Clostridium difficile is a strictly anaerobic, spore-forming bacterium that is linked to over 250,000 infections annually in the United States. One of the greatest challenges facing C. difficile research has been the lack of genetic tools. This limited repertoire is due, in part, to the anaerobic nature of C. difficile. For example, most fluorescent protein reporters require O2 for chromophore maturation. Here, we demonstrate that O2-dependent fluorescent proteins produced anaerobically can acquire fluorescence after cells are fixed with cross-linkers to preserve native patterns of protein localization. This was shown using the blue and the red codon-optimized fluorescent proteins, CFPopt and mCherryOpt, respectively. Little is known about cell division in C. difficile. Here we identify and characterize a three-gene operon encoding cell division proteins found only in C. difficile and a small number of closely related bacteria. These proteins were named MldA, MldB, and MldC, for midcell localizing division proteins. MldA is predicted to be a membrane protein with coiled-coil domains and a peptidoglycan-binding SPOR domain. MldB and MldC are predicted to be cytoplasmic proteins; MldB has two predicted coiled-coil domains, while MldC lacks obvious conserved domains or sequence motifs. Mutants of mldA or mldB had morphological defects, including loss of rod shape (a curved cell phenotype) and inefficient separation of daughter cells (a chaining phenotype). Fusions of CFPopt to MldA, MldB, and MldC revealed that all three proteins localize sharply to the division site. Mutants lacking the Mld proteins are severely attenuated for pathogenesis in a hamster model of C. difficile infection. Because all three Mld proteins are essentially unique to C. difficile, they could be exploited as targets for antibiotics that combat C. difficile without disrupting the intestinal microbiome. C. difficile pathogenesis is mediated primarily by two large exotoxins called Toxin A (TcdA) and Toxin B (TcdB). Transcription of tcdA and tcdB depends on TcdR, an alternative sigma factor for RNA polymerase. Previous studies have shown both toxins are produced upon entry into stationary phase, and that this response is mediated in part by the CodY repressor, which senses GTP and branched chain amino acids. Here we used mCherryOpt as a reporter of gene expression to visualize toxin expression at the level of individual cells. This approach led to the unexpected discovery that only a subset of cells in the population induces expression of tcdA (and tcdB under specific conditions). In other words, toxin production is a “bistable” phenotype. Further experiments indicated TcdR plays a central role in mediating bistability, while CodY makes a minor but still significant contribution to bistability. Why it is advantageous for only a subset of C. difficile cells to produce toxin is not known, but one interesting possibility is related to conflicting requirements for transmission to a new host. Some cells produce toxin to provoke diarrhea while other cells differentiate into spores that can survive exposure to air.

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No description available.

This research sought to determine which induction condition resulted in the greatest GST-GFP fusion protein expression. It will hopefully serve as a guide for future researchers trying to produce their own recombinant protein containing GST and GFP-tags. The CDNB Enzyme Assay was used to determine the quantity of GST-GFP fusion protein present and tested three variables: IPTG concentration, duration, and temperature of induction. The findings showed that IPTG concentration, temperature, and induction duration all had a significant impact on protein expression. Induction temperatures of 20 °C and 25 °C showed better protein expression at IPTG concentrations of 1.0 mM IPTG over 0.1 mM IPTG. Induction durations of 20 hours and 5 hours were better than 3 hours. The 25 °C condition had the greatest protein expression, followed by 20 °C condition. In a follow-up experiment, using 1.0 mM IPTG and 20- hour induction duration, the 20 °C condition showed higher GST activity. Analysis of the 20 °C Western blots revealed the presence of possibly truncated/degraded fusion protein (not seen in the 25 °C Western blots). Future experimenters should use C-terminal GST-tags, as opposed to N-terminal GST-tags, to prevent accidental purification of truncated proteins in addition to the target protein. If this is not possible, then the recommendation is to use the 25 °C temperature for induction, as this temperature had similar GST-GFP fusion protein expression and resulted in much cleaner Western blots.

Cell dedifferentiation is a cell fate regression process in which the cell fate memory of a differentiated cell is erased, leading to regain stem cell characteristics. Auxin regulates both cell dedifferentiation and differentiation in plants. It is unknown how auxin controls the two opposite processes. Here the minimal auxin requirements for cell dedifferentiation were found, molecular markers associated with the cell dedifferentiation event were identified. When cellular auxin concentration exceeds the level of meristem cell, most differentiated cells undergo dedifferentiation. In differentiated cells, the polar auxin efflux system prevents cell dedifferentiation by reducing auxin accumulation, particularly in the presence of exogenous auxin. Classic plant tissue culture experiments have shown that exposure of cell culture to a high auxin to cytokinin ratio promotes root formation and a low auxin to cytokinin ratio leads to shoot regeneration. Since the auxin level is highly elevated in the shoot meristem tissues, it is unclear how a low auxin to cytokinin ratio promotes the regeneration of shoots. To identify genes mediating the cytokinin and auxin interaction during organogenesis in vitro, three allelic mutants that display root instead of shoot regeneration in response to a low auxin to cytokinin ratio are identified using a forward genetic approach in Arabidopsis. Molecular characterization shows that the mutations disrupt the AUX1 gene, which has been reported to regulate auxin influx in plants. Meanwhile, it was found that cytokinin substantially stimulates auxin accumulation and redistribution in calli and some specific tissues of Arabidopsis seedlings. In the aux1 mutants, the cytokinin regulated auxin accumulation and redistribution is substantially reduced. These results suggest that auxin elevation and other changes stimulated by cytokinin, instead of low auxin or exogenous auxin directly applied, is essential for shoot regeneration. In this study, as a part of interaction between auxin and cytokinin it was identified that the induction of ARR5 and ARR6 expression by cytokinin is subjected to the regulation of auxin. The expression of ARR5 and ARR6 follows a mutual exclusive pattern in response to the induction of exogenous auxin in Arabidopsis seedlings and calli. The results suggest that auxin interacts with the cytokinin via a gene and tissue specific induction of the negative regulators in the cytokinin signaling pathway.

No description available.

Streptococcus mutans is a secondary colonizer of the dental plaque biofilm and is the primary causative agent of dental caries. Sugar metabolism is central to S. mutans growth and survival. S. mutans produces lactic acid as an end product of sugar metabolism, which results in dissolution of the tooth enamel, leading to dental cavities. Sucrose metabolism also results in the formation of extracellular dextrans that are a key component of the extracellular matrix that encases the bacteria in the biofilm. The availability of sugars is dependent on diet, on competition with other bacteria and on the location of the bacteria within the dental plaque. I hypothesize there are distinct subpopulations of S. mutans within biofilms that respond differently to environmental conditions. I have identified several genetic markers that are helping us identify and characterize some of these subpopulations, and how they react to starvation and to the restoration of nutrients in single species biofilms of S. mutans. Two of the loci that were identified as markers via microarray analysis are rpsT and pdh. rpsT encodes a small ribosomal protein which is strongly expressed during exponential growth, when the cells are producing high levels of ribosomes. The other marker, pdh, is a four-gene operon encoding the pyruvate dehydrogenase complex; pdh is upregulated in late stationary phase. Our laboratory has recently shown that expression of the pdh operon is important for long-term survival in stationary phase, where a subpopulation (~0.5%) is dividing, forms long chains and expresses pdh. In the current studies, rpsT and pdh promoters driving expression of gfp were used to identify the exponential phase subpopulation (rpsT) and a subpopulation capable of surviving in late stationary phase (pdh). In addition, I developed an unstable variant of GFP by fusing a proteolytic tag sequence to the C-terminus of GFP (encoded by ugfp). When the rpsT promoter was inserted upstream, uGFP was produced and subsequently degraded within about 1.5 hours of translation. This behavior allowed us to distinguish exponentially growing cells, as the signal diminishes once the cells entered stationary phase. In biofilms that had been starved for 10 days, there was no expression of PrpsTugfp. I observed that when sucrose was added to these biofilms, some bacteria within the biofilm microcolonies underwent fast exponential-like growth indicated by expression of PrpsT-ugfp. Within 24 hours of the sucrose addition, most growth had ceased and fluorescence had decreased. Using a Ppdh-gfp construct in bacteria in 10-day starved biofilms, fluorescence was observed in long chains of cells within the biofilms indicating slow growth. I hypothesized that the pdh-expressing cells were capable of responding to sucrose restoration and would be one of the principal subpopulations to do so. However, when sucrose was added, these fluorescing chains did not exhibit any growth, while other non-fluorescing bacteria within the biofilm clearly responded to the sucrose by growing. This was unexpected since inactivating the pdh operon leads to drastically reduced survival. It is concluded that pdh plays a role in long term survival, but pdhexpressers do not appear to respond to sugar restoration. This led me to hypothesize that the pdh-expressing population is interacting with other populations of cells in some capacity, enabling them to survive. To determine if this was the case, we performed a mixed culture experiment with wild-type S. mutans and the pdh null mutant. I observed that when these two strains were grown in co-culture, the pdh null mutant survived at low levels, for over 30 days, while this mutant by itself typically did not survive past ten days. This result indicates that the wild-type strain was able to interact with the mutant, leading to increased survival. In biofilms, it seems possible that the pdh-expressing cells secrete a substance or directly interact with other cells, somehow promoting their survival in the starved biofilm. The fluorescent constructs appear to mark distinct populations of cells that respond in different ways to sugar availability, suggesting that S. mutans forms a mixed population of cells able to grow in the presence of sugar or survive prolonged sugar starvation. These studies demonstrate that indeed subpopulations of cells do exist within biofilms, and their interactions may be more complex than previously thought. / Microbiology and Immunology

The interaction between Arabidopsis and its natural downy mildew pathogen, Hyaloperonospora arabidopsidis (Hpa), provides a model for understanding how oomycetes colonize plants. Hpa is a model organism for many highly destructive oomycete pathogens and transcriptomics of this interaction have been well-documented. However, the material in these studies has been derived from infected leaves that contain a mix of pathogen-proximal and pathogen-distal plant cells. The most direct interactions between Arabidopsis and Hyaloperonospora arabidopsidis occur in haustoriated cells- where the pathogen can secrete effectors and acquire nutrients needed for successful colonization and reproduction. These cells are difficult to isolate due to their limited number and ephemeral nature. I have developed a method to isolate the translatome (i.e., mRNAs associated with ribosomes) of pathogen-proximal cells. This method utilizes translating ribosome immuno-purification technology (TRAP), regulated by both pathogen-responsive and tissue-specific promoters, to isolate mRNAs that are being translated in pathogen-proximal cells. Compared to "bulk" transcriptomics of material isolated from homogenized leaves, this method will enrich for transcripts that are differentially expressed, and translated, in pathogen-proximal cells. From this method, RNA was isolated in amount and quality sufficient for sequencing. This sequencing data will enable the discovery of plant genes that may be manipulated by the pathogen to suppress defense responses and extract nutrients. / Ph. D. / The interactions between plants and the pathogens that feed on them are complex and at times difficult to study. Among the many different types of plant pathogens, oomycetes (a class of fungus-like organisms) are especially destructive. Using Arabidopsis and its natural downy mildew pathogen, Hyaloperonospora arabidopsidis (Hpa) as model for understanding how oomycetes colonize plants, I hope to learn more about plant-pathogen interactions. Hpa is a model organism for many highly destructive oomycete pathogens and several aspects of this interaction have been well-documented. However, the material in these studies has been derived from infected leaves that contain a mix of plant cells that are both in direct contact with the pathogen, or from uninfected areas of the plant. The most direct interactions between Arabidopsis and Hpa occur in cells that have been invaginated with a pathogen feeding structure called a haustorium. These cells are difficult to isolate due to their limited number and ephemeral nature. I have developed a method to isolate the translatome (i.e., mRNAs that are being translated by and are associated with ribosomes) of pathogen-proximal cells. This method utilizes translating ribosome immuno-purification technology (TRAP), regulated by both pathogen-responsive and tissue-specific promoters, to isolate mRNAs that are being translated in pathogen-proximal cells. Compared to “bulk” transcriptomics of material isolated from homogenized leaves, this method will enrich for transcripts that are differentially expressed, and translated, in pathogen-proximal cells. From this method, RNA was isolated in amount and quality sufficient for sequencing. This sequencing data will enable the discovery of plant genes that may be manipulated by the pathogen to suppress defense responses and extract nutrients.

No presente estudo, a diversidade bacteriana endofítica fixadora de nitrogênio foi pesquisada utilizando métodos microbiológicos e moleculares. Isolaram-se microrganismos de raiz, caule e folha de cana-de-açúcar. Análises por seqüenciamento do 16S rDNA identificaram 150 endófitos. No total, foram identificados 18 gêneros. Destes, apenas 4 estavam presentes em cana-de-açúcar submetida aos 3 tratamentos. A maior diversidade de gêneros foi encontrada em cana sob adubação orgânica: 10 gêneros, em cana sob adubação inorgânica foram 11 gêneros e 8 em cana sem adubação. A maior parte dos gêneros pertence à família Enterobacteriaceae, como Klebsiella, Pantoea e Enterobacter. A enzima endoglicanase foi produzida por 82% dos isolados de cana sob adubação orgânica, (54%) inorgânica e (48%) sem adubação. Quanto à atividade de pectinase: 42%, 60% e 36% foram apresentadas por isolados de cana orgânica, inorgânica e sem adubação, respectivamente. A capacidade de solubilizar fosfatos inorgânicos foi detectada em 76,6% dos isolados, sendo a maior capacidade de solubilização de fosfatos encontrada em bactérias isoladas de cana-de-açúcar sob adubação orgânica (71%), de cana submetida a adubação convencional (78%) e sem adubação (88%). Foram realizados estudos de colonização em plântulas de cana-de-açúcar com 4 endofitos geneticamente modificados (EGMs) capazes de expressar os genes gfp e dsred. A avaliação da colonização na cana pela microscopia de fluorescência, mostrou que os (EGMs) gfp e dsred colonizaram as raízes e caules das plantas inoculadas, sem causar qualquer sintoma de doença. / In the present study, endophytic bacterial diversity has been searched using both microbiologic and molecular methods. Microorganisms were isolated from sugarcane root, shoot and leaf. 150 isolates were identified by 16S rDNA sequencing. 18 genera were found and only 4 were present in sugarcane submitted to the three treatments. The greatest genera diversity was found in sugarcane submited to organic fertlization: 10 genera in sugarcane submitted to inorganic fertilization were found 11 genera and 8 genera in sugarcane without fertilization. Great part of the found genera belongs to the Enterobacteriaceae family: Klebsiella, Pantoea and Enterobacter. Some physiological characteristics were determined in the isolates. Endoglucanase was produced by 82% of the isolates from sugarcane submitted to organic fertilization. Lower activities were found in bacteria isolated from inorganic fertilization and no fertilization, respectively 54% and 48%. As far as pectinase activity is concerned, a percentage of 42%, 60% and 36% was presented by the isolates from organic fertilization, inorganic fertilization and no fertilization, respectively. The hability of phosphate solubilization was detected in 76.6% of the isolates. In sugarcane under organic fertilization a percentage of 71% was found, in bacteria from inorganic fertilization, 78%, and without fertilization, 88%. Plant colonization was determined using sugarcane plantlets inoculated with four genetically modified bactéria (GMEs), able to express the genes gfp and dsred. The colonization was evaluated by fluorescence microscopy, which showed that the endophytic bacteria expressing gfp and dsred genes had invaded roots and shoots from inoculated plants, without causing any disease symptom.

A divisão celular nas bactérias requer a formação do divisomo, um complexo protéico que tem como o primeira etapa a polimerização da proteína FtsZ, seguida pela associação de 15 outras proteínas conhecidas. Os mecanismos envolvidos na regulação espacial do divisomo são bem caracterizados, mas o controle temporal da divisão celular em relação a outros eventos do ciclo, como a replicação do cromossomo, segue controversa. Neste trabalho, aplicamos a metodologia de microscopia de série temporal para estudar duas questões fundamentais do processo de divisão: a montagem do complexo que executa a divisão e a possibilidade da oscilação periódica na expressão de um ou mais genes envolvidos em divisão possa participar do controle temporal da montagem do divisomo. Para investigar se há oscilação da expressão gênica, construímos inicialmente variantes instáveis GFP através da adição de sequências peptídicas C-terminais que encaminham para a degradação em B. subtilis e utilizamos estes repórteres para criar fusões transcricionais sob o controle de promotores de genes centrais do processo de divisão. Depois de otimizar as condições de microscopia de série temporal com fusões transcricionais usando a variante instável GFPAISV, observamos que a autofluorescência de B. subtilis interferia nas nossas quantificações. Como forma de contornar a autofluorescência, construímos então fusões transcricionais com duas variantes de YFP (proteína fluorescente amarela) e optamos por trabalhar com Ypet-AISV. A análise de filmes de células individuais, tanto com fusões a GFPAISV como a Ypet-AISV, indicou que apenas o promotor do operon ftsL-pbpB apresentava um padrão de oscilação significativamente diferente de um promotor artificial usado como controle negativo. Esta hipótese, no entanto, não foi confirmada por medidas estáticas de populações de células nas quais correlacionamos intensidade de fluorescência com posição no ciclo celular. Portanto, nossos dados não foram capazes de evidenciar flutuações na expressão dos genes ftsL-pbpB, minCD, ftsZ, ftsA e zapA ao longo do ciclo celular. Para estudar a cinética de montagem divisomo foram realizados experimentos de microscopia de série temporal de FtsZ-mCherry e Pbp2B-GFP, onde observamos que a associação de Pbp2B ao divisomo ocorre 3 minutos após a formação do anel de FtsZ em meio rico e 4 minutos em meio mínimo. Também realizamos experimentos de microscopia de série temporal com uma cepa contendo FtsZ-YFP e DivIVA-CFP, determinando que DivIVA é incorporado ao divisomo 16 minutos após a formação do anel de FtsZ em meio rico e 20 minutos em meio mínimo. Estes dados confirmam que a montagem do divisomo ocorre em três etapas, e não duas, como anteriormente proposto. / Cell division in bacteria requires the formation of the divisome, a protein complex that has as the first step polymerization of FtsZ, followed by the assembly of 15 other known proteins. The mechanisms that underlie spatial regulation of divisome assembly have been largely elucidated, but the temporal control that ties the timing of cell division to other cell cycle events, such as chromosomal replication, remains surrounded by controversy. In this work, we use time-lapse microscopy to address two issues in B. subtilis cell division: the timing of divisome assembly, and the possibility that a periodic oscillation in expression of one or more genes essential for divisome assembly may play a role in defining the timing of cell division. To study the possibility of oscilation in gene expression, we have first built unstable variants of GFP by adding to its C-terminus peptide sequences that target the protein for degradation and used those variants to build transcriptional fusions to access the promoter activity of core cell division genes. After optimizing time-lapse conditions with transcriptional fusions to cell divison genes with the unstable GFPAISV, we observed that B. subtilis autofluorescence was an issue to our quantifications. To improve our signal-to-noise ratio, we built transcriptional fusions with two variants of YFP (Yellow Fluorescent Protein), and decided to work with Ypet. In our single-cell analysis for GFPAISV and for Ypet-AISV, only the ftsL operon promoter presented an oscilating pattern different from our negative control. This was not confirmed, however, when we attempted to correlate fluorescence signal with cell cycle position in static single-cell measurements. Thus, we conclude that that there are no fluctuations in ftsL, pbpB, minCD, ftsZ, ftsA or zapA gene expression throughout the cell cycle. To study divisome assembly we performed time-lapse microscopy of FtsZ-mCherry and Pbp2B-GFP, and determined that the association of Pbp2B occurs 3 minutes after FtsZ polymerization in rich medium and 4 minutes in minimal medium. We also performed time-lapse microscopy with FtsZ-YFP and DivIVA-CFP, determining that DivIVA is incorporated to the divisome in 16 minutes after FtsZ polymerization in rich medium and 20 minutes in minimal medium. This data confirms the assembly of the divisome in three steps rather than two, as previously proposed.