The Yersinia enterocolitica pYV virulence plasmid contains multiple intrinsic DNA bends which melt at 37°C

Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, Idaho 83843, USA.
Journal of Bacteriology (Impact Factor: 2.69). 07/1999; 181(14):4198-204.
Source: PubMed

ABSTRACT Temperature has a pleiotropic effect on Yersinia enterocolitica gene expression. Temperature-dependent phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis (</=30 degrees C) and virulence plasmid-encoded Yop secretion (37 degrees C). The mechanism by which temperature exerts this change in genetic programming is unclear; however, altered gene expression by temperature-dependent changes in DNA topology has been implicated. Here, we present evidence that the Y. enterocolitica virulence plasmid, pYV, undergoes a conformational transition between 30 and 37 degrees C. Using a simplified two-dimensional, single-gel assay, we show that pYV contains multiple regions of intrinsic curvature, including virF, the positive activator of virulence genes. These bends are detectable at 30 degrees C but melt at 37 degrees C, the temperature at which the cells undergo phenotypic switching. We also show that pACYC184, a plasmid used as a reporter of temperature-induced changes in DNA supercoiling, has a single region of intrinsic bending detected by our assay. Topoisomers of pACYC184, with and without this bend, isolated from Y. enterocolitica were resolved by using chloroquine gels. The single bend has a dramatic influence on temperature-dependent DNA supercoiling. These data suggest that the Y. enterocolitica pYV plasmid may undergo a conformational change at the host temperature due to melting of DNA bends followed by compensatory adjustments in superhelical density. Hence, changes in DNA topology may be the temperature-sensing mechanism for virulence gene expression in Y. enterocolitica and other enteric pathogens.

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Available from: Scott A Minnich, Sep 08, 2014
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    • "In some cases, temperature is directly sensed through DNA: for example, in the enteropathogen Shigella, a temperature-dependent change in local DNA topology affects the transcriptional control exerted by the H-NS regulator of the virF promoter region, that drives the expression of the first positive activator of the virulence cascade only at permissive temperature (T > 32°C) (Falconi et al., 1998; Prosseda et al., 2004). A strict correlation between DNA curvature and thermoregulated expression has also been reported in other microorganisms , like Yersinia pestis, E. coli, and Clostridium perfringens (Katayama et al., 1999; Rohde et al., 1999; Madrid et al., 2002). (iv) In a very few cases, the transcriptional regulators themselves behave as intrinsic heat-sensing protein thermometers: they change conformation and, therefore, their relative binding affinity for DNA, according to the temperature, thereby affecting transcription of genes belonging to their regulon. "
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    ABSTRACT: Bacteria exploit different strategies to perceive and rapidly respond to sudden changes of temperature. In Helicobacter pylori the response to thermic stress is transcriptionally controlled by a regulatory circuit that involves two repressors, HspR and HrcA. Here we report that HrcA acts as a protein thermometer. We demonstrate that temperature specifically modulates HrcA binding to DNA, with a complete and irreversible temperature-dependent loss of DNA binding activity at 42°C. Intriguingly, although the reduction of HrcA binding capability is not reversible in vitro, transcriptional analysis showed that HrcA exerts its repressive influence in vivo, even when the de novo repressor synthesis is blocked after the temperature challenge. Accordingly, we demonstrate the central role of the chaperonine GroESL in restoring the HrcA binding activity, lost upon heat challenge. Together our results establish HrcA as a rare example of intrinsic temperature sensing transcriptional regulator, whose activity is posttranscriptionally modulated by the GroESL chaperonine.
    Molecular Microbiology 04/2014; DOI:10.1111/mmi.12600 · 5.03 Impact Factor
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    • "When combined with the results shown in Figure 2, these data provide clear indication that the Ysa T3SS is expressed in a contact dependent manner during infection of HeLa cells. In contrast to yspP, yopH appears to have similar expression levels in both spent DMEM and during HeLa cell infection, which would appear to contradict the results shown in Figure 2. It should be noted, however, that expression of the Ysc T3SS has been shown to be induced at 37°C, due to changes in the secondary structure of the virulence plasmid (Rohde et al. 1999) and the ribosome-binding site of the transcriptional activator LcrF (Bohme et al. 2012) at that temperature. "
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    ABSTRACT: Yersinia enterocolitica biovar 1B maintains two type III secretion systems (T3SS) that are involved in pathogenesis, the plasmid encoded Ysc T3SS and the chromosomally encoded Ysa T3SS. In vitro, the Ysa T3SS has been shown to be expressed only at 26°C in a high-nutrient medium containing an exceptionally high concentration of salt - an artificial condition that provides no clear insight on the nature of signal that Y. enterocolitica responds to in a host. However, previous research has indicated that the Ysa system plays a role in the colonization of gastrointestinal tissues of mice. In this study, a series of Ysa promoter fusions to green fluorescent protein gene (gfp) were created to analyze the expression of this T3SS during infection. Using reporter strains, infections were carried out in vitro using HeLa cells and in vivo using the mouse model of yersiniosis. Expression of green fluorescent protein (GFP) was measured from the promoters of yspP (encoding a secreted effector protein) and orf6 (encoding a structural component of the T3SS apparatus) in vitro and in vivo. During the infection of HeLa cells GFP intensity was measured by fluorescence microscopy, while during murine infections GFP expression in tissues was measured by flow cytometry. These approaches, combined with quantification of yspP mRNA transcripts by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), demonstrate that the Ysa system is expressed in vitro in a contact-dependent manner, and is expressed in vivo during infection of mice.
    12/2013; 2(6). DOI:10.1002/mbo3.136
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    • "Consistent with this hypothesis is the demonstration that thermo-induced Yop expression coincided with variations of pYV DNA supercoiling (Rohde et al., 1994). Five years later, the same authors showed that these dramatic topological changes could be initiated by the melting of local DNA bends as a consequence of a temperature shift from 30°C to 37°C (Rohde et al., 1999). However, in contrast to the ysc genes involved in the synthesis of the Yop secretion apparatus itself, transcription of most yops– as well as other pYV-borne genes such as ylpA yadA, sycE (Skurnik and Toivanen, 1992; Wattiau and Cornelis, 1993) and the virC operon (Michiels et al., 1991)– has been shown to require an additional regulator: LcrF (Yother et al., 1986; Lambert de Rouvroit et al., 1992). "
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    ABSTRACT: In response to the ever-present need to adapt to environmental stress, bacteria have evolved complex (and often overlapping) regulatory networks that respond to various changes in growth conditions, including entry into the host. The expression of most bacterial virulence factors is regulated; thus the question of how bacteria orchestrate this process has become a recurrent research theme for every bacterial pathogen, and the three pathogenic Yersinia are no exception. The earliest studies of regulation in these species were prompted by the characterization of plasmid-encoded virulence determinants, and those conducted since have continued to focus on the principal aspects of virulence in these pathogens. Most Yersinia virulence factors are thermally regulated, and are active at either 28 degrees C (the optimal growth temperature) or 37 degrees C (the host temperature). However, regulation by this omnipresent thermal stimulus occurs through a wide variety of mechanisms, which generally act in conjunction with (or are modulated by) additional controls for other environmental cues such as pH, ion concentration, nutrient availability, osmolarity, oxygen tension and DNA damage. Yersinia's recent entry into the genome sequencing era has given scientists the opportunity to study these regulators on a genome-wide basis. This has prompted the first attempts to establish links between the presence or absence of regulatory elements and the three pathogenic species' respective lifestyles and degrees of virulence.
    Current issues in molecular biology 08/2005; 7(2):151-77. · 6.00 Impact Factor
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