Publications

4372

ABSTRACT “ Candidatus Pelagibacter ubique,” an abundant marine alphaproteobacterium, subsists in nature at low ambient nutrient concentrations and may often be exposed to nutrient limitation, but its genome reveals no evidence of global regulatory mechanisms for adaptation to stationary phase. High-resolution capillary liquid chromatography coupled online to an LTQ mass spectrometer was used to build an accurate mass and time (AMT) tag library that enabled quantitative examination of proteomic differences between exponential- and stationary-phase “ Ca . Pelagibacter ubique” cells cultivated in a seawater medium. The AMT tag library represented 65% of the predicted protein-encoding genes. “ Ca . Pelagibacter ubique” appears to respond adaptively to stationary phase by increasing the abundance of a suite of proteins that contribute to homeostasis rather than undergoing a major remodeling of its proteome. Stationary-phase abundances increased significantly for OsmC and thioredoxin reductase, which may mitigate oxidative damage in “ Ca . Pelagibacter,” as well as for molecular chaperones, enzymes involved in methionine and cysteine biosynthesis, proteins involved in ρ-dependent transcription termination, and the signal transduction enzyme CheY-FisH. We speculate that this limited response may enable “ Ca . Pelagibacter ubique” to cope with ambient conditions that deprive it of nutrients for short periods and, furthermore, that the ability to resume growth overrides the need for a more comprehensive global stationary-phase response to create a capacity for long-term survival.

In recent years, lily (Lilium spp.) has become an important ornamental crop in diverse regions of Mexico. Since 2005, unusual symptoms have been observed on lily plants grown from imported bulbs in both greenhouse and production plots at San Pablo Ixayo, Boyeros, and Tequexquinauac, Mexico State. Symptoms included a zigzag line pattern on leaves, dwarfism, enlargement of stems, shortened internodes, leaves without petioles growing directly from bulbs, air bulbils, death of young roots, atrophy of flower buttons, and flower abortion. Symptoms were experimentally reproduced on healthy lily plants by graft inoculation. Total DNA was extracted from 50 diseased, 10 symptomless, and 10 graft-inoculated plants by the method of Dellaporta et al. (2). DNA samples were analyzed for phytoplasma presence by two different nested PCR assays. One assay employed ribosomal RNA gene primer pair P1/P7 followed by R16F2n/R16R2 (1), whereas ribosomal protein (rp) gene primer pairs rpF1/rpR1 and rp(I)F1A/rp(I)R1A (4) were used in a second assay. A DNA fragment approximately 1.2 kb long was consistently amplified from all symptomatic plant samples only by both assays. A comparative analysis of 16S rDNA sequences (Genbank Accession Nos. EF421158–EF421160 and EU124518–EU124520) and rp gene sequences (EU277012–EU277014), derived from PCR products, revealed that phytoplasma infecting lily were most similar (99.9% to 16S rDNA and 99.7% to rp) to carrot phytoplasma sp. ca2006/5 and also were similar (99.8% to 16SrDNA and 99.2% to rp) to broccoli phytoplasma sp. br273. Both carrot and broccoli phytoplasmas were classified as members of aster yellow 16S rDNA restriction fragment length polymorphism subgroup 16SrI-B (3). Although infection of lilies by aster yellows (‘Ca. phytoplasma asteris’) subgroup 16SrI-B and 16SrI-C was reported from the Czech Republic and Poland, to our knowledge, this is the first report of ‘Ca. phytoplasma asteris’-related strains associated with lily plants in Mexico. References: (1) R. F. Davis et al. Microbiol. Res. 158:229, 2003. (2) S. L. Dellaporta et al. Plant Mol. Biol. Rep. 1:19, 1983. (3) B. Duduk et al. Bull. Insectol. 60 2:341, 2007. (4) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004.

ABSTRACTThe chromosome sequence of “CandidatusPhytoplasma australiense” (subgrouptuf-Australia I;rp-A), associated with dieback in papaya, Australian grapevine yellows in grapevine, and several other important plant diseases, was determined. The circular chromosome is represented by 879,324 nucleotides, a GC content of 27%, and 839 protein-coding genes. Five hundred two of these protein-coding genes were functionally assigned, while 337 genes were hypothetical proteins with unknown function. Potential mobile units (PMUs) containing clusters of DNA repeats comprised 12.1% of the genome. These PMUs encoded genes involved in DNA replication, repair, and recombination; nucleotide transport and metabolism; translation; and ribosomal structure. Elements with similarities to phage integrases found in these mobile units were difficult to classify, as they were similar to both insertion sequences and bacteriophages. Comparative analysis of “Ca.Phytoplasma australiense” with “Ca.Phytoplasma asteris” strains OY-M and AY-WB showed that the gene order was more conserved between the closely related “Ca.Phytoplasma asteris” strains than to “Ca. Phytoplasma australiense.” Differences observed between “Ca.Phytoplasma australiense” and “Ca.Phytoplasma asteris” strains included the chromosome size (18,693 bp larger than OY-M), a larger number of genes with assigned function, and hypothetical proteins with unknown function.

To characterize potentially important surface-exposed proteins of the phytoplasma causing chrysanthemum yellows (CY), new primers were designed based on the conserved regions of 3 membrane protein genes of the completely sequenced onion yellows and aster yellows witches’ broom phytoplasmas and were used to amplify CY DNA. The CY genes secY, amp, and artI, encoding the protein translocase subunit SecY, the antigenic membrane protein Amp and the arginine transporter ArtI, respectively, were cloned and completely sequenced. Alignment of CY-specific secY sequences with the corresponding genes of other phytoplasmas confirmed the 16S rDNA-based classification, while amp sequences were highly variable within the ‘Candidatus Phytoplasma asteris’. Five CY partial sequences were cloned into the pRSetC expression vector, and 3 of the encoded protein fragments (Amp 64/651, Amp 64/224, ArtI 131/512) were expressed as fusion antigens for the production of CY-specific polyclonal antibodies (A416 against Amp 64/224; A407 against ArtI 131/512). A416 recognized, in Western blots, the full-length Amp from CY-infected plants (periwinkle, daisy) and insect vectors ( Euscelidius variegatus , Macrosteles quadripunctulatus ). A416 also reacted to European aster yellows, to primula yellows phytoplasmas, to northern Italian strains of ‘Ca. Phytoplasma asteris’ from lettuce and gladiolus, but it did not react to American aster yellows phytoplasma.

Summary Amoeba‐resisting bacteria (ARB) such as Legionella spp. are currently regarded as potential human pathogens living in the environment. To detect ARB from both human and environmental samples, co‐culture with amoebae has been demonstrated as an efficient tool. However, using this procedure, mostly water from cooling towers and hospital water supplies have been investigated as the possible reservoir of ARB. In the present study, we studied ARB population in 77 environmental water samples including rivers, fountains, lakes and domestic wells in the south of France. As a result, a total of 244 isolates corresponding to 89 different species of ARB, but not Legionella spp., were identified. Ability to grow within and/or to be lytic for amoebae was revealed for the first time for several human pathogens . Six isolates are likely to be the members of a new or uncharacterized genus/species. An anaerobic bacterium, Clostridium frigidicarnis was demonstrated to be lytic for amoebae. This preliminary work demonstrates that the water environment in the vicinity of humans is a reservoir of ARB, including well‐known pathogens for which amoebae and/or water was not recognized earlier as a possible reservoir.

Citrus huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The disease is associated with three different species of fastidious α-proteobacteria, namely ‘Candidatus Liberibacter asiaticus’, Ca. L. americanus, and Ca. L. africanus (1). ‘Ca. L. asiaticus’ was first detected in South Florida in 2005 and has spread throughout the citrus-growing areas of Florida. ‘Ca. L. asiaticus’ is transmitted naturally by the Asian citrus psyllid, Diaphorina citri, and can also be transmitted by graft propagation and via various species of dodder (Cuscuta). HLB affects most if not all citrus and citrus relatives within the family Rutaceae (2), including the ornamental shrub Murraya paniculata (4). In addition, ‘Ca. L. asiaticus’ and ‘Ca. L. americanus’ can infect tobacco (Nicotiana xanthi) and periwinkle (Catharanthus roseus) (1,4). Here we report that ‘Ca. L. asiaticus’ can infect tomato (Lycopersicon esculentum) cvs. Manapal and FL47. Manapal and FL 47 plants grown from seed were placed adjacent to ‘Ca. L. asiaticus’-infected sweet orange (Citrus sinensis) plants with dodder (Cuscuta pentagona) already well established on them. Young dodder shoots still connected to the citrus were draped over the tomato plants and subsequently also became attached to the tomato stems. After 1 month, the tomato plants were detached from the citrus and most of the dodder removed. One month later, these tomato plants started to show vein clearing and subsequently the mature leaves became thicker and leathery. Some leaves showed blotchy mottle symptoms and some fruits became lopsided in a manner similar to HLB symptom expression on citrus. PCR amplification of the ‘Ca. L. asiaticus’ 16S rDNA with primers OI1/OI2c and the β-operon with primers A2/J5 (1) revealed the presence of ‘Ca. L. asiaticus’ DNA. Sequence analysis confirmed that the sequences of the cloned amplicons were identical to those from the HLB-infected citrus source plant. Both conventional and quantitative real-time PCR (3) revealed a much lower abundance of ‘Ca. L. asiaticus’ DNA in tomato as compared with ‘Ca. L. asiaticus’-infected citrus or periwinkle, indicating that ‘Ca. L. asiaticus’ bacteria multiplied at a lower titer in these tomato cultivars. References: (1) J. M. Bové, J. Plant Pathol. 88:7, 2006. (2) S. E. Halbert et al. Fla. Entomol. 87:330, 2004. (3) W. Li et al. J. Microbiol. Methods 66:104, 2006. (4) L. Z. Zhou et al. Plant Dis. 91:227, 2007.