Transcriptional Analysis of Long-Term Adaptation ofYersinia enterocoliticato Low-Temperature Growth

Abstract

To analyze the transcriptional response of Yersinia enterocolitica cells to prolonged growth at low temperature, a collection of luxCDABE transposon mutants was cultivated in parallel at optimal (30°C) and suboptimal (10°C) temperatures and screened for enhanced promoter activities during growth until entering stationary phase. Among 5,700 Y. enterocolitica mutants, 42 transcriptional units were identified with strongly enhanced or reduced promoter activity at 10°C compared to 30°C, and changes in their transcriptional levels over time were measured. Green fluorescent protein fusions to 10 promoter regions confirmed the data. The temporal order of induction of the temperature-responsive genes of Y. enterocolitica was deduced, starting with the expression of cold shock genes cspA and cspB and the elevated transcription of a glutamate-aspartate symporter. Subsequently, cold-adapted cells drastically up-regulated genes encoding environmental sensors and regulators, such as UhpABC, ArcA, and methyl-accepting chemotaxis protein I (MCPI). Among the most prominent cold-responsive elements that were transcriptionally induced during growth in early and middle exponential phase are the insecticidal toxin genes tcaA and tcaB, as well as genes involved in flagellar synthesis and chemotaxis. The expression pattern of the lateexponential-to early-stationary-growth phase is dominated by factors involved in biodegradative metabolism, namely, a histidine ammonia lyase, three enzymes responsible for uptake and utilization of glycogen, the urease complex, and a subtilisin-like protease. Double-knockout mutants and complementation studies demonstrate inhibitory effects of MCPI and UhpC on the expression of a putative hemolysin transporter. The data partially delineate the spectrum of gene expression of Y. enterocolitica at environmental temperatures, providing evidence that an as-yet-unknown insect phase is part of the life cycle of this human pathogen.

Presence of a functional flagellar cluster Flag-2 and low-temperature expression of flagellar genes in Yersinia enterocolitica W22703

“…We recently reported the identification of luxCDABE insertions into the genome of Y. enterocolitica strain W22703 that resulted in increased light emission at 10 u C as compared to 30 u C (Bresolin et al, 2006a). Among mutants with strongly elevated light emission, we identified 21 clones harbouring transposon insertions in 12 different genes associated with motility and chemotaxis (Table 1; Fig.…”

“…2c), suggesting a putative role of the Flag-2 genes in virulence towards mammals. The temperature-dependent activity of enzymes involved in bioluminescence was also taken into account by using a correction factor derived from our own experimental data and the Arrhenius prediction (Bresolin et al, 2006a). This approach gave an optimal temperature of 15-20 u C for the expression of all genes investigated (data not shown).…”

“…In Y. enterocolitica, low temperature is a key environmental factor for the expression of several genes important for survival outside mammalian hosts (Bresolin et al, 2006a;Kapatral et al, 2004) as well as for the coordinate expression and assembly of the flagellar structure (Kapatral & Minnich, 1995;Kapatral et al, 2004;Rohde et al, 1994). So far, all functional flagellar genes of Y. enterocolitica have been found to be located in the common flagellar cluster 1 (Flag-1), which is also required for full virulence (Young et al, 2000).…”

“…Identification and characterization of transposon insertion sites was performed by inverse PCR (Bresolin et al, 2006a). Briefly, 400 ng chromosomal DNA of each transposon mutant was completely digested with ClaI, HindIII or SspI (Fermentas), enzymes were heat-inactivated and fragments were treated with T4 DNA ligase (Invitrogen) to allow self-ligation, resulting in circular molecules.…”

“…The chromosomal DNA was then precipitated with 500 ml 2-propanol, washed in ethanol, dried and dissolved in 500 ml TE buffer (10 mM Tris/HCl, 1 mM Na 2 EDTA, pH 7.4) containing 1 ml RNase (10 mg ml 21 ). Transposon mutagenesis using pUT mini-Tn5 luxCDABE Km2 and mating with E. coli S17.1 lpir as the donor strain has been described elsewhere (Bresolin et al, 2006a). PCRs were carried out with Taq polymerase (Fermentas) using the following programme: one cycle at 95 uC for 2 min; 30 cycles at 95 uC for 10 s, at the appropriate annealing temperature for 30 s, and at 72 uC for 45-180 s depending on the expected fragment length; one cycle at 72 uC for 10 min.…”