Tesi sul tema "Anaerobic bacteria Molecular aspects"

Le méthane, un des principaux gaz à effet de serre, est majoritairement produit et consommé par l'activité métabolique de microorganismes affiliés aux domaines des Archaea et des Bacteria. Afin d'appréhender le cycle biogéochimique du méthane, il est essentiel d'identifier l'ensemble des acteurs impliqués dans ce dernier ainsi que les facteurs environnementaux modulant leurs activités. Les lacs d'eau douce constituent une source importante de méthane, car, dans ces écosystèmes, les conditions environnementales favorisent la méthanogenèse au détriment d'autres processus terminaux de la dégradation anaérobie de la matière organique. Au cours de cette thèse, les études sur les communautés impliquées dans le cycle biogéochimique du méthane ont été conduites dans la colonne d'eau et les sédiments anoxiques du Lac Pavin (Auvergne), unique lac méromictique de France. Cet écosystème a été choisi comme site d'étude en raison des fortes concentrations en méthane présentes dans sa couche d'eau profonde qui contrastent avec les faibles émissions de ce gaz vers l'atmosphère. Ces observations géochimiques suggèrent une intense activité de production et de consommation du méthane, offrant un cadre pertinent pour l'étude des communautés ciblées. Les approches moléculaires visant à caractériser la structure spatiale, la composition, les zones d'activité et les facteurs (ascendants et descendants) potentiellement impliqués dans la régulation des communautés de méthanogènes et de méthanotrophes ont été, au cours de ce travail, systématiquement associées à des approches culturales et microcalorimétriques afin d'acquérir des données sur la physiologie des microorganismes impliqués dans le cycle du méthane. Les résultats obtenus mettent en évidence que les communautés de méthanogènes sont distribuées sur l'ensemble de la colonne d'eau anoxique et dans la strate superficielle des sédiments profonds. Ce groupe métabolique, essentiellement représenté par des espèces affiliées aux Methanosaetaceae et aux Methanoregulaceae, est particulièrement actif dans la zone benthique qui constituerait la source principale de méthane dans cet écosystème. Une nouvelle espèce méthanogène, Methanobacterium lacus, a été isolée de ces sédiments et décrite, et vient enrichir le faible nombre d'espèces méthanogènes isolées à ce jour à partir des lacs d'eau douce. L'étude écophysiologique de cette souche suggère que la température pourrait en partie expliquer la faible représentativité des Methanobacteriales dans cet écosystème. Une partie du méthane semble être directement consommée dans la zone anoxique (pélagique et benthique). L'existence de ce processus d'oxydation anaérobie, soutenu par les approches microcalorimétriques, pourrait être, dans les sédiments profonds, sous la dépendance de lignées candidates archéennes dont la physiologie reste encore énigmatique. Le remplacement progressif des méthanogènes par 2 lignées candidates d'archaea (MBG-D et MCG) le long du profil sédimentaire suggère qu'elle se développe dans des niche contrastées. La régulation putative des communautés archéennes par les virus a été analysée. Cette étude est la première à rapporter la présence de particules virales de type "archaeovirus" dans un environnement non-extrême (en termes de température, pH et salinité) ainsi que des particules virales pouvant représentées de nouvelles familles de virus. Une activité virale intense est suggérée dans ces sédiments par le nombre important de cellules infectées, comparativement à d'autres sédiments, et par le changement concomitant de la structure de la communauté virale et procaryotique avec la profondeur. Bien qu'une partie du méthane soit probablement oxydée en anaérobiose, la consommation de ce métabolite est principalement dépendante de l'activité de méthanotrophes aérobies dominées par des espèces affiliées au genre Methylobacter, un des principaux genres de méthanotrophes rencontré en milieu d'eau douce. (...)

Bibliography: leaves 189-205.
xi, 205 leaves : ill. ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
The aim of this PhD project is to screen feral goat rumen fluid for the presence of new organisms that may play a role in the detoxification of tannins and to investigate their mechanisms of action. An enrichment experiment is conducted to screen rumen fluid for anaerobic bacteria capable of growing in the presence of high levels of "Acacia" condensed tannin. Four morphologically-distinct bacteria are isolated, confirming that resistance is a property shared by more than one organism. One isolate is chosen at random for further characterisation and is identified as a strain of "Selenomonas ruminantium" subspecies "ruminantium". It is arbitrarily designated strain K2. "Selenomonas ruminantium" K2 is shown to be not only tannin-resistant but also able to grow on tannic acid. It is proposed that this bacterium obtained energy for growth from tannic acid. The thesis examines the molecular mechanisms controlling tannin resistance or tannin degradation in rumen microorganisms.
Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 1997

Bibliography: leaves 189-205.
xi, 205 leaves : ill. ; 30 cm.
The aim of this PhD project is to screen feral goat rumen fluid for the presence of new organisms that may play a role in the detoxification of tannins and to investigate their mechanisms of action. An enrichment experiment is conducted to screen rumen fluid for anaerobic bacteria capable of growing in the presence of high levels of "Acacia" condensed tannin. Four morphologically-distinct bacteria are isolated, confirming that resistance is a property shared by more than one organism. One isolate is chosen at random for further characterisation and is identified as a strain of "Selenomonas ruminantium" subspecies "ruminantium". It is arbitrarily designated strain K2. "Selenomonas ruminantium" K2 is shown to be not only tannin-resistant but also able to grow on tannic acid. It is proposed that this bacterium obtained energy for growth from tannic acid. The thesis examines the molecular mechanisms controlling tannin resistance or tannin degradation in rumen microorganisms.
Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 1997

In this thesis, the hypothesis that photoautotrophic Fe(II)-oxidizing bacteria catalyzed the deposition of Banded Iron Formations (BIFs), an enigmatic class of ancient sedimentary rocks is explored. Ecophysiological, geochemical, genetic and biochemical approaches are taken to elucidate the molecular mechanism of photoautotrophic Fe(II) oxidation in an effort to identify molecular biosignatures that are unique to this metabolism and capable of being preserved BIFs. In an ecophysiological approach, we show that Fe(II) oxidation by these phototrophs proceeds at appreciable rates in the presence of high concentrations of H2 when CO2 is abundant. These findings substantiate a role for the involvement of these phototrophs in BIF deposition under the presumed geochemical conditions of the Archean. In a geochemical approach, we find that although phylogenetically distinct phototrophs fractionate Fe isotopes in a way that is consistent with Fe isotopic values found in Precambrian BIFs, it is unlikely that this fractionation can be used as a biosignature for this metabolism given its similarity to fractionations produced by abiotic Fe(II) oxidation reactions. In two distinct genetic approaches, we identify genes involved in Fe(II) oxidation in Rhodopseudomonas palustris TIE-1 and Rhodobacter SW2. Genes identified in TIE-1 encode a predicted integral membrane protein that appears to be part of an ABC transport system and a putative CobS, an enzyme involved in cobalamin (vitamin B12) biosynthesis. Candidate genes on a cloned fragment of the Rhodobacter SW2 genome that confer Fe(II) oxidation activity to a non-oxidizing strain include those predicted to encode permeases and a protein with potential redox capability. Finally, in a preliminary biochemical approach, c-type cytochromes and other proteins that are exclusive or more highly expressed under Fe(II) growth conditions in TIE-1 and SW2 are identified in SDS-PAGE gels. The work described here furthers our search for a biosignature unique to photoautotrophic Fe(II) oxidation by providing mechanistic information on this metabolism.

Tese de dout., Ciências Biotecnológicas (Biotecnologia Ambiental), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010
The objective of the research described in this thesis was the identification and characterization of anaerobic bacterial communities with high metal resistance and ability for metal removal, thus with potential for application in bioremediation processes. A sulphate-reducing bacteria (SRB) consortium resistant to high concentrations of heavy metals (Fe, Cu, Zn), similar to those typically present in acid mine drainage (AMD), was obtained from a wastewater treatment plant. Moreover, this consortium showed ability to use wine wastes as carbon and electron source. The phylogenetic analysis of the dsr gene sequence revealed that this consortium contains species of SRB affiliated to Desulfovibrio fructosovorans, Desulfovibrio aminophilus and Desulfovibrio desulfuricans. Wine wastes as carbon source for SRB activity were applied with success in a bioremediation process for the treatment of artificial AMD. TGGE fingerprinting and phylogenetic analysis showed that the composition of the community in the bioreactor fed with wine wastes remained stable during the whole time of operation and its bacterial diversity was higher than the community in the bioreactor fed with ethanol. Several microbial communities were investigated for their ability to remove uranium (VI) and additionally the impact of U(VI) on SRB communities was explored. Although the original communities were mainly composed by SRB, after uranium exposure these bacteria were not detected in the communities. The highest efficiency for U(VI) removal was observed with a consortium from a soil collected in Monchique thermal place. Moreover this community also showed ability to remove Cr(VI). However when U(VI) was replaced by Cr(VI) several differences in the structure of the bacterial community were observed. The mechanism of U(VI) removal by this consortium was also investigated and was found that U(VI) removal occurred by enzymatic reduction and bioaccumulation. Phylogenetic analysis of 16S rRNA showed that this community was mainly composed by bacteria closely related to Sporotalea genus and Rhodocyclaceae family.
Fundação para a Ciência e a Tecnologia(FCT)

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban Institute Of Technology, 2005 xxiii, 172 p. : ill. ; 30 cm
The provision of safe and sanitary water is a constitutional right and above all, a necessity of life. As a result of the rapid urbanisation and the past policies of apartheid, a large population of South Africa dwell in informal settlements, where there is very little hope of development, as the government does not possess the resources that are necessary for a full-scale sanitation programme. Therefore, on-site treatments have been considered to provide sanitation in these dense peri-urban areas. The anaerobic baffled reactor (ABR) is one such sanitation system. This reactor utilises the phenomenon of anaerobic digestion to degrade substrates. One of the major disadvantages of any anaerobic treatment processes is the extreme sensitivity of the bacterial communities, thus inducing slow recovery rates following toxic shocks. Therefore, an understanding of these microbial consortia is essential to effectively control, operate and optimise the anaerobic reactor. Fluorescence in situ hybridization, 4’,6-diamidino-2-phenylindole (DAPI) staining and DNA sequencing techniques were applied to determine the microbial consortium, as well as their reactions to daily operating conditions. With an understanding of these populations and their responses to perturbations within the system, it is possible to construct an anaerobic system that is successful in its treatment of domestic wastewater. In situ hybridizations were conducted for three operating periods, each characterised by specific flow rates. Results showed Eubacterial population dominance over the Archaeal population throughout both of the operating periods investigated. However, these cells cumulatively consisted of 50% of the total biomass fraction, as determined by DAPI staining. Group-probes utilised revealed a high concentration of fermentative acidogenic bacteria, which lead to a decrease in the pH values. It was noted that the ABR did not separate the acidogenic and methanogenic phases, as expected. Therefore, the decrease in pH further inhibited the proliferation of Archaeal acetoclastic methanogens, which were not present in the second operating period. DNA sequencing results revealed the occurrence of the hydrogenotrophic Methanobacterium and Methanococcus genera and confirmed the presence of Methanosarcina. Sequencing of the bacterial DNA confirmed the presence of the low G+ C Gram Positives (Streptococcus), the high G+C Gram Positives (Propionibacterium) and the sulfate reducing bacteria (Desulfovibrio vulgaris). However, justifications were highly subjective due to a lack of supportive analytical data, such as acetate, volatile fatty acids and methane concentrations. Despite this, findings served to add valuable information, providing details on the specific microbial groups associated with ABR treatment processes.

博士
國立中興大學
土壤環境科學系所
100
In the present study, the systematic classification and molecular detection of hydrocarbon degrading and plant growth promoting rhizobacteria were established. The hydrocarbons degrading bacteria were isolated from oil contaminated samples and the degrading capability was analyzed. Dehydrogenase and lipase were used to estimate the microbial activity within oil contaminated soil. The strategy of bioaugmentation was demonstrated that isolates Azospirillum picis CC-TAR-3T, Azospirillum rugosum CC-AFH-6T, Azospirillum formosense CC-Nfb-7T, Novosphingobium olei CC-TPE-1T, and Sphingomonas formonsensis CC-Nfb-2T could increase the enzymatic activity and maintain the stable communities in biotreatmental processes. Besides, the biochemical characteristics and plant promoting ability of PGPR with oil degrading ability were studied. In addition to the abilities to degrade hydrocarbon pollutant of different strains, others have nitrogen fixating, tricalcium phosphate solubilizing, protein decompositing, cellulose decompositing and siderophore producing capabilities. These bacteria were able to be applied to oil-contaminated soil, and the phytoremediation strategy was potential to be combined to integrate the bioremediation processes. The molecular detection technique for the genus Azospirillum was established, the novel designed genus-specific primer pair (Azo494-F/Azo756-R) was successfully used to distinguish the closest related genus Rhodocista spp. and Skermanella spp. With PCR-DGGE, FISH, and real-time PCR technologies, the genus-specific primer was demonstrated with 2.7 pg μL-1 detection limits, containing 6.6 × 102 CFU per gram soil. The detection technique could be used to rapid determinate, identify and develop the novel nature bioresource in the environmental samples.

Interacting microbial associations capable of utilizing thiosulphate as an energy source were enriched/isolated from activated sludge, landfill site [mal covering soil and soil from an acid mine water drainage site. The isolates were designated Lf-I, Ws-2 and Am-3, respectively. Although hydrogen sulphide was the target molecule for gas biofiltration, thiosulphate, which is a key oxidized intermediate, was used in this study due to the difficulty of working with a toxic gas. Together with thiosulphate oxidation, the microbial associations were assessed for their abilities to oxidize dissolved sulphide to elemental sulphur. Physiological analyses (temperature, pH and substrate concentration optimization) were made with closed and open cultures while morphological characterization and species compositional changes were monitored by light and scanning electron microscopy (SEM). To investigate further functional and structural responses to physiological changes, denaturing-gradient gel electrophoresis (DGGE) separation of PCR-amplified 16S DNA gene fragments and Biolog GN microtitre plates were used. The associations were found to be active metabolically between 0 and 35°C, 15 and 50°C, and 15 and 45°C, with optimum temperatures of 25, 40 and 35°C for Lf-l, Ws-2 and Am-3, respectively. The optimum pH range for microbial association Lf-l was between 3 and 4. The maximum specific growth rates of associations Lf-l , Ws-2 and Am-3 were 0.08, 0.06 and 0.03 h~l , respectively. Components of all three Gram negative rod-dominated associations were motile and displayed anaerobiosis. During open culture cultivation the species complement of Lf-l , as determined by morphological analysis, changed. The same association oxidized sulphide (40 ppm) to sulphur although Ws-2 and Am-3 did not have this capacity. Biolog GN plates detected pH-effected species compositional changes in Lf-l and these were confirmed by DGGE. The same technique showed that enrichment had occurred in the Biolog GN wells. Species composition changes also resulted in response to different pH values (2 to 9), temperatures (5 to 40°C) and dilution rates (0.003 to 0.09 h-1 ), but activity changes were not always accompanied by population profile changes.
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

M. Tech. (Department of Biotechnology, Faculty of Engineering and Technology) Vaal University of Technology
The Klip River has suffered severe anthropogenic effects from industrial, agricultural, mining and domestic activities. As a result harmful contaminants such as heavy metals have accumulated in the river, causing microorganisms inhabiting the environment to develop mechanisms to protect them from the harmful effects of the contaminants. The current study deals with the isolation and characterization of heavy metal resistant bacteria isolated from the Klip River Catchment. Water and sediment samples were collected from 6 sites of the Klip River, and the Vaal Barrage (control). In-situ parameters, such as pH, turbidity, salinity, conductivity, temperature and dissolved oxygen were determined. Lead, iron, cadmium, nickel, zinc and copper concentrations of water were determined by atomic absorption spectroscopy. For bacterial analysis sediment and water samples were collected in sterile glass jars and bottles respectively. Heavy metal resistant bacterial isolates were screened on heavy metal constituted Luria Bertani (LB) agar. Biochemical profiles of the isolates were constructed using the API 20E® strips, antibiotic susceptibility tests were done and growth studies were carried out using spectrophotometric methods. The isolates were identified using 16SrDNA sequencing and alignment. A partial sequence of the copper resistance gene pcoA was amplified from strains Lysinibacillus sp. KR25 [KJ935917], and Escherichia coli KR29 [KJ935918]. The pcoR gene was amplified from E. coli (KR29) and the partial sequence for the chromate resistance gene chrB, was amplified from Pseudomonas sp. KR23 [KJ935916]. The gene fragments were then sequenced and translated into protein sequences. The partial protein sequences were aligned with existing copper and chromate resistance proteins in the Genbank and phylogenetic analysis was carried out. The physico-chemical properties of the translated proteins were predicted using the bioinformatics tool Expasy ProtParam Program. A homology modelling method was used for the prediction of secondary structures using SOPMA software, 3D-protein modelling was carried out using I-TASSER. Validation of the 3D structures produced was performed using Ramachandran plot analysis using MolProbity, C-score and TM-scores. Plasmid isolation was also carried out for both the wild type strains and cured derivatives and their plasmid profiles were analysed using gel electrophoresis to ascertain the presence of plasmids in the isolates. The cured derivatives were also plated on heavy metal constituted media. Antibiotic disc diffusion tests were also carried out to ascertain whether the antibiotic resistance determinants were present on the plasmid or the chromosome. The uppermost part of the Klip River had the lowest pH and thus the highest levels of heavy metal concentrations were recorded in the water samples. Turbidity, salinity and specific conductivity increased measurably at Site 4 (Henley on Klip Weir). Sixteen isolates exhibiting high iron and lead resistance (4 mM) were selected for further studies. Antibiotic susceptibility tests revealed that the isolates exhibited multi-tolerances to drugs such as Ampicillin (10 μg/ml), Amoxcyllin (10 μg/ml), Cephalothin acid (30 μg/ml), Cotrimoxazole (25 μg/ml), Neomycin (30 μg/ml), Streptomycin (10 μg/ml), Tetracycline (30 μg/ml), Tobramycin (10 μg/ml) and Vancomycin (30 μg/ml). Growth studies illustrated the effect of heavy metals on the isolates growth patterns. Cadmium and chromium inhibited the growth of most of the microorganisms. The following strains had high mean specific growth rates; KR01, KR17, and KR25, therefore these isolates have great potential for bioremediative applications. Using 16SrDNA sequencing the isolates were identified as KR01 (Aeromonas hydrophila), KR02 (Bacillus sp.), KR04 (Bacillus megaterium), KR06 (Bacillus subtilis), KR07 (Pseudomonas sp), KR17 (Proteus penneri), KR18 (Shewanella), KR19 (Aeromonas sp.), KR22 (Proteus sp.), KR23 (Pseudomonas sp.), KR25 (Lysinibacillus sp.), KR29 (Escherichia coli), KR44 (Bacillus licheniformis) and KR48 (Arthrobacter sp.). Three heavy metal resistance genes were detected from three isolates. The pcoA gene was amplified from strains Lysinibacillus sp KR25, and Escherichia coli KR29; pcoR gene from E. coli KR29 and the chrB gene, from Pseudomonas sp. KR23. The genes encoding for heavy metal resistance and antibiotic resistance were found to be located on the chromosome for both Pseudomonas sp. (KR23) and E.coli (KR29). For Lysinibacillus (KR25) the heavy metal resistance determinants are suspected to be located on a mobile genetic element which was not detected using gel electrophoresis. The translated protein sequence for pcoA_25 showed 82% homology with the copper resistant protein form Cronobacter turicensis [YP003212800.1]. Sequence comparisons between the pcoR partial protein sequence found in E. coli KR29 showed 100% homology with 36 amino acids (which was 20% of the query cover) from a transcriptional regulatory protein pcoR found in E. coli [WP014641166.1]. For the chrB partial protein sequence detected in Pseudomonas sp. (KR23), 97% of the query sequence showed 99% homology to a vitamin B12 transporter btuB in Stenotrophus sp. RIT309.

Anaerobic digestion (AD) is becoming a widely adopted technology for conversion of organic waste and nutrient-rich fertiliser production due to its cost-effectiveness and sustainability. In this study, a batch experiment was conducted using five different types of food waste and cow dung (CD). No significant difference was observed among the four substrates that produced the highest methane (P<0.05). Based on the batch experiment results, two substrates were selected for semi-continuous digestion and the highest methane yield (67%) was obtained from co-digestion (CO). PCR-DGGE results revealed higher bacterial and archaeal diversity indices in CO as compared to mono-digestion of CD and mixed food waste. The high-throughput sequence analyses revealed that the Operational Taxonomic Units (OTUs) belonging to the phyla Bacteroidetes, followed by Firmicutes, Actinobacteria and Proteobacteria, were dominant in all treatments. The enhanced methane production in CO could be attributed to the neutral pH and partial shift of archaea from Methanosaeta to Methanosarcina. The digestate and fresh CD were screened for plant growth promoting bacteria (PGPB), nutrient and heavy metal content. The dung contained higher concentrations of heavy metals (P<0.05) and potential pathogens in comparison to the digestate. The use of digestate may, therefore, enhance soil fertility with minimal negative environmental effects.
School of Agriculture and Life Sciences
M. Sc. (Life Sciences)

Bacteria adhere despite severe mechanical perturbations. In Gram-positive bacteria, this adhesion is dependent upon a set of extracellular proteins, most notably pili, that have a unique abundance of internal disulfide, isopeptide, and thioester bonds. How these cell adhesion proteins manage to withstand such mechanical assaults, and what role these internal covalent bonds play to that end, remain open questions. Herein, we apply single-molecule force spectroscopy to delve into the mechanical behavior of three Gram-positive pilus proteins. We find that structural components of the Actinomyces oris and Corynebacterium diphtheriae pili have isopeptide-delimited extensions at extreme mechanical forces. This behavior enables efficient energy dissipation under high mechanical loads. Meanwhile, the pilus tip adhesin of Streptococcus pyogenes can covalently bind to targets via its internal thioester bond. We find that reactions with this internal thioester bond are reversible, and that both the nucleophilic bond cleavage and its spontaneous reformation are negatively force-dependent, inhibited at forces above ~30 pN and above ~7 pN, respectively. Based on these observations, we propose a model of shear-enhanced covalent adhesion for Gram-positive bacteria. Finally, we move from single-molecule characterization to application as we explore the potential of a peptide competitors to modulate the folding and function of bacterial virulence factors.

Indiana University-Purdue University Indianapolis (IUPUI)
Borrelia burgdorferi, a bacterium which causes Lyme disease, is maintained in nature through a cycle involving two distinct hosts: a tick vector and a mammalian host. To adapt to these two diverse environments, B. burgdorferi undergoes dramatic alterations in its surface lipoprotein. Two essential lipoproteins, outer surface protein A (OspA) and outer surface protein C (OspC), are reciprocally regulated throughout the B. burgdorferi lifecycle. Very little is known about the regulation of OspA. These studies elucidate the regulatory mechanisms controlling the expression of OspA. Various truncations of the ospA promoter were created and then studied in our novel in vitro model of ospA repression or grown within the host-adapted model. A T-Rich region of the ospA promoter was determined to be a cis-element essential for both the full expression and full repression of ospA.

Indiana University-Purdue University Indianapolis (IUPUI)
Lyme disease, the most prevalent vector-borne illness in the United States, is a multisystem inflammatory disorder caused by infection with the spirochete Borrelia burgdorferi (Bb). This spirochete is maintained in nature through an enzootic cycle involving ticks and small mammals. The Bb genome encodes a large number of surface lipoproteins, many of which are expressed during mammalian infection. One of these lipoproteins is the major outer surface protein C (OspC) whose production is induced during transmission as spirochetes transition from ticks to mammals. OspC is required for Bb to establish infection in mice and has been proposed to facilitate evasion of innate immunity. However, the exact biological function of OspC remains elusive. Our studies show the ospC-deficient spirochete could not establish infection in NOD-scid IL2rγnull mice that lack B cells, T cells, NK cells, and lytic complement, whereas the wild-type spirochete was fully infectious in these mice. The ospC mutant also could not establish infection in SCID and C3H mice that were transiently neutropenic during the first 48 h post-challenge. However, depletion of F4/80+ phagocytes at the skin-site of inoculation in SCID mice allowed the ospC mutant to establish infection in vivo. In phagocyte-depleted SCID mice, the ospC mutant was capable to colonize the joints and triggered neutrophilia during dissemination in a similar pattern as wild-type bacteria. We then constructed GFP-expressing Bb strains to evaluate the interaction of the ospC mutant with phagocytes. Using flow cytometry and fluorometric assay for phagocytosis, we found that phagocytosis of GFP-expressing ospC mutant spirochetes by murine peritoneal macrophages and human THP-1 cells was significantly higher than parental wild-type Bb strains, suggesting that OspC has an anti-phagocytic property. This enhancement in phagocytosis was not mediated by MARCO and CD36 scavenger receptors and was not associated with changes in mRNA levels of TNFα, IL-1β, and IL-10. Phagocytosis assays with HL60 neutrophil-like cells showed that uptake of Bb strains was independent to OspC. Together, our findings reveal that F4/80+ phagocytes are important for clearance of the ospC mutant, and suggest that OspC promotes spirochetes' evasion of macrophages in the skin of mice during early Lyme borreliosis.

Symbioses between microbes and multicellular eukaryotes are found in all biomes, and encompass a spectrum of symbiotic lifestyles that includes parasitism and disease, commensalism, and mutually beneficial interdependent host-microbe relationships. Regardless of outcome, these symbiotic lifestyles are governed by a complex molecular "courtship" between microbe and potential host. This courtship is the primary determinant of the host range of a given microsymbiont. Host immunity poses a formidable barrier to the establishment of host-microbe relationships, and the majority of microbial suitors will be thwarted by it. Only by successfully "wooing" the host cell's immune defenses with the appropriate molecular signals can a microsymbiont successfully colonize its host. A strategy common to microsymbionts across the spectrum of symbiotic lifestyles and host organisms is the delivery of microbial-encoded effector proteins into the cytoplasm of host cells to manipulate the host cell's molecular machinery for the purposes of subverting host immunity. Bacteria, in particular, have adapted a number of secretion systems for this purpose. The most well-characterized of these is the type III secretion system (T3SS), a molecular apparatus that specializes in injecting type III effector (T3Es) proteins directly into host cells. The work in this thesis focuses on T3Es of plant-associated bacteria, with particular emphasis on mutualistic bacteria. We present evidence that collections of T3Es from Sinorhizobium fredii and Bradyrhizobium japonicum are, in stark contrast to those of phytopathogenic bacteria, in a co-evolutionary equilibrium with their hosts. This equilibrium is characterized by highly conserved T3E collections consisting of many "core" T3Es with little variation in nucleotide sequence. The T3Es of Mesorhizobium loti MAFF303099 suggest a completely different picture of the evolution of T3Es. MAFF303099 recently acquired its T3SS locus, and the work in this thesis provides an evolutionary snapshot of a mutualist that is innovating a T3E collection primarily through horizontal gene transfer. Collectively, this work represents the first comprehensive catalog of T3Es of rhizobia and, in the case of Sinorhizobium and Bradyrhizobium, the first evidence of purifying selection for T3Es.
Graduation date: 2012

Indiana University-Purdue University Indianapolis (IUPUI)
Candida albicans and Candida glabrata are opportunistic human pathogens that are the leading cause of fungal infections, which are increasingly becoming the leading cause of sepsis in immunosuppressed individuals. C. glabrata in particular has become a significant concern due to the increase in clinical isolates that demonstrate resistance to triazole antifungal drugs, the most prevalent treatment for such infections. Triazole drugs target the ERG11 gene product and prevent C-14 demethylation of the first sterol intermediate, lanosterol, preventing the production of the pathways end product ergosterol. Ergosterol is required by yeast for cell membrane fluidity and cell signaling. Furthermore, C. glabrata, and not C. albicans, has been reported to utilize cholesterol as a supplement for growth. Although drug resistance is known to be caused by an increase in expression of drug efflux pumps, we hypothesize a second mechanism: that the overuse of triazole drugs has lead to the increase of resistance by C. glabrata through a 2-step process: 1) the accumulation of ergosterol auxotrophic mutations and 2) mutants able to take up exogenous cholesterol anaerobically in the body acquire a second mutation allowing uptake of cholesterol aerobically. Two groups of sterol auxotrophic C. glabrata clinical isolates have been reported to take up sterol aerobically but do not produce a sterol precursor. Sterol auxotrophs have been created in C. glabrata by disrupting different essential genes (ERG1, ERG7, ERG11, ERG25, and ERG27) in the ergosterol pathway to assess which ergosterol mutants will take up sterols aerobically. Random and site-directed mutagenesis was also completed in ERG25 of Saccharmoyces cerevisiae. The ERG25 gene encodes a sterol C-4 methyloxidase essential for sterol biosynthesis in plants, animals, and yeast. This gene functions in turn with ERG26, a sterol C-3 dehydrogenase, and ERG27, a sterol C-3 keto reductase, to remove two methyl groups at the C-4 position on the sterol A ring. In S. cerevisiae, ERG25 has four putative histidine clusters, which bind non-heme iron and a C-terminal KKXX motif, which is a Golgi to ER retrieval motif. We have conducted site-directed and random mutagenesis in the S. cerevisiae wild-type strain SCY876. Site-Directed mutagenesis focused on the four histidine clusters, the KKXX C-terminal motif and other conserved amino acids among various plant, animal, and fungal species. Random mutagenesis was completed with a procedure known as gap repair and was used in an effort to find novel changes in enzyme function outside of the parameters utilized for site-directed mutagenesis. The four putative histidine clusters are expected to be essential for gene function by acting as non-heme iron binding ligands bringing in the oxygen required for the oxidation-reduction in the C-4 demethylation reaction.