be in the prespore is actually present in that compartment and the remaining 70% is in the mother cell. Thus, both cell types contain unequal DNA content. This study focused on the effect of this genetic asymmetry on sigma F-directed gene expression, and exploited this effect in order to study aspects of sigma F to sigma E inter-compartmental signaling. Perturbed signaling resulted in the discovery of a novel twin-spore forming morphology, which was further characterized. A DNA translocase is required to translocate the remaining portion of the chromosome from the mother cell to the prespore. The replication terminus region of the chromosome was observed to be the last to enter the prespore and thus, sigma F-directed genes showed delayed and reduced expression when moved to a terminus-proximal location. The studies indicate that this positional regulation of sigma F-directed gene expression is attributed to both delayed entry and inhibition in sigma F activity at late stages of sporulation. Moreover, the next prespore-specific sigma factor, sigma G, could have a role in inhibiting sigma F. The link between sigma F and sigma E activation is the spoIIR locus, which is transcribed in the prespore from a sigma F-directed promoter soon after the formation of the asymmetric septum. Inactivation of the structural genes for sigma F or sigma E or SpoIIR results in the formation of a second septum at the opposite pole; development proceeds no further, resulting in an "abortively disporic" phenotype. The second septum is formed about 20 min after the first, and sigma E activity is required to prevent its formation. As a sigma F-directed gene, spoIIR is subject to `positional regulation': a delay in spoIIR expression caused by moving it from its origin proximal position to the chromosome terminus, is sufficient to delay sigma E activation and block spore formation, giving the abortively disporic phenotype. The effects of delaying and enhancing spoIIR expression were tested. The changes delayed sigma E activation, and many organisms formed a septum at both ends. However, both prespores in these organisms were able to develop into mature spores (twins). Extra rounds of chromosome replication occured during twin formation, so that each twin had a chromosome and the mother cell had either one or two chromosomes. This over-initiation of chromosome replication is a prerequisite for twin spore formation. Moreover, the studies showed that mother cells of twin forming organisms were longer than those containing single spores; image analysis showed that mother cell length correlates with chromosome content. In contrast to twin spore formation, during normal spore development, there is usually one copy of the chromosome in the prespore and one in the mother cell, with no growth of either compartment. Therefore, the system allowed investigating regulation of chromosome replication and growth of the mother cell. The studies showed that replication and growth are permitted because of the absence of active sigma E and of reduced levels of transcription directed by the master regulator for entrance to spore formation, Spo0A. The results indicate that the burst of Spo0A-directed expression along with activation of sigma E provide mechanisms to block replication and growth of the mother cell. / Microbiology and Immunology

<p> The increase in microbes resistant to a wide array of antibiotics has led to the need for the development of novel antimicrobials. However in order to develop new antimicrobials, novel pathways need to be targeted. Teichoic acid is an anionic polymer covalently attached to the cell wall of Gram-positive bacteria. Recent research has demonstrated that teichoic acid genes are indispensable to the viability of Bacillus subtilis. This makes teichoic acid biosynthetic proteins ideal candidates for the development of a new antimicrobial. Of the teichoic acid glycerol phosphate (tag) genes involved in the biosynthesis of teichoic acid in B. subtilis 168, a conditional deletion mutant of tagD, whose protein product encodes the proposed glycerol-3-phosphatecytidylyltransferase, has been previously constructed and was shown to have a lethal phenotype upon depletion of TagD. This was used in a microarray analysis to find genes that were transcriptionally up-regulated upon the depletion of TagD in B. subtilis 168. Ten candidate genes were selected from those up-regulated and used in the design of a novel, real-time, cell-based luminescent reporter system that responds to lesions in wall biosynthesis. Characterization of these reporter systems in tag gene deletion backgrounds and an examination of their response to antibiotics of various mechanism of action led to the identification of our candidate reporter system P ywac, a robust reporter of both lesions in teichoic acid and peptidoglycan synthesis. In a proof-of-principle screen, the use of Pywac as a reporter of lesions in the cell wall was validated. This reporter system is unique in that it combines conventional genetics with a high throughput capacity. It will not only be amenable for screening small molecules to find inhibitors that impinge on teichoic acid biosynthesis, but it can also be used to probe genetic interactions in B. subtilis. </p> / Thesis / Master of Science (MSc)