Isolation of partial toxR gene of Vibrio harveyi and design of toxR-targeted PCR primers for species detection.

AIMS: To differentiate Vibrio harveyi from closely related Vibrio species by toxR sequence analysis and design primers for the specific detection of the shellfish pathogen. METHODS AND RESULTS: The partial toxR homologue from the shellfish pathogen V. harveyi was isolated by PCR using degenerate primers. The 578-bp toxR fragment from V. harveyi, that exhibited highest homology with partial toxR of V. parahaemolyticus (68%), is predicted to encode for a polypeptide with 192 amino acid residues. Alignment of the V. harveyi toxR nucleotide and deduced amino acid sequence with those from other Vibrio species revealed the presence of the fairly characteristic conserved transcription activation and transmembrane domain as well as the divergent membrane tether region that may be targeted for the development of species-specific oligonucleotide primers. Consequently, PCR primers that could amplify a 390-bp gene fragment in V. harveyi were designed by targeting portions of the V. harveyi toxR that display variability with toxR sequences from other Vibrio species. The 390-bp-amplicon was detected in all V. harveyi strains examined except in the nontarget bacteria and unexpectedly, in two shrimp-derived strains (VIB 391 and STD 3-101) from Thailand and Ecuador. Results show that strains exhibiting the 390-bp amplicon mostly belong to the same cluster based on previous amplified fragment length polymorphism data while strains which were previously unclustered or unclassified did not display the 390-bp PCR product. CONCLUSIONS: The toxR sequence variation could differentiate V. harveyi from closely related Vibrio species. A PCR protocol amplifying a 390-bp fragment of the V. harveyi toxR was established and could be useful in the specific and rapid detection of the species. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular approaches reported in this study could facilitate the early diagnosis and surveillance of luminous vibriosis in hatchery-reared fish and shellfish species through rapid identification and specific detection of causal agent.

Transposon mutagenesis reinforces the correlation between Mycoplasma pneumoniae cytoskeletal protein HMW2 and cytadherence.

A new genetic locus associated with Mycoplasma pneumoniae cytadherence was previously identified by transposon mutagenesis with Tn4001. This locus maps approximately 160 kbp from the genes encoding cytadherence-associated proteins HMW1 and HMW3, and yet insertions therein result in loss of these proteins and a hemadsorption-negative (HA-) phenotype, prompting the designation cytadherence-regulatory locus (crl). In the current study, passage of transformants in the absence of antibiotic selection resulted in loss of the transposon, a wild-type protein profile, and a HA+ phenotype, underscoring the correlation between crl and M. pneumoniae cytadherence. Nucleotide sequence analysis of crl revealed open reading frames (ORFs) orfp65, orfp216, orfp41, and orfp24, arranged in tandem and flanked by a promoter-like and a terminator-like sequence, suggesting a single transcriptional unit, the P65 operon. The 5' end of orfp65 mRNA was mapped by primer extension, and a likely promoter was identified just upstream. The product of each ORF was identified by using antisera prepared against fusion proteins. The previously characterized surface protein P65 is encoded by orfp65, while the 190,000 Mr cytadherence-associated protein HMW2 is a product of orfp216. Proteins with sizes of 47,000 and 41,000 Mr and unknown function were identified for orfp41 and orfp24, respectively. Structural analyses of HMW2 predict a periodicity highly characteristic of a coiled-coil conformation and five leucine zipper motifs, indicating that HMW2 probably forms dimers in vivo, which is consistent with a structural role in cytadherence. Each transposon insertion mapped to orfp216 but affected the levels of all products of the P65 operon. HMW2 is thought to form a disulfide-linked dimer, formerly designated HMW5, and examination of an hmw2 deletion mutant confirms that HMW5 is a product of the hmw2 gene.

Identification of a possible cytadherence regulatory locus in Mycoplasma pneumoniae.

Transposon mutagenesis was used to analyze Mycoplasma pneumoniae cytadherence. Mycoplasmas were electroporated with Tn4001, and transformants were identified by antibiotic selection using gentamicin. The resulting colonies were screened for hemadsorption (HA) as an indicator for cytadherence. Six HA- colonies from independent transformations were isolated, filter cloned, and characterized in more detail. Southern hybridization analysis revealed that all six transposon insertions mapped to the same 252-kbp ApaI fragment and 19.5-kbp XhoI fragment. More detailed analysis localized the insertion to two adjacent EcoRI fragments. This site is distinct from the locus containing the genes for the high-molecular weight cytadherence-accessory proteins HMW1 and HMW3, and yet these proteins were absent from the protein profiles of all six transformants. To determine if transposon insertion was responsible for the HA- phenotype, reversion frequencies of the transformants were assessed after passage in the presence of antibiotic selection. In contrast to a spontaneously arising HMW-deficient variant, which reverted to an HA+ phenotype readily, no HA+ revertants were identified for any of the six transformants. These observations suggest that a potential regulatory locus that may be important in the expression of the HMW cytadherence-accessory proteins has been identified.

Transformation of Mycoplasma pneumoniae with Tn4001 by electroporation.

Mycoplasma pneumoniae was transformed with the Staphylococcus aureus transposon Tn4001 by electroporation. A transformation frequency of 10(-3) to 10(-5)/colony-forming unit was observed using 30.0 microgram plasmid DNA and 10(7)-10(8) M. pneumoniae colony-forming units. DNA hybridization analyses using standard and pulsed field agarose gel electrophoresis confirmed chromosomal insertion of the transposon, apparently by a transpositional mechanism into random sites. These studies demonstrate the functionality of Tn4001 in M. pneumoniae and suggest its potential as a genetic tool in this mycoplasma.