Low temperature-induced insecticidal activity of Yersinia enterocolitica

Sequence analysis of a genomic island of Y. enterocolitica W22703

A transposon mutant library was established in Y. enterocolitica W22703 using the plasmid pUT mini-Tn5 luxCDABE Km2. Two independent mutants exhibiting higher levels of light emission at 10°C were identified. The insertion point of the transposon was determined by inverse polymerase chain reaction (PCR). The translated nucleotide sequences flanking the mini-Tn5 insertion site of both mutants showed significant similarities to parts of the Tc proteins of P. luminescence and Y. pestis. Surprisingly, no homologies to the genome sequence of Y. enterocolitica strain 8081v were found. Starting from the two transposon insertion sites of the mutants W22703-tcaA(134)::Tn5lux and W22703-tcaB1(152)::Tn5lux, the surrounding chromosomal region of 20 403 bp was sequenced as described in Experimental procedures. When compared with the genome sequence of strain 8081v, the left hand junction of the nucleotide sequence revealed to be identical to open reading frames (ORFs) YE3795, YE3796 and YE3797, encoding a HlyD family secretion protein, a putative lipoprotein and a putative LysR-like transcriptional regulatory protein respectively. To define the 3′-end of tc-PAI^Ye, we amplified and sequenced a fragment of approximately 3 kb comprising the last 462 nucleotides of tccC and the last 519 nucleotides of tldD (YE3798) encoding a putative DNA gyrase modulator. The sequence data show that the strain-specific region had been inserted between ORFs YE3797 and YE3798 with respect to the 8081v genome. The annotation of the whole nucleotide sequence revealed 10 strain-specific ORFs that are schematically represented in Fig. 1. The ORFs were named according to the similarity of their amino acid sequences to the proteins encoded by the toxin genes tcaA, tcaB, tcaC and tccC from P. luminescens and Yersinia spp. as outlined in Table 1. According to a sequence deletion of approximately 200 bp as compared with Y. pestis, the tcaB locus is divided into two ORFs that were therefore named tcaB1 and tcaB2. Five genes, tcaA, tcaB1, tcaB2, tcaC and tccC, encode conserved proteins that belong to the three TcaAB-like, TcaC-like and TccC-like insect toxin elements. The genes coding for TcaABC-like and the TccC-like elements identified here are separated by ORFs encoding two phage-related proteins and a putative exported protein. tccC is followed by approximately 1500 bp containing ORF11 that encodes a hypothetical protein. The gene tcaA is preceded by two genes showing homologies to putative LysR-like regulators. Due to the potential role of these proteins in the regulation of tc-like genes, we provisionally termed the two ORFs tcaR1 and tcaR2. The tcaC sequence shows a frameshift at nucleotide 314 that introduces a stop codon at the position indicated in Fig. 1, but the expression of a functional C-domain of 1433 amino acids cannot be excluded. The frameshift is not present, however, in the respective region of the Y. enterocolitica strains NCTC10460 and H270/78, but was also found in H324/78. No similarities to tRNAs, integrases or IS elements could be found. The strain-specific sequence has an average G+C composition of 45.4% which is slightly lower than that of the genome sequence of strain 8081v (47.3%). We propose to name the 19 kb region that spans from tcaR1 to ORF11, the tc-like pathogenicity island of Y. enterocolitica, abbreviated tc-PAI^Ye.

Distribution of homologous tc genes in Y. enterocolitica biovars

The complete tc-PAI^Ye sequence was obtained from Y. enterocolitica strain W22703 and no nucleotide similarities to the genome sequence of strain 8081v were found, with the exception of the putative regulator gene tcaR1. To gain further information on the distribution of tc-PAI^Ye and tc-like genes in five biogroups of Y. enterocolitica, we subjected a total of 29 strains to PCR analysis using primers specific for tcaA, tcaB1, tcaB2 and tcaC. The strains had been isolated from humans, the environment, unprocessed food or domestic animals. The results shown in Table 2 indicate that all four toxin genes are present in biovars 2–4. Interestingly, no PCR fragments were obtained from chromosomal DNA of biovars 1A and 1B. The observation of a biovar-specific distribution of genes encoding Tcs was further confirmed by Southern blot analysis under standard and low-stringency conditions using a DNA probe amplified from tcaB1. The respective 428 bp fragment is highly conserved between Y. enterocolitica, Y. pseudotuberculosis and P. luminescens. This fragment hybridized to chromosomal DNA of all tested Y. enterocolitica biovars 2–4, as well as to Y. pseudotuberculosis and P. luminescens (data not shown), but not to DNA of biovars 1A and 1B, demonstrating the presence of a homologue of tcaB1 in the respective strains (Fig. 2). No hybridization of the tcaB1 probe to chromosomal DNA of the non-pathogenic species Yersinia ruckeri, Y. kristensenii, Y. frederiksenii, Y. intermedia and Y. aldovae was observed.

Low temperature-dependent transcription of tcaA

Mutant W22703-tcaA(134)::Tn5lux was initially identified by screening a mutant library for genes with upregulated expression during growth at 10°C compared with 30°C. To gain a further insight into the temperature-dependent expression of the tc genes of Y. enterocolitica, the transcriptional activity of tcaA at different temperatures was investigated. Cultures of W22703-tcaA(134)::Tn5lux were grown overnight at 30°C, diluted as described and incubated at 10°C, 15°C, 20°C, 25°C, 30°C and 37°C. Bioluminescence was measured over all growth phases until cells reached stationary phase (Fig. 3A). No light emission above background was observed during growth at 37°C, suggesting a complete block of tcaA expression at this temperature. Induction of luciferase activity as a reporter for tcaA transcription, however, was observed when the growth temperature was gradually decreased to 10°C. The absolute, relative light units (RLU) at optical density at 405 nm (OD₄₀₅) of ∼1.0 ranged from 2742 (30°C) to 54 158 (25°C) and 341 912 (15°C), indicating a striking increase of tcaA expression at temperatures below 30°C. By relating RLU to the growth phases (RLU/OD₄₀₅), initial induction of bioluminescence was observed at OD₄₀₅ of 0.2 at 15°C, and transcriptional activity steadily increased until cells entered the stationary phase. A similar expression profile was derived from mutant W22703-tcaB1(152)::Tn5lux (data not shown).

As the activity of an enzyme decreases with lower temperatures, the ‘real’ light emission can be determined as the product of the RLU value and a temperature-dependent factor. To determine this factor with respect to the assumed temperature optimum of the luciferase at 37°C, 76 independent mutants that had not shown temperature-dependent luxCDABE transcription were taken from the transposon library, and their light emission was measured at different temperatures. For each factor, the mean of five RLU values at four different time points was divided by the respective OD₄₀₅ values, resulting in the approximated correction factors 1.6, 2.5, 3.1, 5.1 and 19.7 at 30°C, 25°C, 20°C, 15°C and 10°C. A temperature optimum of 10–15°C for tcaA transcription was determined by multiplication of the respective correction factors with the RLU of cells reaching the stationary phase at OD₄₀₅ of ∼1.1 (Fig. 3B). The logarithm of the quotient of the corrected RLU values at 10°C (2142 493) and 37°C (52) is 4.62, indicating that the expression of tcaA increased by more than four orders of magnitude when Y. enterocolitica was grown at low temperature compared with mammalian body temperature.

Insecticidal activity

As model organisms to test for insecticidal activity M. sexta and Pieris brassicae were selected. M. sexta was chosen as the tc-PAI^Ye genes showed greatest similarity to the Tc elements of P. luminescens that are active against this insect. X. nematophilus and P. brassicae combination served as a highly sensitive control. The culture supernatants or cell extracts recovered from Y. enterocolitica cultures grown at 30°C did not show insecticidal activity to M. sexta above that of the negative control. However, cell extracts from the two Y. enterocolitica strains grown at 10°C showed a significant level of insecticidal activity against M. sexta, with a slightly lower activity of NCTC10460 compared with strain W22703. This effect might rather be the result of sequence variations within the genes encoding the enzymatically active toxins, than of the frameshift in tcaC. The culture supernatants from the strains grown at 10°C and 30°C did not show insecticidal activity. As expected, culture supernatants and cell extracts of X. nematophilus displayed good insecticidal activity towards P. brassicae, and no detectable insecticidal activity towards M. sexta was observed. Wild-type strain 8081v that lacks tc-PAI^Ye and any tc-like gene in its genome showed no insecticidal activity towards either of the insect tested. When the mutants W22703-tcaA(134)::Tn5lux and W22703-tcaB1(152)::Tn5lux were tested for activity against M. sexta, the results indicated that insertional knockout of tcaA, but not tcaB1, resulted in the loss of insecticidal activity when extracts were gained from 10°C cultures (Table 3). As the transposon unit contains two putative transcriptional terminators downstream of luxCDABE and the kanamycin resistance cassette, polar effects of an insertion in tcaA on genes within a putative operon could not be excluded. Therefore, a non-polar tcaA deletion mutant was constructed that showed no insecticidal activity in the bioassay. The in trans complementation of tcaA restored the wild-type insecticidal activity. In each case, we used strains transformed with plasmid pACYC184 to confirm that the observed effects are unequivocally a result of the cloned fragment. Table 3 summarizes these data. Taken together, tcaA, but not tcaB, is essential for the observed toxic activity of Y. enterocolitica strains against M. sexta larvae.

Bacterial strains, plasmids and growth conditions

Bacterial strains used in this study were taken from the strain collection of the Abteilung Mikrobiologie, ZIEL, Weihenstephan, or have been obtained from the Institut für Hygiene und Umwelt, Hamburg, Germany. The sources of other strains and plasmids are indicated in Table 4. All cultures were grown in Luria–Bertani (LB) broth (10 g l⁻¹ tryptone, 5 g l⁻¹ yeast extract, 5 g l⁻¹ NaCl), or on LB agar (LB broth supplemented with 1.5% agar). E. coli was grown at 37°C, and X. nematophilus and Y. enterocolitica at 30°C or as indicated. If necessary, kanamycin (50 µg ml⁻¹), streptomycin (50 µg ml⁻¹), tetracycline (12 µg ml⁻¹) or nalidixic acid (20 µg ml⁻¹) was added to the media. Strains were frozen in LB containing 13% glycerol at −70°C.

General molecular techniques

DNA manipulation and isolation of chromosomal DNA was performed according to standard procedures (Sambrook and Russell, 2001), or to the manufacturer's protocol. Transposon mutagenesis using pUT mini-Tn5 luxCDABE Km2 was carried out essentially by the method of Winson et al. (1998) using E. coli strain S17.1 λpir. PCR was carried out with Thermoprime Taq polymerase (ABgene, Hamburg, Germany) and the following programme: 95°C for 2 min; 30 cycles at 95°C for 10 s, specific annealing temperature for 30 s, and 72°C for 20–180 s depending on the expected fragment length; and a final extension of 72°C for 10 min. Chromosomal DNA (100 ng) was used as template for PCR amplification. Conjugational transfer was performed using the mobilizing E. coli strain SM10 as the donor for the matings (Simon et al., 1983; Herrero et al., 1990).

Sequencing of insecticidal toxin genes

Chromosomal DNA (400 ng) of transposon mutants W22703-tcaA(134)::Tn5lux and W22703-tcaB1(152)::Tn5lux was completely digested with ClaI and HindIII. Fragments were treated with T4 DNA ligase (Gibco, CA, USA) to generate circular molecules by self-ligation, and inverse PCR (Ochman et al., 1988) was performed using transposon-specific primer pairs IF1 (5′-tgttccgttgcgctgcccgg-3′)/ClaIR1 (5′-gcgca tcgggcttcccatac-3′) and INV1F (5′-gttgcgctgcccggattacag-3′)/INV2R (5′-gaaatcaccatgagtgacgactg-3′) derived from the I-end of mini-Tn5. The PCR fragments obtained were sequenced initially using the primers IF1 and INV1F. Chromosomal walking was performed by inverse PCR using the restriction enzymes SphI, HpaI, EcoRI, MunI and DraI (MBI Fermentas) and sequencing with island-specific primers. In addition, a 1053 bp fragment of tcaC was amplified from the chromosomal DNA of strains NCTC10460, H270/78 and H324/78 with the primers toxIF21 (5′-ctgttgcaaagcctgagc-3′) and toxIR17 (5′-gtggaacatcaagacttgc-3′), and sequenced. SequiServe (Vaterstetten, Germany) and Biolux (Stuttgart, Germany) performed sequencing.

Southern blotting and DNA hybridization

Chromosomal DNA was completely digested with EcoRI, size fractionated by agarose gel electrophoresis (0.8% agarose), denaturated and transferred to Biodyne^® Plus membrane (PALL, Dreieich, Germany) using the VacuGene™XL vacuum blotting system (AmershamPharmacia, Freiburg, Germany) as recommended by the manufacturer. Hybridization, washing and detection of the digoxigenin (DIG)-labelled probe (Roche, Mannheim, Germany) were performed according to the manufacturer's instructions. A 428 bp fragment was generated by PCR using the primer combination toxIFI (5′-ggtgctgaagtcaacacc-3′) and toxIR2 (5′-aggaacttcctgactgcg-3′) that binds to nucleotides 144–572 of tcaB1. The standard conditions for hybridization of this probe were 15 h at 38°C in 5× SSC containing 50% formamide, followed by two high-stringency washing steps for 15 min each at 65°C in 0.1× SSC and 0.1% SDS. To apply low-stringency conditions, hybridization was performed at 30°C, and washing was carried out for 15 min at room temperature in 2× SSC and 0.1% SDS.

Construction of W22703ΔtcaA and pACYC184/F37.R43(s)

To construct an in-frame deletion of tcaA, two fragments of 825 bp and 808 bp were amplified using the oligonucleotide pairs tcaA.delF1 (5′-cagccgtacgaccgc-3′)/tcaA.delR1 (5′-gagaattcacgttctttatttggcatagctac-3′) and tcaA.delF2 (5′-gagaattcttacaaataacaataaaaaac-3′)/tcaA.delR2 (5′-actgcattctcagtgag-3′), and ligated via the introduced EcoRI sites. Following nested PCR with the oligonucleotides tcaA.nestedAB (5′-cgggatccttaaattaactgatagag-3′) and tcaA.nestedCD (5′-cgggatccaatatagtcggccgc-3′) and the ligation mixture as a template, the resulting fragment was cloned into pKNG101 via BamHI, giving rise to pKNG101ΔtcaA. This construct was transformed from SM10 into W22703 by conjugation, and streptomycin-resistant bacteria growing at 30°C harbouring the chromosomally integrated plasmid were isolated. Making use of levansucrase encoded by sacB as a positive marker for plasmid excision, bacteria were selected on agar plates containing 5% sucrose, and streptomycin-sensitive clones were screened by PCR with appropriate primers to identify mutant W22703ΔtcaA in which the second recombination step resulted in the deletion of tcaA. The gene deletion was confirmed by sequencing. The first 18 nucleotides at the 5′-end and the last 36 nucleotides of the 3′-end of tcaA were retained to maintain translation start signals and possible translational coupling with tcaB1. To complement W22703ΔtcaA, the complete coding sequence of tcaA and 291 nucleotides of its upstream sequence were amplified with the oligonucleotides toxIF37 (5′-ccggaattcgaaaaaggtgctg gac-3′) and toxIR43 (5′-ccggaattctgagtgggtaagttcatc-3′) and cloned into pACYC184 via EcoRI. In the resulting recombinant plasmid, pACYC184/F37.R43(s), the direction of tcaA transcription corresponds to that of the disrupted plasmid gene encoding the chloramphenicol acetyltransferase. Cloning of this construct was performed in E. coli strain DH5α and then transferred into W22703ΔtcaA by electroporation.

Measuring transcriptional profiles using a luxCDABE reporter

Bioluminescence measurements were performed in 96-well plates (Greiner Bio-one, Frickenhausen, Germany). Fresh cultures were prepared by diluting 10 µl of each overnight culture into 190 µl of LB medium and overnight shaking at 30°C and 350 r.p.m. Cultures of the transposon mutants were then diluted 1:4000 in microtitre plates filled with 200 µl of temperated LB medium. The strains were then grown at the appropriate temperature with shaking (500 r.p.m.) until reaching stationary phase. Bioluminescence (490 nm) and OD (405 nm) from all plates were measured in parallel every hour using a Wallac VICTOR² 1420 multilabel counter (Perkin Elmer Life Sciences, Turku, Finland). Bioluminescence was recorded as RLU.

Insect bioassay

Culture supernatants and cell lysates of Y. enterocolitica and X. nematophilus were prepared according to Sergeant et al. (2003) with minor modifications. Bacterial cells from overnight cultures were inoculated in 300 ml of LB and grown at 30°C and 10°C for 28 h and 48 h respectively. Cells were collected by centrifugation (7000 g for 15 min at 4°C) and the culture supernatants filter-sterilized (0.2 µm pore size) and stored at 4°C. The cell pellet was washed three times by resuspension in 10 ml of PBS (10 mM phosphate buffer, pH 7.4; 2.7 mM KCl; 137 mM NaCl). The final pellet was resuspended in 3 ml of PBS and lysed by sonication at 25% power for 30 s (Sonopuls HD2200, Bandelin electronic, Berlin). Sonication was repeated four times with a 1 min interval on ice between each step. The cell extract was filtered (0.2 µm pore size) and stored at 4°C. The protein concentration was measured with the bicinchoninic acid protein assay kit (Pierce, Rockford, III) following the manufacturer's instructions. The protein concentration of the cell extract samples was normalized to 0.2 µg ml⁻¹, while culture supernatant samples were used neat. For each a 50 µl sample was spread onto an agar-based artificial diet (David and Gardiner, 1965) which contained streptomycin (20 µg ml⁻¹), cefataxime and tetracycline (each at 100 µg ml⁻¹). To each container (4.5 cm in diameter), 10 larvae were added, and the assay pots were incubated at 25°C (16 h day length period), with a relative humidity of 80% for 120 h. Larvae of P. brassicae and M. sexta were used in these assays, and daily recordings of larvae mortality were made. Three independent replicate assays were carried out. A positive result was scored if over 50% of larvae were dead by the end of the assay, and a negative result if less than 5% of larvae were dead as occasional mortality was detected in control samples. E. coli cells, and separately water, were used as controls for the assay. General visual observations on larval size were also noted.

Bioinformatics

For the analysis of the nucleotide and amino acid sequences obtained, the Husar Analysis Package (version 4.0; http://genome.dkfz-heidelberg.de), the genome sequence annotation for Y. enterocolitica (Sanger Institute, http://www.sanger.ac.uk/Projects/Y_enterocolitica) and databases at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) were used. Sequence similarity was determined using the blast algorithm. The nucleotide sequence of the tc-PAI^Ye recently found in strain T83 is available under Accession No. AY647257. The nucleotide sequences reported here will appear in the EMBL, GenBank and DDBJ databases under the Accession No. AJ920332.