Browsing by Author "Williams, Joshua W."

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    Multi-tiered Regulation of luxR Provides Precise Timing and Maintenance of the Quorum Sensing Response of Vibrio fischeri
    Williams, Joshua W. (Virginia Tech, 2009-06-04)
    The quorum-sensing response of Vibrio fischeri involves a complex network of genes (encoding regulatory proteins as well as sRNAs), that govern host-association and production of bioluminescence. A key regulator of this system is LuxR, which is the transcriptional activator of the lux operon as well as several other genes in. LuxR also autoregulates its own transcription, which we have shown causes bistability and hysteresis in the quorum-sensing response. This behavior allows the system to maintain a stable and robust response in the face of environmental fluctuation or decreases in external autoinducer concentration caused by other sources. There are many factors that are known to regulate luxR expression, including the ArcA redox-responsive regulator, the cAMP-CRP secondary metabolism regulator, and components of the quorum-sensing pathway like LitR. Because of this, LuxR levels are critical in both the timing of quorum-sensing induction, as well as the maintenance of the response over time. This makes it a potential target for multiple levels of regulation in response to factors such as environmental and metabolic conditions, as well as other components of the quorum-sensing network. Another important global regulatory protein in V. fischeri (and most other species of Gram-negative proteobacteria) is the post-transcriptional regulator CsrA. CsrA controls processes involved in carbon storage and utilization, as well as the transition from exponential to stationary phase growth. We have demonstrated that CsrA is regulated by two sRNAs (CsrB1 and CsrB2) in V. fischeri. Because CsrA regulates changes in cell behavior and is an important metabolic regulator, there is a good possibility that it has some interactions with the quorum-sensing regulon, whose endproduct, bioluminescence, creates a large metabolic demand from the cell. In an effort to determine at which point in the quorum-sensing regulatory network CsrA regulation is important, epistasis experiments were designed using factorial design, which is a subset of statistical analysis of variance (ANOVA). This method was used to generate a high degree of confidence in the data, so that even minor interactions in the regulatory networks could be established. By altering the levels of CsrA expression in various mutant strains of V. fischeri, we have demonstrated that CsrA acts by an unknown mechanism to increase the transcription of luxR when the quorum-sensing regulator LitR is absent. Our results also demonstrated that CsrA mediates this effect through repression of ArcA activity, which is known to act directly on the luxR and luxI intergenic region as a repressor. This indicates that CsrA may bypass the upstream parts of the quorum-sensing regulatory cascade that lead to litR activation, so that LitR and LuxR may be regulated differently in response to certain conditions. This work has shown that the interactions between global regulons can coordinately control the amount of quorum-sensing induction by affecting the level of LuxR in the cell. The balance of these regulatory networks allows the cell to tightly regulate the quorum-sensing response. Thus, LuxR serves as a critical regulatory hub in the cell, at which multiple signals can be integrated in order to generate the appropriate cellular response.
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    Prediction of CsrA-regulating small RNAs in bacteria and their experimental verification in Vibrio fischeri
    Kulkarni, Prajna R.; Cui, Xiaohui; Williams, Joshua W.; Stevens, Ann M.; Kulkarni, Rahul V. (2006-01-01)
    The role of small RNAs as critical components of global regulatory networks has been highlighted by several recent studies. An important class of such small RNAs is represented by CsrB and CsrC of Escherichia coli, which control the activity of the global regulator CsrA. Given the critical role played by CsrA in several bacterial species, an important problem is the identification of CsrA-regulating small RNAs. In this paper, we develop a computer program (CSRNA_FIND) designed to locate potential CsrA-regulating small RNAs in bacteria. Using CSRNA_FIND to search the genomes of bacteria having homologs of CsrA, we identify all the experimentally known CsrA-regulating small RNAs and also make predictions for several novel small RNAs. We have verified experimentally our predictions for two CsrA-regulating small RNAs in Vibrio fischeri. As more genomes are sequenced, CSRNA_FIND can be used to locate the corresponding small RNAs that regulate CsrA homologs. This work thus opens up several avenues of research in understanding the mode of CsrA regulation through small RNAs in bacteria.