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 M r cytadherence-associated protein HMW2 is a product of orfp216. Proteins with sizes of 47,000 and 41,000 M r 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.Mycoplasma pneumoniae attachment to host respiratory epithelium is a multifactorial process, the complexity of which is only beginning to be appreciated (20). This cell wall-less prokaryote is a leading cause of pneumonia in older children and young adults. Colonization requires binding to host cell receptors (cytadherence) by mycoplasma surface proteins localized primarily to a differentiated terminal structure termed the attachment organelle (reviewed in reference 34). The membrane proteins P1 (169 kDa [19]) and P30 (30 kDa [2]) are thought to be directly involved in receptor recognition (34), although the evidence for P30 is limited. Their functionality is dependent upon accessory proteins, including HMW1-HMW3 and A, B, and C (20, 34), which collectively maintain the proper distribution and/or disposition of the adhesins in the mycoplasma membrane (1, 20). In addition, proteins P65 (32) and P200 (33) share characteristic structural features with HMW1 and HMW3, suggesting a common function. Most are elements of a cytoskeleton-like network in M. pneumoniae, consistent with a possible scaffolding role (20). In particular, HMW3 is a major componen...

No abstract

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 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 insertions 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. Attachment of the cell wall-less prokaryote Mycoplasma pneumoniae to the mucosal epithelium (cytadherence) is a crucial step in the successful colonization of the human respiratory tract, leading to atypical pneumonia and tracheobronchitis (8, 12). Cytadherence is generally mediated by a differentiated terminal structure (1, 4, 11, 18), seen as a membranebound extension of the mycoplasma cell, at what is the leading end as mycoplasmas move by gliding motility (6). The cytadhesin proteins P1 (1, 19) and P30 (2, 3, 7) are clustered at this attachment organelle (1, 4, 11, 18), apparently through their interaction with a complex network of cytadherence-accessory proteins (22, 23). These include the high-molecular-weight proteins HMW1 to HMW5, which are components of a cytoskeleton-like scaffolding network in the mycoplasma cell (37, 38). HMW3 appears to be a structural element of the attachment organelle, where it may anchor the adhesin proteins at the tip structure (38). HMW1 and HMW4, on the other hand, are associated exclusively with the filamentous extensions of the mycoplasma cell (37). While HMW1 and HMW4 are phosphorylated by an ATP-dependent Ser/Thr kinase (9, 26), their function remains unclear. The expression of HMW1 to HMW5 by M. pneumoniae is coordinately regulated (22, 23). Spontaneously arising variants lacking HMW1 to HMW5 are avirulent, cytadhere very poorly, and fail to cluster the adhesin P1 at the attachment organelle, which has an altered shape from that seen in wild-type mycoplasmas (14, 15, 22, 23). This is a reversible phenomenon, with reacquisition of HMW1 to HMW5 corresponding to a return to a virulent, cytadherence-positive phenoty...

The Vibrio harveyi hemolysin gene (vhh), which encodes for a virulence factor involved in pathogenicity to fish and shellfish species, may be targeted for species detection or strain differentiation. Primers designed for this gene were used in detection studies of V. harveyi strains from various hosts. One primer set among four tested, could amplify the expected gene fragment in PCR using templates from all 11 V. harveyi strains studied. Detection of the presence of the hemolysin gene could therefore serve as a suitable detection marker of Vibrio harveyi isolates potentially pathogenic to fish and shrimps.