Publications

4698

In India, satkara (Citrus macroptera) is found in the northeastern regions of Shella and Dowki near Cherrapunji in Meghalaya, the Jampui Hills of Tripura and Mizoram, and the Chendel District of Manipur. Locally, it is called satkara because ‘sat’ refers to multiples of seven and the fruit generally contains fourteen segments. The fruit is used in the preparation of pickles and its oil is used in the perfume industry. In January 2007, in Behliangchhip, Jampui Hills, Tripura, India, we noticed seven satkara trees of 26 showing leaf mottling and yellowing in young shoots, symptoms typical of huanglongbing (HLB). HLB is a destructive citrus disease caused by a nonculturable, phloem-limited alpha-proteobacterium of the genus ‘Candidatus Liberibacter’. Among the three known ‘Ca. Liberibacter’ species (Ca. Liberibacter asiaticus, Ca. L. africanus, and Ca. L. americanus), only Ca. L. asiaticus has been reported in India (1) but not from satkara. To identify the pathogen, total DNA was extracted and purified from 200 mg of leaf midribs from five symptomatic and five symptomless trees with the DNeasy Plant Mini Kit (Qiagen, Gmbh, Hilden, Germany) as per the manufacturer's instructions. Samples were tested for ‘Ca. L. asiaticus’ by PCR with primer sets OI1/OI2c targeting the 16S rDNA locus (3) and A2/J5 targeting the beta-operon locus of ribosomal proteins (2). DNA samples from all symptomatic leaves were positive with both primer sets. The OI1/OI2c primers generated an amplicon of approximately 1,160 bp and digestion of the amplicon by XbaI yielded two DNA fragments of approximately 640 bp and 520 bp, suggesting the presence of ‘Ca. L. asiaticus’(3). The A2/J5 primer set generated an ~703-bp amplicon, indicating ‘Ca. L. africanus’ was not present. No DNA was amplified from the asymptomatic satkara trees. One each of the OI1/OI2c and A2/J5 amplicons was purified, cloned (pGEM-T Easy vector, Promega, Hampshire, UK) and sequenced (Bangalore Genei, Bengaluru, Karnataka, India). The derived sequences, 1167 bp for the OI1/OI2c amplicon and 703 bp for the A2/J5 amplicon, have been deposited in the GenBank database under Accession Nos. GQ369792 and GU074017, respectively. Sequence comparison revealed that GQ369792 and GU074017 shared >99% identity to the corresponding regions of ‘Ca. L. asiaticus’ in the GenBank database. No DNA was amplified by PCR with primer set GB1/GB2 (4) specific for ‘Ca. L. americanus’. To our knowledge, this is the first report of molecular identification of ‘Ca. L. asiaticus’ infecting satkara in India. References: (1) A. K. Das. Curr. Sci. 87:1183, 2004. (2) A. Hocquellet et al. Mol. Cell. Probes 13:373, 1999. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (4) D. C. Teixeira et al. Mol. Cell. Probes 19:173, 2005.

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.

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).

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.