Publications

4468

ABSTRACTMolecular analysis of bacteria enriched under in situ-like conditions and mechanically isolated by micromanipulation showed that a hitherto-uncultivated microaerophilic bacterium thriving in oxygen-sulfide counter-gradients (R. Thar and M. Kühl, Appl. Environ. Microbiol. 68:6310-6320, 2000) is affiliated with the ε-subdivision of theProteobacteria. The affiliation was confirmed by the use of whole-cell hybridization with newly designed specific oligonucleotide probes. The bacterium belongs to a new genus and received the provisional name “CandidatusThioturbo danicus.”

ABSTRACT “ Helicobacter heilmannii ” (proposed name) type 1 colonizes the human stomach. It has been shown to be identical to“ Candidatus Helicobacter suis,” a Helicobacter species colonizing the stomachs of >60% of slaughter pigs. This bacterium has not been isolated in vitro until now. Antibiotic susceptibility testing of “ Candidatus Helicobacter suis” has not been carried out so far. For the present study, a mouse model was adopted to evaluate the antibiotic susceptibility of this organism. Mice infected with“ Candidatus Helicobacter suis” were treated with amoxicillin and omeprazole, a therapy which is used to treat H. heilmannii infections in humans. Two different isolates of“ Candidatus Helicobacter suis” were tested. The excretion of bacterial DNA was assessed during treatment, using PCR on fecal samples. At the end of the experiment, 8 days after the cessation of treatment, the presence of infection was evaluated using a urease test and a PCR test on stomach samples. A marked decrease in the excretion of bacterial DNA was observed a few days after the onset of treatment, and the level remained low until the end of the experiment. A difference in susceptibility between the two“ Candidatus Helicobacter suis” isolates was pointed out. The in vivo mouse model infected with“ Candidatus Helicobacter suis” will be useful for further screening of potential therapeutic regimens.

ABSTRACT Huanglongbing (yellow dragon disease) is a destructive disease of citrus. The etiological agent is a noncultured, phloem-restricted alpha-proteobacterium, “ Candidatus Liberibacter africanus” in Africa and “ Candidatus Liberibacter asiaticus” in Asia. In this study, we used an omp -based PCR-restriction fragment length polymorphism (RFLP) approach to analyze the genetic variability of “ Ca . Liberibacter asiaticus” isolates. By using five different enzymes, each the 10 isolates tested could be associated with a specific combination of restriction profiles. The results indicate that the species “ Ca . Liberibacter asiaticus,” even within a given region, may comprise several different variants. Thus, omp -based PCR-RFLP analysis is a simple method for detecting and differentiating “ Ca . Liberibacter asiaticus” isolates.

During 2003, surveys of sugarcane yellow leaf disease and papaya bunchy top-like disease were carried out on plantations in Havana province, Cuba, to determine the roles of weeds and Auchenorrhyncha insects in the epidemiology of these diseases. More than 250 plant and insect samples were collected and indexed by using a nested PCR for phytoplasma 16S rDNA with the generic primer pairs P1/P7 and R16F2n/R16R2. The PCR products were further characterized by restriction fragment length polymorphism using HaeIII, AluI, Sau3AI, Tru9I, HhaI, HpaII and TaqI endonucleases, giving patterns that distinguished them from those of the other reference phytoplasmas analysed. Phylogenetic analysis of 16S rRNA gene sequences identified the phytoplasmas present in sugarcane (Saccharum officinarum L.), Cynodon dactylon L., Conyza canadensis L. Cronq., Sorghum halepense L. Pers., Macroptilium lathyroides L. Urb., Saccharosydne saccharivora (Westwood) and Cedusa spp., and those in papaya (Carica papaya L.) and Empoasca papayae, as two novel provisional phytoplasma species. We propose that these phytoplasmas should be given Candidatus status, as ‘Candidatus Phytoplasma graminis' and ‘Candidatus Phytoplasma caricae’, respectively.

Phytopathogenic mollicutes, which include spiroplasmas and phytoplasmas, are cell wall-less bacteria that parasitize plant hosts and insect vectors. Knowledge of the evolution of these agents is important in understanding their biology. The availability of the first complete phytoplasma and several partial spiroplasma and phytoplasma genome sequences made possible an investigation of evolutionary relationships between phytopathogenic mollicutes and other micro-organisms, especially Gram-positive bacteria, using a comparative genomics approach. Genome data from a total of 41 bacterial species were used in the analysis. Sixty-one conserved proteins were selected from each species for the construction of a hypothetical phylogenetic tree. The genes encoding these selected proteins are among a core of genetic elements that constitute a hypothetical minimal genome. The proteins were concatenated into five superproteins according to their functional categories, and phylogenetic trees were reconstructed using distance, parsimony and likelihood methods. Phylogenetic trees based on the five sets of concatenated proteins were congruent in both clade topology and relative branching length.Spiroplasma kunkeliiand phytoplasmas clustered together with other mollicutes, forming a monophyletic group. Phytoplasmas diverged from spiroplasmas and mycoplasmas at early stages in the evolution of mollicutes. Branch lengths on the phylogenetic trees were noticeably longer in theMollicutesclade, suggesting that the genes encoding the five sets of proteins evolved at a greater rate in this clade than in other clades. This observation reinforces the concept that mollicutes have rapidly evolving genomes.