A Consensus Sequence for Binding of SmcR, a Vibrio vulnificus LuxR Homologue, and Genome-wide Identification of the SmcR Regulon

Abstract

Quorum sensing has been implicated as an important global regulatory system controlling the expression of numerous virulence factors in bacterial pathogens. In the present study, DNA targets of SmcR, a Vibrio vulnificus LuxR homologue, were selected from a random pool of DNA fragments by using a cycle selection procedure consisting of in vitro DNA-SmcR interaction, purification of SmcR-DNA complexes, and PCR amplification of SmcR-bound DNA. The amplified DNA fragments were cloned and analyzed separately by electrophoretic mobility shift assay to verify the specific binding of SmcR to the DNA. The DNA sequences bound by SmcR were determined by DNase I footprinting, and alignment of the resulting 29 sequences revealed a 22-bp consensus SmcR-binding sequence, 5-TTAT-TGATWWRWTWNTNAATAA-3 (where W represents A or T, R is G or A, and N is any nucleotide), with an 8-bp (TTATT-GAT) inverted repeat. The consensus sequence revealed greater efficiency for the binding of SmcR than the SmcR-binding sequence previously identified within P vvpE . Mutational analysis demonstrated that the 9th and 10th bases from the center are the most essential for SmcR binding. A genome-wide search using the consensus sequence predicted that at least 121 genes are under the control of SmcR, and 10 of these newly identified SmcR regulon members were verified as being regulated by SmcR in V. vulnificus as well as in vitro. The consensus sequence and newly identified genes should be of use for elucidating the regulatory mechanism of SmcR and provide further insight into the role of the quorum sensing in V. vulnificus pathogenesis.Bacterial pathogenicity is multifactorial and a complex phenomenon that involves the products of many genes, collectively called virulence factors (1). Expression of many of these virulence factors is coordinately controlled by a common global regulatory system in response to environmental signals. This coordinate regulation facilitates cooperation of the virulence factors and is crucial for the overall success of the infectious microorganisms during pathogenesis (2). Many bacteria monitor their cell population densities through the exchange of diffusible signal molecules (autoinducer (AI) 4 ) that accumulate extracellularly (for recent reviews, see Refs. 3 and 4). This type of communication, termed quorum sensing, has been recognized as a global regulatory system controlling the expression of numerous virulence factors in bacterial pathogens (for recent reviews, see Refs. 5 and 6). When the concentration of the various AIs increases to critical levels, a signal transduction cascade triggered through cognate receptors alters the expression of over 50 genes or operons (7).The cell density-dependent regulation of bioluminescence in Vibrio harveyi is frequently used as a model for quorum sensing. V. harveyi LuxR is the transcriptional activator of the luminescence operon, and its synthesis is controlled by the levels of three autoinducers, AI-1, AI-2, and CAI-1 (4). To date, homologues of LuxR, which are postulated to regu...

Iron and Quorum Sensing Coordinately Regulate the Expression of Vulnibactin Biosynthesis in Vibrio vulnificus

“…In this study, we showed that SmcR represses the transcription of the vvs genes by directly binding to the promoter region. The SmcR binding site (5Ј-AATGAT-GTTAATGATAATTATT-3Ј) in the vvsA promoter is a mirror image of the known SmcR binding consensus sequence (5Ј-TTATTGATWWRWTWNTNAATAA-3Ј) (18). The vvsA site has a 22-bp inverted repeated sequence just like the consensus sequence, suggesting that the size and the inverted repeat feature may be more important for SmcR binding than the base pairs per se.…”

“…5B), indicating that binding of SmcR to the vvsA promoter is specific. A 22-bp consensus sequence for SmcR binding has been proposed based on examination of promoter regions of V. vulnificus genes directly regulated by SmcR (5Ј-TTATTGATWWR-WTWNTNAATAA-3Ј; W represents A or T, R represents G or A, N represents any nucleotide) (18). However, a similar sequence is not apparent in the vvsA promoter region.…”

“…The signal is funneled to the regulator SmcR, the homolog of LuxR in V. harveyi (15), which subsequently modulates the expression of target genes such as vvpE and vvpH, encoding metalloprotease and hemolysin, respectively (14,16,17). A 22-bp consensus SmcR binding sequence was identified in the upstream regions of target genes: 5Ј-TTATTGATWWRWTWNTNAATAA-3Ј (where W represents A or T, R is G or A, and N is any nucleotide) (18).…”

Quorum Sensing and Silencing in Vibrio parahaemolyticus

“…HapR regulates approximately 4.1% of the V. cholerae El Tor genome (73). For V. vulnificus, a 121-gene SmcR regulon has been predicted by using a consensus binding sequence that was generated by DNA footprinting assays (35). A screen of random genomic V. harveyi DNA fused to promoterless green fluorescent protein (GFP) identified 71 autoinducer-regulated genes, all of which were LuxR dependent (64).…”

AphA and LuxR/HapR reciprocally control quorum sensing in vibrios

“…We made progress in determining how V. harveyi LuxR controls gene expression by defining its DNA-binding site and decoding its regulon (Waters and Bassler 2006;Lee et al 2008;Pompeani et al 2008), but we understand comparatively little about AphA in V. harveyi. However, the mechanisms of AphA repression and activation have received attention in V. cholerae, which should guide our continued analyses (Kovacikova and Skorupski 2001;Kovacikova et al 2003Kovacikova et al , 2004Kovacikova et al , 2005Kovacikova et al , 2010De Silva et al 2005;Yang et al 2009).…”