Bacterial cells were once treated as membrane-enclosed bags of cytoplasm: a homogeneous, undifferentiated suspension in which polymers (proteins, nucleic acids, etc.) and small molecules diffused freely to interact with each other. Biochemical studies have determined the molecular mechanisms underlying the biological processes of metabolism, replication and transcription-translation, etc. However, recent advancements in optical techniques armed with fluorescent tags for proteins and nucleic acids have increased our ability to peer into the interior of live bacterial cells. This has revealed an organized layout of multi-protein complexes, or molecular machines, dedicated to specific functions at defined sub-cellular locations; the timing of their assembly and/or rates of their activity being determined by available nutrition and environmental signals from the niche occupied by the organism. In the present study, we have attempted to identify the intracellular location and organization of the molecular machines assembled for protein synthesis (ribosomes), DNA replication (replisomes) and cell division (divisome) in different bacteria. We have used the model system Escherichia coli as well as Helicobacter pylori and mycobacterial strains (Mycobacterium marinum and Mycobacterium smegmatis), which grow at different rates and move to dormancy late into stationary phase Bacterial nucleoid plays a major role in organizing the location and movement of active ribosomes, replisomes and placement of divisome. While the active ribosomes appear to follow the dynamic folds of the bacterial nucleoid during cell growth in E. coli, inactive ribosomes appear to accumulate near the periphery. The replisome in H. pylori was visualized as a sharp, single focus upon SSB and DnaB co-localization in growing helical rods but disassembled into diffused fluorescence when the cells attained non-replicative coccoid stage. Our investigation into mycobacterial life-cycle revealed unique features such as an absence of a dedicated mid-cell site for divisome assembly and endosporulation upon entry into stationary phase. In brief, we present the cell cycle-dependent subcellular organization of molecular machines in bacteria.

The bacteria Streptomyces venezuelae produce the novel antibiotic jadomycin. The study of growth characteristics and metabolic behavior of the bacteria are necessary to scale up antibiotic production and facilitate further research. In this study, a method for producing consistent inoculum was developed that showed good repeatability when used in growth trials. The rod shaped spores of Streptomyces venezuelae were determined to be approximately 0.8 x 0.2 ?m with a smooth surface type. The effects of temperature and pH on bacterial growth and substrate consumption were examined in a 7 L bioreactor. Of the range of parameters tested (28, 32, 36 °C, and media pH of 5, 7 and 9), 32 °C with a media pH of 7 yielded the highest rate of growth (µmax of 1.43 hours-1 with a lag time of 7.7 hours). The results of all trials showed that free glucose was consumed before the maltose, which was the major sugar substrate in the media. The initiation of exponential bacterial growth occurred after rapid consumption of free glucose. A heat balance analysis was also performed over the bioreactor to identify the heat generated through agitation, losses over the vessel and the heat of metabolism from Streptomyces venezuelae. Under normal operating parameters 33 - 24 % of the heat generated through mixing was lost with the exhaust gas, while 56 - 64 % was lost through the bioreactor wall. The heat of mixing was calculated to be 1.62 J•s-1 while the maximum amount of heat generated by Streptomyces venezuelae metabolism and activity during a growth trial was 2.28 J•s-1 for 60 x 109 CFU?mL-1.

The chicken eggshell possesses physical and chemical barriers to protect the embryo from pathogens. The avian eggshell cuticle is the outmost layer of the eggshell whose protein constituents remain largely unknown. Since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination, we hypothesize that cuticle protein components play an important role in microbial resistance. In our study, at least 47 proteins were identified by LC/MS/MS in the non-calcified cuticle layer. Similar to Kunitz-like protease inhibitor (also annotated as ovocalyxin-25, OCX-25) and ovocalyxin-32 (OCX-32) were two of most abundant proteins of the cuticle proteins. Some proteins that have antimicrobial activity were also detected in the proteomic results, such as lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor. This study represents the first comprehensive report of the cuticle proteome. Since the sequence similarity of the kunitz motif in OCX-25 is similar to that of BPTI, it is predicted that it will have the same trypsin inhibitory and antimicrobial activity against Gram-positive and/or Gram-negative bacteria. In order to test the antimicrobial property and trypsin inhibitor activity of OCX-25, cuticle proteins were extracted by 1N HCl. Antimicrobial activity was monitored using the Bioscreen C instrument; and antimicrobial activity was identified primarily against Staphylococcus aureus. Trypsin inhibitor activity was studied by using a specific trypsin assay, and the assay indicated that the cuticle proteins could inhibit the reaction of trypsin and substrate. Therefore, the current research has provided some insight into the antimicrobial and enzymatic aspects of the cuticle proteins, and its function for egg protection. Eggshell membranes are another important component of the chicken eggshell.Due to its insoluble and stable properties, there are still many questions regarding formation and constituents of the eggshell membranes. The purpose of our study was to identify eggshell membrane proteins, particularly these responsible for its structural features, by examining the transcriptome of the white isthmus during its formation. Bioinformatics tools were applied to analyze the differentially expressed genes as well as their encoded proteins. Some interesting proteins were encoded by the over-expressed genes in the white isthmus during the formation of eggshell membranes, such as Collagen X, and similar to spore coat protein SP75. These proteins may have potential applications. Our study provides a detailed description of the chicken white isthmus transcriptome during formation of the eggshell membranes; it could lead to develop the strategies to improve food safety of the table egg.

Title from second page of PDF document. Includes bibliographical references.

Spores are single-celled dispersal units surrounded by a wall of the highly resistant biopolymer sporopollenin. All land plants produce spores. Spore development is described in Physcomitrella patens, a moss with single-celled spores, and Dendroceros, a hornwort with multicellular spores. Correlated light, fluorescence and immuno-electron microscopy localizes callose in the aperture of developing spores in the model moss Physcomitrella. Twelve copies of callose synthase genes were annotated bioinformatically and compared with Arabidopsis callose synthase genes. This study identifies a suspect gene involved in moss spore exine development. Unicellular spores of Dendroceros following meiosis remain in tetrads, fill the intercapsular space, and are surrounded by a convoluted, homogeneous electron-opaque outer wall and narrow fibrillar inner wall. No precise pattern of cell division leads to multicellular spores of variable shape and cell number. Evolution of precocious endospory in epiphytic hornworts is a means to protect nascent spores while it develops biochemical and structural machinary to withstand drying. To advance knowledge of genetic control of spore wall development, the sequenced genome of Physcomitrella is probed using a bioinformatic approach to decipher the evolution of five selected genes putatively involved in spore wall formation. Those encoding for callose synthase provide the most complete results. Callose involvement in spore development is a plesiomorphic feature of land plants. Phylogenomic analysis of callose synthases in land plants with sequenced genomes revealed a single moss callose synthase basal in a clade containing the only Arabidopsis callose synthase implicated in exine development of pollen walls as well as two clades of moss specific callose synthase proteins. A predicted protein-protein interactome was constructed to investigate the protein landscape in Physcomitrella for proteins involved in sporogenesis. Orthologous genes were identified between Physcomitrellaand several other species to map orthologous interactions and predict the first bryophyte interactome. The Physcomitrella predicted protein-protein interactome contains 41,936 unique interactions for 4062 different proteins, none of which are associated with sporogenesis. Rather the most conserved interactions among proteins were those associated with metabolic processes. The utility of predicted protein interactions to infer biological roles, providing provisional molecular roadmaps is demonstrated to generate hypotheses for experimental approaches.

Early blight (Alternaria solani Sorauer)is a major foliar disease of potatoes in most growing regions of the world and is underestimated in South Africa. This project studies the epidemiology and control of the disease in South Africa. A decision support system (DSS) for early blight in South Africa was developed and evaluated in field trials. This early blight DSS is the first such system to be developed in South Africa and once incorporated with the late blight model, will represent innovative technology for use in the South African potato industry. Trends in weather variables and concentrations of airborne conidia of A. solani were monitored. Distinct seasonal variation was noted. Peaks in spore concentration coincided with periods favourable for spore formation and dispersal; most notable was diurnal periodicity and interrupted wetting periods. The results obtained from these measurements will be useful in improving early blight DSSs for southern Africa. Isolates of A. solani from various potato-growing regions in South Africa were characterized using virulence, vegetative compatibility (VC) and random amplified microsatellite (RAMS) primers. Neither the virulence assays nor VC tests sufficiently characterised the population. Analysis of RAMS profiles revealed 27% genetic diversity among the isolates. This value is similar to diversity values obtained by previous authors studying A. solani, however, it is relatively high for an asexually reproducing fungus. There was no evidence for geographical clustering of isolates, indicating that isolates are widespread across South Africa. A survey on control practices and grower perceptions of early blight in South Africa was conducted using a questionnaire. These questionnaires were distributed to growers from 10 potato-growing regions in South Africa. Results highlighted the most popular control methods and cultivars in the South African potato industry. The majority of respondents indicated that they would use an accurate, cost-effective early blight DSS, and that more research is necessary on early blight in South Africa. A survey on control practices and grower perceptions of early blight in South Africa was conducted using a questionnaire. These questionnaires were distributed to growers from 10 potato-growing regions in South Africa. Results highlighted the most popular control methods and cultivars in the South African potato industry. The majority of respondents indicated that they would use an accurate, cost-effective early blight DSS, and that more research is necessary on early blight in South Africa. Estimated crop losses ranged from 1% - 60%, with an average of approximately 20%. This is the first comprehensive epidemiological study to be conducted on early blight in South Africa and has highlighted the need for further research. / Thesis (DPhil (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

The chicken eggshell possesses physical and chemical barriers to protect the embryo from pathogens. The avian eggshell cuticle is the outmost layer of the eggshell whose protein constituents remain largely unknown. Since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination, we hypothesize that cuticle protein components play an important role in microbial resistance. In our study, at least 47 proteins were identified by LC/MS/MS in the non-calcified cuticle layer. Similar to Kunitz-like protease inhibitor (also annotated as ovocalyxin-25, OCX-25) and ovocalyxin-32 (OCX-32) were two of most abundant proteins of the cuticle proteins. Some proteins that have antimicrobial activity were also detected in the proteomic results, such as lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor. This study represents the first comprehensive report of the cuticle proteome. Since the sequence similarity of the kunitz motif in OCX-25 is similar to that of BPTI, it is predicted that it will have the same trypsin inhibitory and antimicrobial activity against Gram-positive and/or Gram-negative bacteria. In order to test the antimicrobial property and trypsin inhibitor activity of OCX-25, cuticle proteins were extracted by 1N HCl. Antimicrobial activity was monitored using the Bioscreen C instrument; and antimicrobial activity was identified primarily against Staphylococcus aureus. Trypsin inhibitor activity was studied by using a specific trypsin assay, and the assay indicated that the cuticle proteins could inhibit the reaction of trypsin and substrate. Therefore, the current research has provided some insight into the antimicrobial and enzymatic aspects of the cuticle proteins, and its function for egg protection. Eggshell membranes are another important component of the chicken eggshell.Due to its insoluble and stable properties, there are still many questions regarding formation and constituents of the eggshell membranes. The purpose of our study was to identify eggshell membrane proteins, particularly these responsible for its structural features, by examining the transcriptome of the white isthmus during its formation. Bioinformatics tools were applied to analyze the differentially expressed genes as well as their encoded proteins. Some interesting proteins were encoded by the over-expressed genes in the white isthmus during the formation of eggshell membranes, such as Collagen X, and similar to spore coat protein SP75. These proteins may have potential applications. Our study provides a detailed description of the chicken white isthmus transcriptome during formation of the eggshell membranes; it could lead to develop the strategies to improve food safety of the table egg.

The Mesozoic is punctuated by several rapid global warming events that are marked by the worldwide deposition of organic-rich marine sediments. These events, known as oceanic anoxic events (OAEs), are characterised by intervals where the worldwide carbon cycle suffers a disruption due to major palaeoceanographic and climatic shifts, leading to anoxic marine environments and the creation of black shales. For this study, the Oceanic Anoxic Event 1a (OAE 1a), which occurred during the Early Cretaceous Aptian age (~120 Ma) was analysed. It was likely triggered by the Greater Ontong Java underwater volcanic event and is associated with major changes in marine environments and ecosystems, including nekton and plankton turnover, and sea water composition changes. The impact of this event on terrestrial land-based ecosystems is, however, less well understood. Here I document well preserved and diverse spore-pollen assemblages spanning OAE 1a from southern Sweden by examining the Höllviken I core. Before the OAE, palynofloras are dominated by conifers, suggestive of a relatively mild and dry coastal environment. At the onset of the OAE a fern spike occurs, where there is a shift to early successional stage vegetation. Gymnosperm diversity and abundance sharply decrease, and the palynofloral assemblages become dominated by ferns, indicating a shift to warm and wet conditions. Gymnosperms gradually recover thereafter, but the formerly abundant conifer pollen Classopollis does not recover and remains rare. Dinoflagellate cysts and microforaminiferal test linings increase in abundance after OAE 1a, suggesting a higher degree of marine influence. These findings show that OAE 1a had a substantial impact on the composition and diversity of high latitude terrestrial vegetation and marine plankton communities. / <p>The work for this thesis was financially supported by the Erasmus Mundus Joint Master Degree PANGEA programme.</p>

1) Einleitung 2) Pathogenese und Klinik des Botulismus 3) Vorkommen von C. botulinum und des Botulismus 4) Beeinflussung von Spore, vegetativer Zelle und BoNT 5) Diagnostik, Therapie und Prophylaxe 6) Diskussion

Objectives: Dental instrument sterilization with steam autoclaves is critical to maintaining infection control standards in dental practice, and preventing patient-to-patient transmission of pathogenic bacteria and viruses. The American Dental Association and the United States Centers for Disease Control and Prevention recommend, and many state dental laws require, weekly use of biological spore tests to verify dental instrument sterilization outcomes. However, the most widely used biological spore test needs microbial culture incubation for 2 days after autoclave exposure, which limits swift identification of sterilization failure. To address this issue, this study evaluated the reliability of a new rapid biological spore test for determining the sterilization efficacy of dental steam autoclaves within 20 minutes. Methods: Two commercial biological spore tests were evaluated in Temple University dental school steam autoclaves, 1.) the Steris Celerity 20 Steam Biologic Indicator with a 20-minute outcome time requirement, and 2.) the 3M Attest 1262 Biological Indicator with a 48-hour outcome time requirement. Both biological spore tests employed live thermoresistant Geobacillus stearothermophilus spores as an indicator of whether sterilization conditions in steam autoclaves were met or not. To compare their efficacy, a total of 157 pairs of the two biological spore tests were placed into dental steam autoclaves with dental instrument cassettes, and subjected to manufacturer-recommended steam autoclave temperature and air pressure operating conditions for an adequate sterilization time of 15 minutes. Two additional groups of 10 pairs each of the two biological indicators were subjected to appropriate steam autoclave temperature and air pressure settings, but only for aborted non-sterilizing time periods of 10 and 5 minutes, respectively. Subsequent aseptic processing and laboratory incubation of both biological indicators was initiated within 2-24 hours, and followed manufacturer recommendations. After autoclave exposure, Steris Celerity 20 Steam Biologic Indicator test ampoules were incubated in a specialized instrument for 20 minutes at 57 °C, which also spectrophotometrically evaluated the microbial culture medium for fluorescent α- glucosidase enzyme signal changes. No change in fluorescent intensity represented successful sterilization, whereas increased fluorescence indicated survival of viable G. stearothermophilus spores germinating into vegetative bacterial cells after failed sterilization. 3M Attest 1262 Biological Indicator ampoules were incubated for 48 hours in a laboratory heating block at 57 °C, after which a pH-based color change in the microbial culture broth was visually assessed. No change in the color of the culture broth (purple color remains) indicated successful sterilization, whereas development of a yellow color in the culture broth, as a result of viable G. stearothermophilus spore germination into vegetative bacterial cells, denoted failed sterilization. Results: A total of 354 biological indicators were exposed to dental steam autoclaves sterilization cycles, incubated for either 20 minutes or 48 hours, and evaluated for G. stearothermophilus spore growth. The Steris Celerity and 3M Attest biological spore tests both uniformly detected successful sterilization, with no G. stearothermophilus spore growth, after 15 minutes of steam autoclave exposure at manufacturer recommended steam autoclave temperature and air pressure operating conditions. This provided 100% agreement, and no statistically significant difference in the prevalence of successful sterilization outcomes, between 157 pairs of both biological indicator types after 15 minutes of steam autoclave exposure. Similarly, both biological spore test systems were also in complete agreement after only 5 minutes of steam autoclave exposure, with 100% of both biological indicators positive for G. stearothermophilus spore growth, indicating failed sterilization. In contrast, after 10 minutes of steam autoclave exposure, there was a complete lack of agreement between the two types of biological indicators. All 10 Steris Celerity spore tests were positive, whereas all 10 3M Attest ampoules were negative, for G. stearothermophilus spore growth after 10 minutes of steam autoclave exposure. Relative to this disagreement, a non-biological chemical indicator strip that was part of the Steris biological indicator test system failed to have a darkened bar develop and extend into the “Accept (OK)” portion of the strip for all Steris Celerity spore tests exposed to either 5 minutes or 10 minutes of steam autoclave exposure, indicating that adequate autoclave steam, temperature and/or time parameters had not been attained for sterilization. Conclusions: The Steris Celerity biological spore test was successful in rapidly determining the sterilization efficacy of dental steam autoclaves within only a 20-minute incubation time period, as compared to 48 hours of incubation required by the widely-used 3M Attest biological spore test. As a result, this rapid assay offers earlier detection of steam autoclave sterilization failure before potentially contaminated dental instruments are used in clinical patient care. The alarming failure of 3M Attest biological spores to grow after a non-sterilizing 10-minute steam autoclave exposure time warrants further product evaluation. / Oral Biology