Publications

4364

ABSTRACTNitrogen is one of the major nutrients limiting microbial productivity in the ocean, and as a result, most marine microorganisms have evolved systems for responding to nitrogen stress. The highly abundant alphaproteobacterium “CandidatusPelagibacter ubique,” a cultured member of the orderPelagibacterales(SAR11), lacks the canonical GlnB, GlnD, GlnK, and NtrB/NtrC genes for regulating nitrogen assimilation, raising questions about how these organisms respond to nitrogen limitation. A survey of 266Alphaproteobacteriagenomes found these five regulatory genes nearly universally conserved, absent only in intracellular parasites and members of the orderPelagibacterales, including “Ca. Pelagibacter ubique.” Global differences in mRNA and protein expression between nitrogen-limited and nitrogen-replete cultures were measured to identify nitrogen stress responses in “Ca.Pelagibacter ubique” strain HTCC1062. Transporters for ammonium (AmtB), taurine (TauA), amino acids (YhdW), and opines (OccT) were all elevated in nitrogen-limited cells, indicating that they devote increased resources to the assimilation of nitrogenous organic compounds. Enzymes for assimilating amine into glutamine (GlnA), glutamate (GltBD), and glycine (AspC) were similarly upregulated. Differential regulation of the transcriptional regulator NtrX in the two-component signaling system NtrY/NtrX was also observed, implicating it in control of the nitrogen starvation response. Comparisons of the transcriptome and proteome supported previous observations of uncoupling between transcription and translation in nutrient-deprived “Ca.Pelagibacter ubique” cells. Overall, these data reveal a streamlined, PII-independent response to nitrogen stress in “Ca.Pelagibacter ubique,” and likely otherPelagibacterales, and show that they respond to nitrogen stress by allocating more resources to the assimilation of nitrogen-rich organic compounds.IMPORTANCEPelagibacteralesare extraordinarily abundant and play a pivotal role in marine geochemical cycles, as one of the major recyclers of labile dissolved organic matter. They are also models for understanding how streamlining selection can reshape chemoheterotroph metabolism. Streamlining and its broad importance to environmental microbiology are emerging slowly from studies that reveal the complete genomes of uncultured organisms. Here, we report another remarkable example of streamlined metabolism inPelagibacterales, this time in systems that control nitrogen assimilation.Pelagibacteralesare major contributors to metatranscriptomes and metaproteomes from ocean systems, where patterns of gene expression are used to gain insight into ocean conditions and geochemical cycles. The data presented here supply background that is essential to interpreting data from field studies.

Abstract Background Huanglongbing (HLB) is a highly destructive citrus disease which threatens citrus production worldwide and ‘Candidatus Liberibacter asiaticus’ (Las), a non-culturable phloem-limited bacterium, is an associated causal agent of the disease. To better understand the physiological and molecular processes involved in host responses to Las, 2-DE and mass spectrometry analyses, as well as ICP spectroscopy analysis were employed to elucidate the global protein expression profiles and nutrient concentrations in leaves of Las-infected grapefruit plants at pre-symptomatic or symptomatic stages for HLB. Results This study identified 123 protein spots out of 191 spots that showed significant changes in the leaves of grapefruit plants in response to Las infection and all identified spots matched to 69 unique proteins/peptides. A down-regulation of 56 proteins including those associated with photosynthesis, protein synthesis, and metabolism was correlated with significant reductions in the concentrations of Ca, Mg, Fe, Zn, Mn, and Cu in leaves of grapefruit plants in response to Las infection, particularly in symptomatic plants. Oxygen-evolving enhancer (OEE) proteins, a PSI 9 kDa protein, and a Btf3-like protein were among a small group of proteins that were down-regulated in both pre-symptomatic and symptomatic plants in response to Las infection. Furthermore, a Las-mediated up-regulation of 13 grapefruit proteins was detected, which included Cu/Zn superoxide dismutase, chitinases, lectin-related proteins, miraculin-like proteins, peroxiredoxins and a CAP 160 protein. Interestingly, a Las-mediated up-regulation of granule-bound starch synthase was correlated with an increase in the K concentrations of pre-symptomatic and symptomatic plants. Conclusions This study constitutes the first attempt to characterize the interrelationships between protein expression and nutritional status of Las-infected pre-symptomatic or symptomatic grapefruit plants and sheds light on the physiological and molecular mechanisms associated with HLB disease development.

In May of 2012, eggplant (Solanum melongena) plants in an experimental research plot located at Zamorano in the Department of Francisco Morazán, Honduras, were observed with symptoms that included leaf chlorosis and cupping, overall stunting, and production of small and malformed fruits. The research plot was planted next to a commercial tomato field heavily infested with the psyllid Bactericera cockerelli, a vector of ‘Candidatus Liberibacter solanacearum’ (1,2,3). This bacterium severely affects potato and other solanaceous species and is the putative causal agent of zebra chip disease (2,3). The plot was planted with the eggplant variety ‘China’ and about 25% of the plants were symptomatic. A total of 10 eggplant samples, including five symptomatic and five asymptomatic plants, were collected from the plot. Total DNA was extracted from the leaf tissue of each of the collected plants with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (1). The DNA samples were then tested by PCR using specific primer sets OA2/OI2c and OMB 1482f/2086r to amplify a portion of 16S rDNA and the outer membrane protein (OMB) genes, respectively, of ‘Ca. L. solanacearum’ (1,2). OMB gene and 16S rDNA fragments of 605 and 1,168 bp, respectively, were amplified from the DNA of two of the five (40%) symptomatic plants with each primer set, indicating the presence of ‘Ca. L. solanacearum.’ No ‘Ca. L. solanacearum’ was detected in the five asymptomatic plants with either primer sets. DNA amplicons with both primer sets were cloned from the DNA of the two ‘Ca. L. solanacearum’-positive plant samples and four clones of each of the four amplicons were sequenced. BLASTn analysis of the 16S rDNA resulted in two independent but related consensus sequences (deposited in GenBank as Accession Nos. KF188224 and KF188225) and were 99% similar to each other. The two sequences showed 99 to 100% identity to a number of 16S rDNA sequences of ‘Ca. L. solanacearum’ in Genbank, including accessions HM245242, FJ811596, and KC768319. For the OMB amplicons, a single consensus sequence was obtained following clone sequencing and was deposited in GenBank as accession KF188229. BLASTn analysis of the sequence indicated that it was 100% identical to several OMB sequences of ‘Ca. L. solanacearum’ in GenBank, including accessions KC768331 and CP002371. To our knowledge, this is the first report of ‘Ca. L. solanacearum’ associated with eggplant in Honduras. Eggplant is an economically important commodity in Central America and serious damage to this crop due to this plant pathogen could expand throughout the region, especially if its insect vector B. cockerelli is not properly managed. ‘Ca. L. solanacearum’ has also caused millions of dollars in losses to potato and several other solanaceous crops in the United States, Mexico, Central America, and New Zealand (2,3). In addition, this bacterium severely damages carrot crops in Europe, where it is transmitted to carrot by the psyllids Trioza apicalis and B. trigonica (3,4). It is imperative that both ‘Ca. L. solanacearum’ and its insect vectors be effectively monitored and managed to minimize their threat to economically important vegetable crops in many parts of the world. References: (1) J. M. Crosslin et al. Southwest. Entomol. 36:125, 2011. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza. Am. J. Pot. Res. 89:329, 2012. (4) J. E. Munyaneza et al. J. Econ. Entomol. 103:1060, 2010.

AbstractBackgroundDuring a two year period, a 27-year-old female veterinarian experienced migraine headaches, seizures, including status epilepticus, and other neurological and neurocognitive abnormalities. Prior to and during her illness, she had been actively involved in hospital-based work treating domestic animals, primarily cats and dogs, in Grenada and Ireland and anatomical research requiring the dissection of wild animals (including lions, giraffe, rabbits, mongoose, and other animals), mostly in South Africa. The woman reported contact with fleas, ticks, lice, biting flies, mosquitoes, spiders and mites and had also been scratched or bitten by dogs, cats, birds, horses, reptiles, rabbits and rodents. Prior diagnostic testing resulted in findings that were inconclusive or within normal reference ranges and no etiological diagnosis had been obtained to explain the patient’s symptoms.MethodsPCR assays targetingAnaplasmaspp.Bartonellaspp. and hemotopicMycoplasmaspp. were used to test patient blood samples. PCR positive amplicons were sequenced directly and compared to GenBank sequences. In addition,Bartonellaalpha Proteobacteria growth medium (BAPGM) enrichment blood culture was used to facilitate bacterial growth andBartonellaspp. serology was performed by indirect fluorescent antibody testing.ResultsAnaplasma platys, Bartonella henselaeandCandidatusMycoplasma haematoparvum DNA was amplified and sequenced from the woman’s blood, serum or blood culture samples. Her serum was variably seroreactive to severalBartonellasp. antigens. Despite symptomatic improvement, six months of doxycycline most likely failed to eliminate theB. henselaeinfection, whereasA. platysandCandidatusM. haematoparvum DNA was no longer amplified from post-treatment samples.ConclusionsAs is typical of many veterinary professionals, this individual had frequent exposure to arthropod vectors and near daily contact with persistently bacteremic reservoir hosts, including cats, the primary reservoir host forB. henselae,and dogs, the presumed primary reservoir host forA. platysandCandidatusMycoplasma haematoparvum. Physicians caring for veterinarians should be aware of the occupational zoonotic risks associated with the daily activities of these animal health professionals.