Publications

4787

Citrus huanglongbing is one of the most destructive diseases of citrus worldwide. The disease is associated with three different species of ‘Candidatus Liberibacter’, of which ‘Ca. L. asiaticus’ is the most widely distributed. An optimized system using ‘Ca. L. asiaticus’-infected periwinkle cuttings was developed to screen chemical compounds effective for controlling the bacterial population while simultaneously assessing their phytotoxicity. The optimal regeneration conditions were determined to be the use of vermiculite as a growth medium for the cuttings, and a fertilization routine using half-strength Murashige and Tucker medium supplemented with both naphthalene acetic acid (4 μg/ml) and indole-3-butyric acid (4 μg/ml). This system allowed a plant regeneration rate of 60.6% for ‘Ca. L. asiaticus’-infected cuttings in contrast to the <1% regeneration rate with water alone. Two chemical agents, penicillin G sodium and 2,2-dibromo-3-nitrilopropionamide (DBNPA), were found to be effective at eliminating or suppressing the ‘Ca. L. asiaticus’ bacterium in this periwinkle regeneration system. When treated with penicillin G sodium at 50 μg/ml, all plants regenerated from ‘Ca. L. asiaticus’-infected cuttings were ‘Ca. L. asiaticus’ negative as determined by both nested polymerase chain reaction (PCR) and quantitative real-time PCR. In addition, DBNPA was also able to significantly reduce the percentage of ‘Ca. L. asiaticus’-positive plants and the titer of the ‘Ca. L. asiaticus’ bacterium at 200 μl/liter.

ABSTRACTProtozoa play host for many intracellular bacteria and are important for the adaptation of pathogenic bacteria to eukaryotic cells. We analyzed the genome sequence of “CandidatusAmoebophilus asiaticus,” an obligate intracellular amoeba symbiont belonging to theBacteroidetes. The genome has a size of 1.89 Mbp, encodes 1,557 proteins, and shows massive proliferation of IS elements (24% of all genes), although the genome seems to be evolutionarily relatively stable. The genome does not encode pathways forde novobiosynthesis of cofactors, nucleotides, and almost all amino acids. “Ca. Amoebophilus asiaticus” encodes a variety of proteins with predicted importance for host cell interaction; in particular, an arsenal of proteins with eukaryotic domains, including ankyrin-, TPR/SEL1-, and leucine-rich repeats, which is hitherto unmatched among prokaryotes, is remarkable. Unexpectedly, 26 proteins that can interfere with the host ubiquitin system were identified in the genome. These proteins include F- and U-box domain proteins and two ubiquitin-specific proteases of the CA clan C19 family, representing the first prokaryotic members of this protein family. Consequently, interference with the host ubiquitin system is an important host cell interaction mechanism of “Ca. Amoebophilus asiaticus”. More generally, we show that the eukaryotic domains identified in “Ca. Amoebophilus asiaticus” are also significantly enriched in the genomes of other amoeba-associated bacteria (including chlamydiae,Legionella pneumophila,Rickettsia bellii,Francisella tularensis, andMycobacterium avium). This indicates that phylogenetically and ecologically diverse bacteria which thrive inside amoebae exploit common mechanisms for interaction with their hosts, and it provides further evidence for the role of amoebae as training grounds for bacterial pathogens of humans.

ABSTRACT ATP/ADP translocases transport ATP across a lipid bilayer, which is normally impermeable to this molecule due to its size and charge. These transport proteins appear to be unique to mitochondria, plant plastids, and obligate intracellular bacteria. All bacterial ATP/ADP translocases characterized thus far have been found in endosymbionts of protozoa or pathogens of higher-order animals, including humans. A putative ATP/ADP translocase was uncovered during the genomic sequencing of the intracellular plant pathogen “ Candidatus Liberibacter asiaticus,” the causal agent of citrus huanglongbing. Bioinformatic analysis of the protein revealed 12 transmembrane helices and predicted an isoelectric point of 9.4, both of which are characteristic of this family of proteins. The “ Ca. Liberibacter asiaticus” gene ( nttA ) encoding the translocase was subsequently expressed in Escherichia coli and shown to enable E. coli to import ATP directly into the cell. Competition assays with the heterologous E. coli system demonstrated that the translocase was highly specific for ATP and ADP but that other nucleotides, if present in high concentrations, could also be taken up and/or block the ability of the translocase to import ATP. In addition, a protein homologous to NttA was identified in “ Ca. Liberibacter solanacearum,” the bacterium associated with potato zebra chip disease. This is the first reported characterization of an ATP translocase from “ Ca. Liberibacter asiaticus,” indicating that some intracellular bacteria of plants also have the potential to import ATP directly from their environment.

A moderately halophilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium, designated strain HRh1T, was obtained from mixed sediment samples from hypersaline chloride–sulfate lakes in the Kulunda Steppe, in south-western Siberia (Russia), using aerobic enrichment culture at 1 M NaCl with thiocyanate as substrate. Cells of the isolate were short, non-motile rods with a Gram-negative type of cell wall. The bacterium was an obligate aerobe capable of chemolithoautotrophic growth with thiocyanate and thiosulfate. With thiosulfate, it grew at NaCl concentrations of 0.2–3.0 M (optimum 0.5 M) and at pH 6.3–8.0 (optimum pH 7.3–7.5). During growth on thiocyanate, cyanate was identified as an intermediate. The dominant cellular fatty acids were C16 : 0and C18 : 1ω7. Phylogenetic analysis based on 16S rRNA gene sequencing placed the isolate in the classGammaproteobacteriaas an independent lineage, with an unclassified marine sulfur-oxidizing bacterium as the closest culturable relative (93 % sequence similarity). A singlecbbLgene (coding for the key enzyme of the Calvin–Benson cycle of autotrophic CO2assimilation) with relatively low similarity to any homologous genes found in chemolithoautotrophs was detected in strain HRh1T. On the basis of our phenotypic and phylogenetic analysis, the halophilic isolate is proposed to represent a new genus and novel species,Thiohalobacter thiocyanaticusgen. nov., sp. nov. The type strain ofThiohalobacter thiocyanaticusis HRh1T(=DSM 21152T=UNIQEM U249T).