Regulatory characteristics of the vibrio vulnificus global regulators, CRP, RpoS, and Fur, essential for its survival and pathogenesis
저자
발행사항
서울 : 韓國外國語大學校 大學院, 2008
학위논문사항
학위논문(박사)-- 韓國外國語大學校 大學院 : 환경학과 2008. 8
발행연도
2008
작성언어
영어
주제어
DDC
589.9
발행국(도시)
서울
형태사항
219 p. : 삽도 ; 26 cm.
일반주기명
지도교수: Lee, Kyu-Ho
참고문헌 수록
소장기관
In pathogenic bacterium, the ability to acquire iron, which is mainly regulated by the ferric uptake regulator (Fur), is essential to maintain growth as well as its virulence. In Vibrio vulnificus, a human pathogen causing gastroenteritis and septicemia, fur gene expression is positively regulated by Fur when the iron concentration is limited (Lee et al., 2003. J. Bacteriol. 185:5891-5896). Footprinting analysis revealed that an upstream region of the fur gene was protected by Fur protein from DNase I under iron-depleted conditions. The protected region, from -142 to -106 relative to the transcription start site of the fur gene, contains distinct AT-rich repeats. Mutagenesis of this repeated sequence resulted in abolishment of binding by Fur. To confirm the role of this cis-acting element in Fur-mediated control of its own gene in vivo, fur expression was monitored in V. vulnificus strains using a transcriptional fusion containing the mutagenized Fur-binding site (furmt::luxAB). Expression of furmt::luxAB showed that it was not regulated by Fur and was not influenced by iron concentration. Therefore, this study demonstrates that V. vulnificus Fur acts as a positive regulator under iron-limited conditions by direct interaction with the fur upstream region.
Bacterial ability to acquire iron is essential to maintain its growth as well as to elicit its virulence, which is regulated by a transcriptional regulator, ferric uptake regulator (Fur). In the previous report (H.?J. Lee et al. 2007 J Bacteriol 189:2629), fur gene expression in Vibrio vulnificus was shown to be activated by a direct binding of iron-free Fur to the upstream region of the fur gene, which includes two direct repeats of 5'-AAATTGT-3'. These finding led us to question if this novel Fur-binding site is unique in autoregulation of fur expression, or it is widely distributed in V. vulnificus genome. Thus, in this study, the V. vulnificus-DNA microarray was utilized to define the Fur-regulon. Total 188 orfs were differentially expressed in ?fur; expression of 38 orfs were significantly reduced in ?fur, and expression of 150 orfs were significantly increased in ?fur. Then, the upstream regions of those orfs were subjected to in silico analysis for the presence of the direct repeats of 5'-AAATTGT-3? or the presence of the classical Fur-box (5?-GATAATGATAATCATTATC-3?). To verify the role of this novel Fur-binding site sequence, 8 genes were chosen for further study. The primer extension assays were performed to identify the promoter regions of the selected genes. Then, transcription fusions including the putative regulatory region for each gene were constructed. Fusion assays showed that expression of each fusion was consistent with the expression patterns determined by DNA microarray experiments. In addition, the putative novel Fur-binding sites were mutagenized and then used for construction of transcriptional fusions. Expression of the mutagenized fusions containing Fur-activated genes were no longer regulated by Fur or an iron chelator, 2, 2'-dipyridyl. However, the fusions containing Fur-repressive genes were not affected by mutation of the sequences homologous the direct repeats of AAATTGT. Instead, these Fur-repressive genes includes the classical Fur-binding sites in their promoter regions. Therefore, the direct repeats of 5'-AAATTGT-3' appears to be the consensus Fur-binding site which is involved in activation by an iron-free Fur.
In the natural environment, microorganisms are often challenged by exposure to various forms of physical stress, including oxidative stresses, osmotic shock, temperature shock, and iron or nutrient starvation. Under these conditions, bacteria turn on expression of certain genes that allow them to cope with the given stresses. One of the effective mechanisms for bacteria to bring about such a major switch in gene expression is a selective utilization of alternative sigma subunits that alter the specificity of core RNA polymerase to promoters of target genes or operons (Ishihama, 2000).
RpoS (?S) that is a subunit of RNA polymerase complex, is involved in gene expression for responses to general stress and the stationary phase, and is highly conserved in bacteria belonging to the ?-subdivision of Proteobacteria. Recent studies demonstrated that expression of rpoS- and ?S-dependent genes are not only induced at the stationary phase but also induced during the exponential phase for responses to diverse stresses (Hengge-Aronis, 1996; Hengge-Aronis et al., 1993; Lee et al., 1995; Loewen and Hengge-Aronis, 1994; Muffler et al., 1997; Muffler et al., 1996). Although ?S protein levels are low during the exponential phase, relatively high levels of rpoS mRNA are present and do not seem to change in response to several stresses that actually result in strongly elevated levels of ?S protein. Therefore, extensive investigation into the mechanism of translational control of rpoS and proteolysis of ?S has been conducted. There are also several studies that have investigated transcriptional regulation of the rpoS gene.
Interestingly, the ferric uptake regulator, Fur, appears to be part of the RpoS regulon (Lee et al., 2003). The Fur protein is a 17-kDa polypeptide that acts as a transcriptional regulator of iron-regulated promoters by Fe2+-dependent DNA binding activity (Bagg and Neilands, 1987). Fur appears to be an abundant protein, which acts as a multimer through protein-protein interactions. The Fur protein can perform multiple functions including regulation, activation, and repression according to iron availability. In some Gram-negative bacteria, the Fur repressor is known to be involved in autoregulation in response to iron (Delany et al., 2002; Delany et al., 2003; Sala et al., 2003).
Vibrio vulnificus, a septicemia-causing pathogenic bacterium, has been found to acquire resistance against various stresses and to elicit expression of virulence factors via a rpoS gene product. In this study, we investigated the transcriptional characteristics of this global regulator. Two distinct transcriptional initiation sites for the rpoS gene, the proximal promoter (Pp) and the distal promoter (Pd), were defined by Northern blot and primer extension experiments. The luxAB-transcriptional fusions containing various lengths of the rpoS upstream region indicated that Pd is a major promoter for the rpoS expression. Western blot analysis showed that RpoS amounts were inversely correlated with the intracellular levels of 3',5'-cyclic monophosphate (cAMP). The expressions from both Pd and Pp were increased in the cya or the crp mutants. An exogenous addition of cAMP to the cya mutant resulted in repressed expression of rpoS. In addition, rpoS expression was significantly lowered in the cpdA mutant in which the level of cAMP was elevated due to a deficiency in 3',5'-cAMP phosphodiesterase. cAMP-CRP complex was shown to bind to two rpoS promoters by electrophoretic mobility shift assays. The alteration of the putative CRP-binding site on each rpoS promoter, via a site-directed mutagenesis, abolished the binding of cAMP-CRP as well as the regulation by cAMP-CRP.
Therefore, this study clearly shows a relationship between the intracellular cAMP level and the degree of rpoS expression, and further demonstrates, for the first time, the direct binding of cAMP-CRP complex to rpoS upstream regions, which results in repression of rpoS gene expression.
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