Publications

4439

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.

‘Candidatus Liberibacter asiaticus’ is the most prevalent Liberibacter sp. associated with huanglongbing (HLB) in Brazil. Within São Paulo state (SP), HLB has spread more rapidly to and reached higher incidence in regions with relatively mild (cooler) summer temperatures. This suggests that climate can influence disease spread and severity. ‘Ca. L. asiaticus’ titers on soft, immature leaves from infected ‘Valencia’ sweet orange plants exposed to different temperature regimes and adult Diaphorina citri fed for 48 h on these plants for ‘Ca. L. asiaticus’ acquisition were determined by quantitative polymerase chain reaction in two experiments. The first experiment included plants with three levels of infection, three incubation periods (IPs), and air temperatures favorable (14.6 to 28°C) and unfavorable (24 to 38°C) to ‘Ca. L. asiaticus’. The second included plants with severe late-stage infections, 10 IPs (based on 3-day intervals over 27 days), and three air temperature regimes (12 to 24, 18 to 30, and 24 to 38°C). Overall, ‘Ca. L. asiaticus’ titers and the percentages of ‘Ca. L. asiaticus’-positive psyllids were lower in plants maintained at the warmer temperature regime (24 to 38°C) than in plants maintained in the cooler regimes. The results suggest that the lower incidence and slower spread of ‘Ca. L. asiaticus’ to warmer regions of SP are related to the influence of ambient temperatures on titers of ‘Ca. L. asiaticus’ in leaves.