Global analysis of the transcriptional regulation of Sinorhizobium meliloti cell cycle progression and study of cell cycle regulation during symbiosis with Medicago sativa

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references.The complex [alpha]-proteobacterial cell cycle regulatory network is essential not only for faithful replication and segregation of the genome, but also to coordinate unique cellular differentiation events that have evolved as adaptations to the different lifestyles of this diverse group of bacteria. The soil-dwelling [alpha]-proteobacterium, Sinorhizobium meliloti, not only has to accurately coordinate the replication of its tripartite genome, but also must undergo a dramatic cellular differentiation in order to form an effective symbiosis with the legume Medicago sativa. Preliminary analyses have indicated that plasticity in the S. meliloti cell cycle regulatory network may be essential to symbiosis, but cell cycle research in S. meliloti has been hindered largely by lack of a method to obtain synchronous populations of S. meliloti. In this thesis, I present the first method to generate synchronous cultures of S. meliloti. I performed microarray gene expression analysis on synchronous populations of S. meliloti to gain a global view of transcriptional regulation of cell cycle events. This represents the first work of this kind done in an [alpha]-proteobacterium besides Caulobacter crescentus, which is the current model for [alpha]-proteobacterial cell cycle studies. The importance of transcriptional regulation of cell cycle progression was first discovered in C. crescentus and the work presented in this thesis highlights the conservation of cell cycle regulated gene expression in S. meliloti. I identified 462 cell cycle regulated transcripts in S. meliloti, which included genes involved in vital cell processes such as cell division, flagella biogenesis, replication and segregation of its tripartite genome as well as several putative cell cycle regulators. I compared the set of genes with cell cycle regulated transcripts identified in my analysis with the set identified in C. crescentus to generate a core set of 128 conserved genes demonstrating cell cycle regulated gene expression in both species. To determine which of the S. meliloti genes with cell cycle regulated transcripts might be part of the CtrA and DnaA regulons in S. meiloti, I performed CtrA and DnaA binding motif analysis. To understand the evolutionary significance of these CtrA and DnaA binding motifs, I looked at conservation of these motifs in homologous genes from several related [alpha]-proteobacteria. The results indicated that the putative CtrA regulon might be more evolutionarily constrained than the putative DnaA regulon. Organisms more closely related to S. meliloti or with more similar lifestyles demonstrated a much greater conservation of the CtrA binding motifs identified in S. meliloti. The CtrA binding motifs in S. meliloti identified by my analysis were not at all well conserved in C. crescentus, which was the most distantly related [alpha]-proteobacteria surveyed. These differences in cell cycle regulated transcription and the putative CtrA regulon between S. meliloti and C. crescentus thus appear to represent specific adaptations to the distinctive genome and unique intracellular symbiotic lifestyle of S. meliloti and illustrate the importance of S. meliloti as a model for cell cycle regulation in [alpha]-proteobacteria with similar intracellular lifestyles. The work presented in this thesis also describes the importance of CtrA regulation in S. meliloti during symbiosis with M. sativa. A crucial part of this symbiosis is a striking cellular differentiation (termed bacteroid differentiation), which includes changes in membrane permeability, cell elongation and branching, endoreduplication of the genome and loss of reproductive capacity and therefore a significant deviation from the free-living cell cycle program. Endoreduplication of the genome requires a decoupling of DNA replication and cell division, which could be achieved by down-regulation of the essential master cell cycle regulator CtrA. I tested the effects of CtrA depletion in S. meliloti and found that CtrA depletion induces a bacteroid-like state characterized by elongated and branched cells and highly elevated DNA content. I also show that S. meliloti CtrA has a comparable half-life to C. crescentus CtrA, but regulated proteolysis of CtrA may be different in the two species since we found CtrA proteolysis to be essential in S. meliloti. In addition, I demonstrate that the promoter and coding regions of C. crescentus ctrA cannot complement an S. meliloti ctrA chromosomal deletion during symbiosis even though they can do so in the free-living state. My attempts to identify the defects in the function C. cresentus ctrA promoter or coding region within M. sativa gave surprising results since S. melioti strains expressing C. crescentus CtrA from the S. meliloti ctrA promoter region and vice versa were able to establish an effective symbiosis with M. sativa. I discuss several possibilities to explain this apparent paradox, but further study is required to fully clarify this observation. Taken as a whole, my thesis work represents a significant advancement to the field of cell cycle research in S. meliloti and [alpha]-proteobacteria as a whole. The cell synchronization method I developed will greatly facilitate more comprehensive analysis of cell cycle regulation in S. meliloti. My microarray gene expression analysis provides a global view of cell cycle regulated transcription in S. meliloti, which can be used in more in-depth explorations of specific mechanisms of transcriptional regulation of cell cycle events in S. meliloti. Lastly, my study of CtrA function in S. meliloti establishes the importance of CtrA regulation during symbiosis with M. sativa.by Nicole J. De Nisco.Ph.D

The DivJ, CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti

Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis.National Institutes of Health (U.S.) (Grant GM31010

Cell Cycle Control by the Master Regulator CtrA in Sinorhizobium meliloti

In all domains of life, proper regulation of the cell cycle is critical to coordinate genome replication, segregation and cell division. In some groups of bacteria, e.g. Alphaproteobacteria, tight regulation of the cell cycle is also necessary for the morphological and functional differentiation of cells. Sinorhizobium meliloti is an alphaproteobacterium that forms an economically and ecologically important nitrogen-fixing symbiosis with specific legume hosts. During this symbiosis S. meliloti undergoes an elaborate cellular differentiation within host root cells. The differentiation of S. meliloti results in massive amplification of the genome, cell branching and/or elongation, and loss of reproductive capacity. In Caulobacter crescentus, cellular differentiation is tightly linked to the cell cycle via the activity of the master regulator CtrA, and recent research in S. meliloti suggests that CtrA might also be key to cellular differentiation during symbiosis. However, the regulatory circuit driving cell cycle progression in S. meliloti is not well characterized in both the free-living and symbiotic state. Here, we investigated the regulation and function of CtrA in S. meliloti. We demonstrated that depletion of CtrA cause cell elongation, branching and genome amplification, similar to that observed in nitrogen-fixing bacteroids. We also showed that the cell cycle regulated proteolytic degradation of CtrA is essential in S. meliloti, suggesting a possible mechanism of CtrA depletion in differentiated bacteroids. Using a combination of ChIP-Seq and gene expression microarray analysis we found that although S. meliloti CtrA regulates similar processes as C. crescentus CtrA, it does so through different target genes. For example, our data suggest that CtrA does not control the expression of the Fts complex to control the timing of cell division during the cell cycle, but instead it negatively regulates the septum-inhibiting Min system. Our findings provide valuable insight into how highly conserved genetic networks can evolve, possibly to fit the diverse lifestyles of different bacteria

The DivJ, CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti

Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C.crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S.meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis

Recurrent urinary tract infection and estrogen shape the taxonomic ecology and function of the postmenopausal urogenital microbiome

Article describes how postmenopausal women are severely affected by recurrent urinary tract infection (rUTI). The authors perform shotgun metagenomics and advanced culture on urine from a controlled cohort of postmenopausal women to identify urogenital microbiome compositional and function changes linked to rUTI susceptibility

Label Free, Lateral Flow Prostaglandin E2 Electrochemical Immunosensor for Urinary Tract Infection Diagnosis

A label-free, rapid, and easy-to-use lateral flow electrochemical biosensor was developed for urinary tract infection (UTI) diagnosis in resource challenged areas. The sensor operates in non-faradaic mode and utilizes Electrochemical Impedance Spectroscopy for quantification of Prostaglandin E2, a diagnostic and prognostic urinary biomarker for UTI and recurrent UTI. To achieve high sensitivity in low microliter volumes of neat, unprocessed urine, nanoconfinement of assay biomolecules was achieved by developing a three-electrode planar gold microelectrode system on top of a lateral flow nanoporous membrane. The sensor is capable of giving readouts within 5 min and has a wide dynamic range of 100–4000 pg/mL for urinary PGE2. The sensor is capable of discriminating between low and high levels of PGE2 and hence is capable of threshold classification of urine samples as UTI positive and UTI negative. The sensor through its immunological response (directly related to host immune response) is superior to the commercially available point-of-care UTI dipsticks which are qualitative, have poor specificity for UTI, and have high false-positive rates. The developed sensor shows promise for rapid, easy and cost-effective UTI diagnosis for both clinical and home-based settings. More accurate point-of-care UTI diagnosis will improve patient outcomes and allow for timely and appropriate prescription of antibiotics which can subsequently increase treatment success rates and reduce costs

Global analysis of cell cycle gene expression of the legume symbiont Sinorhizobium meliloti

In α-proteobacteria, strict regulation of cell cycle progression is necessary for the specific cellular differentiation required for adaptation to diverse environmental niches. The symbiotic lifestyle of Sinorhizobium meliloti requires a drastic cellular differentiation that includes genome amplification. To achieve polyploidy, the S. meliloti cell cycle program must be altered to uncouple DNA replication from cell division. In the α-proteobacterium Caulobacter crescentus, cell cycle-regulated transcription plays an important role in the control of cell cycle progression but this has not been demonstrated in other α-proteobacteria. Here we describe a robust method for synchronizing cell growth that enabled global analysis of S. meliloti cell cycle-regulated gene expression. This analysis identified 462 genes with cell cycle-regulated transcripts, including several key cell cycle regulators, and genes involved in motility, attachment, and cell division. Only 28% of the 462 S. meliloti cell cycle-regulated genes were also transcriptionally cell cycle-regulated in C. crescentus. Furthermore, CtrA- and DnaA-binding motif analysis revealed little overlap between the cell cycle-dependent regulons of CtrA and DnaA in S. meliloti and C. crescentus. The predicted S. meliloti cell cycle regulon of CtrA, but not that of DnaA, was strongly conserved in more closely related α-proteobacteria with similar ecological niches as S. meliloti, suggesting that the CtrA cell cycle regulatory network may control functions of central importance to the specific lifestyles of α-proteobacteria.National Institutes of Health (U.S.) (Grant GM31010)National Institutes of Health (U.S.) (Grant P30 ES002109)National Cancer Institute (U.S.) (Award P30 CA14051

DigEST: Digital plug‐n‐probe disease Endotyping Sensor Technology

Abstract In this work, we propose a novel diagnostic workflow—DigEST—that will enable stratification of disease states based on severity using multiplexed point of care (POC) biosensors. This work can boost the performance of current POC tests by enabling clear, digestible, and actionable diagnoses to the end user. The scheme can be applied to any disease model, which requires time‐critical disease stratification for personalized treatment. Here, urinary tract infection is explored as the proof‐of‐concept disease model and a four‐class classification of disease severity is discussed. Our method is superior to traditional enzyme‐linked immunosorbent assay (ELISA) as it is faster and can work with multiple disease biomarkers and categorize diseases by endotypes (or disease subtype) and severity. To map the nonlinear nature of biochemical pathways of complex diseases, the method utilizes an established supervised machine learning model for digital classification. This scheme can potentially boost the diagnostic power of current electrochemical biosensors for better precision therapy and improved patient outcomes