A duplex qPCR for the simultaneous detection of Escherichia coli O157:H7 and Listeria monocytogenes using LNA probes.

UNLABELLED: In this study, a duplex qPCR assay was developed for the needs of the Irish fish industry to screen for the two major food-borne pathogens of fish, Listeria monocytogenes and Escherichia coli O157:H7. The assay can claim positive or negative results for two pathogens in one go in only 20 h including 16 h universal pre-enrichment and compared to traditional ISO approved plate culture methods the labour and the cost involved in testing of one sample is reduced to minimum. The highly specific genomic areas targeted for PCR amplification in the assay are the hly gene for listeriolysin O (LLO) of L. monocytogenes and the stx gene for Shiga-like toxin expressed by E. coli O157:H7. The detection limit of the assay is consistent with the consumer protection limits of 1 pg genomic DNA or 1 CFU 25 g(-1) fish meat (with enrichment) allowing the test to be considered as a substitute to standard plate culture methods. SIGNIFICANCE AND IMPACT OF THE STUDY: The study highlights a novel duplex qPCR for Listeria monocytogenes and Escherichia coli O157:H7 that could be used as an alternative to plate-based ISO or singleplex PCR methods while minimizing the costs. The assay uses rapid DNA extraction methods and locked nucleic acid probes. Sensitivity and specificity are 100 and 98·95% respectively. The potential for quantitative rage of the assay is 10(8) -10(1) CFU ml(-1) .

A complex network regulates expression of eps and other virulence genes of Pseudomonas solanacearum.

We have discovered an unusual and complex regulatory network used by the phytopathogen Pseudomonas solanacearum to control transcription of eps, which encodes for production of its primary virulence factor, the exopolysaccharide EPS I. The major modules of this network were shown to be three separate signal transduction systems: PhcA, a LysR-type transcriptional regulator, an dual two-component regulatory systems, VsrA/VsrD and VsrB/VsrC. Using lacZ fusions and RNA analysis, we found that both PhcA and VsrA/VsrD control transcription of another network component, xpsR, which in turn acts in conjunction with vsrB/vsrC to increase transcription of the eps promoter by > 25-fold. Moreover, gel shift DNA binding assays showed that PhcA specifically binds to the xpsR promoter region. Thus, the unique XpsR protein interconnects the three signal transduction systems, forming a network for convergent control of EPS I in simultaneous response to multiple environmental inputs. In addition, we demonstrate that each individual signaling system of the network also acts independently to divergently regulate other unique sets of virulence factors. The purpose of this complex network may be to allow this phytopathogen to both coordinately or independently regulate diverse virulence factors in order to cope with the dynamic situations and conditions encountered during interactions with plants.

Phenotype conversion in Pseudomonas solanacearum due to spontaneous inactivation of PhcA, a putative LysR transcriptional regulator.

Phenotype conversion (PC) in Pseudomonas solanacearum is the coordinated change in production of extracellular polysaccharide and a variety of extracellular proteins, some of which contribute to virulence. Although PC is normally spontaneous, it is mimicked by transposon inactivation of the phcA locus (S. M. Brumbley and T. P. Denny, J. Bacteriol. 172:5677-5685, 1990). The DNA sequence of a 1.8-kb region from strain AW1 that contains phcA revealed one open reading frame that should encode a polypeptide of 38.6 kDa. The PhcA protein produced in Escherichia coli by using a T7 RNA polymerase expression system was of the predicted size. The deduced amino acid sequence of PhcA is similar to that of some members of the LysR transcriptional activator gene family, especially in the amino terminus, where a putative helix-turn-helix DNA-binding motif was identified. An analogous allele (phcA1) was cloned from the spontaneous PC mutant strain AW1-PC and found to be nonfunctional in complementation studies. When phcA1 was expressed in E. coli, the PhcA1 protein was 35.5 kDa, 3 kDa smaller than PhcA. Sequence analysis of phcA1 and chimeric constructs of phcA and phcA1 confirmed that PhcA1 is truncated by a 2-bp insertion 147 nucleotides upstream of the carboxyl terminus of PhcA. Southern blot analysis of 10 additional independently isolated PC mutants of strain AW1 revealed that two strains have larger insertions (0.2 and 1.0 kb) within phcA. These results suggest that phcA encodes a DNA-binding protein that regulates the transcription of one or more of the genes involved in P. solanacearum virulence and that spontaneous PC can be attributed to one of several different insertions within this locus.

A cloned avirulence gene from Pseudomonas solanacearum determines incompatibility on Nicotiana tabacum at the host species level.

A locus in Pseudomonas solanacearum AW1 responsible for the hypersensitive response (HR) on tobacco was cloned by complementation in the tobacco-pathogenic strain P. solanacearum NC252. The NC252 HR+ transconjugants lost pathogenicity on tobacco, indicating that the cloned locus could restrict the host range of NC252. Restriction enzyme mapping, transposon mutagenesis, and subcloning showed that, at most, 2.0 kilobases of the cloned DNA was required for NC252 transconjugants to elicit HR on tobacco. Site-directed insertional mutagenesis of the wild-type locus in strain AW1 to create AW1-31 eliminated HR activity on tobacco. However, AW1-31 retained pathogenicity on tomato and eggplant, confirming that this locus contains an avirulence gene, designated avrA. In contrast to the wild type, AW1-31 multiplied to almost the same extent as NC252 after infiltration into tobacco leaves. Nevertheless, AW1-31 did not wilt tobacco when stem inoculated, suggesting that additional factors condition host range. AW1 was HR+ on 27 N. tabacum cultivars, whereas AW1-31 was always HR-, strongly suggesting that avrA is specific at the host species level.

Novel Alterations in Plasmid DNA Associated with Aromatic Hydrocarbon Utilization by Pseudomonas putida R5-3.

Subcultures of Pseudomonas putida R5-3 altered their plasmid DNA content in specific ways depending on the particular aromatic hydrocarbon utilized as the sole carbon source. Two indigenous plasmids, 115 and 95 kilobases (kb) in size, were observed in R5-3A, which was derived from R5-3 by growth on minimal medium containing p-methylbenzoate as the sole carbon source. When R5-3A was transferred to medium containing m-xylene or toluene, derivative strains were obtained in which the 95-kb plasmid was lost and a new plasmid of 50 or 60 kb appeared. Reversion to the original plasmid profile of R5-3A was observed when xylene- or toluene-grown cells were returned to medium containing p-methylbenzoate. Restriction enzyme analysis and Southern blot hybridizations of total plasmid DNA indicated deletions and rearrangements of DNA restriction fragments in the derivatives maintained on m-xylene and toluene when compared with the original R5-3A. In the derivatives which retrieved the original plasmid profile, the restriction enzyme fragment pattern was identical to that in the original R5-3A, in that the fragments which were missing after growth on m-xylene or toluene were again present. Southern blot hybridizations revealed that part of the plasmid DNA lost from the original plasmid profile was integrated into the chromosomal DNA of xylene-grown R5-3B and that these plasmid fragments were associated with aromatic hydrocarbon metabolism. Hybridization with pathway-specific DNA fragments from the TOL plasmid pWWO indicated that this 95-kb plasmid contains DNA homologous to the meta-fission pathway genes.

Identification of Pseudomonas alcaligenes chromosomal DNA in the plasmid DNA of the chlorobenzene-degrading recombinant Pseudomonas putida strain CB1-9.

The recombinant Pseudomonas putida strain CB1-9, which acquired the ability to grow on chlorobenzenes, contains a 33-kilobase (kb) plasmid (pKFL3) which lacked homology to an indigenous 15-kb plasmid (pKFL1) in Pseudomonas alcaligenes C-0 parent but was homologous to a 55-kb plasmid (pKFL2) from the P. putida R5-3 parent. Chromosomal DNA of P. alcaligenes C-0 hybridized to probes prepared from pKFL3 but not to probes prepared from pKFL2. A single clone from a genomic library of P. alcaligenes C-0 hybridized to EcoRI-digested pKFL3. Southern blot hybridization with the insert DNA from that clone identified homology with specific restriction enzyme fragments in pKFL3. The ability of the recombinant to utilize 3-chlorobenzoate, chlorobenzene, and 1,4-dichlorobenzene as well as its loss of utilization of xylenes and methylbenzoates appears to be associated with the transfer and integration of chromosomal DNA from P. alcaligenes into a Tol-like plasmid of P. putida R5-3.