Circulating microRNA Profiles for Premature Cardiovascular Death in Patients with Kidney Failure with Replacement Therapy.

INTRODUCTION: Patients with kidney failure with replacement therapy (KFRT) suffer from a disproportionately high cardiovascular disease burden. Circulating small non-coding RNAs (c-sncRNAs) have emerged as novel epigenetic regulators and are suggested as novel biomarkers and therapeutic targets for cardiovascular disease; however, little is known about the associations of c-sncRNAs with premature cardiovascular death in KFRT. METHODS: In a pilot case-control study of 50 hemodialysis patients who died of cardiovascular events as cases, and 50 matched hemodialysis controls who remained alive during a median follow-up of 2.0 years, we performed c-sncRNAs profiles using next-generation sequencing to identify differentially expressed circulating microRNAs (c-miRNAs) between the plasma of cases and that of controls. mRNA target prediction and pathway enrichment analysis were performed to examine the functional relevance of differentially expressed c-miRNAs to cardiovascular pathophysiology. The association of differentially expressed c-miRNAs with cardiovascular mortality was examined using multivariable conditional logistic regression. RESULTS: The patient characteristics were similar between cases and controls, with a mean age of 63 years, 48% male, and 54% African American in both groups. We detected a total of 613 miRNAs in the plasma, among which five miRNAs (i.e., miR-129-1-5p, miR-500b-3p, miR-125b-1-3p, miR-3648-2-5p, and miR-3150b-3p) were identified to be differentially expressed between cases and controls with cut-offs of p < 0.05 and log2 fold-change (log2FC) > 1. When using more stringent cut-offs of p-adjusted < 0.05 and log2FC > 1, only miR-129-1-5p remained significantly differentially expressed, with higher levels of miR-129-1-5p in the cases than in the controls. The pathway enrichment analysis using predicted miR-129-1-5p mRNA targets demonstrated enrichment in adrenergic signaling in cardiomyocytes, arrhythmogenic right ventricular cardiomyopathy, and oxytocin signaling pathways. In parallel, the circulating miR-129-1-5p levels were significantly associated with the risk of cardiovascular death (adjusted OR [95% CI], 1.68 [1.01-2.81] for one increase in log-transformed miR-129-1-5p counts), independent of potential confounders. CONCLUSIONS: Circulating miR-129-1-5p may serve as a novel biomarker for premature cardiovascular death in KFRT.

Association of DNA methylation signatures with premature ageing and cardiovascular death in patients with end-stage kidney disease: a pilot epigenome-wide association study.

Patients with end-stage kidney disease (ESKD) display features of premature aging. There is strong evidence that changes in DNA methylation (DNAm) contribute to age-related pathologies; however, little is known about their association with premature aging and cardiovascular mortality in patients with ESKD. We assayed genome-wide DNAm in a pilot case-control study of 60 hemodialysis patients with (n=30, cases) and without (n=30, controls) a fatal cardiovascular event. DNAm was profiled on the Illumina EPIC BeadChip. Four established DNAm clocks (i.e., Horvath-, Hannum-, Pheno-, and GrimAge) were used to estimate epigenetic age (DNAmAge). Epigenetic age acceleration (EAA) was derived as the residuals of regressing DNAmAge on chronological age (chroAge), and its association with cardiovascular death was examined using multivariable conditional logistic regression. An epigenome-wide association study (EWAS) was performed to identify differentially methylated CpGs associated with cardiovascular death. All clocks performed well at predicting chroAge (correlation between DNAmAges and chroAge of r=0.76-0.89), with GrimAge showing the largest deviation from chroAge (a mean of +21.3 years). There was no significant association of EAAs with cardiovascular death. In the EWAS, a CpG (cg22305782) in the FBXL19 gene had the strongest association with cardiovascular death with significantly lower DNAm in cases vs. controls (PFDR=2.0x10-6). FBXL19 is involved in cell apoptosis, inflammation, and adipogenesis. Overall, we observed more accelerated aging in patients with ESKD, although there was no significant association of EAAs with cardiovascular death. EWAS suggests a potential novel DNAm biomarker for premature cardiovascular mortality in ESKD.

Circulating Microbiota in Cardiometabolic Disease.

The rapid expansion of microbiota research has significantly advanced our understanding of the complex interactions between gut microbiota and cardiovascular, metabolic, and renal system regulation. Low-grade chronic inflammation has long been implicated as one of the key mechanisms underlying cardiometabolic disease risk and progression, even before the insights provided by gut microbiota research in the past decade. Microbial translocation into the bloodstream can occur via different routes, including through the oral and/or intestinal mucosa, and may contribute to chronic inflammation in cardiometabolic disease. Among several gut-derived products identifiable in the systemic circulation, bacterial endotoxins and metabolites have been extensively studied, however recent advances in microbial DNA sequencing have further allowed us to identify highly diverse communities of microorganisms in the bloodstream from an -omics standpoint, which is termed "circulating microbiota." While detecting microorganisms in the bloodstream was historically considered as an indication of infection, evidence on the circulating microbiota is continually accumulating in various patient populations without clinical signs of infection and even in otherwise healthy individuals. Moreover, both quantitative and compositional alterations of the circulating microbiota have recently been implicated in the pathogenesis of chronic inflammatory conditions, potentially through their immunostimulatory, atherogenic, and cardiotoxic properties. In this mini review, we aim to provide recent evidence on the characteristics and roles of circulating microbiota in several cardiometabolic diseases, such as type 2 diabetes, cardiovascular disease, and chronic kidney disease, with highlights of our emerging findings on circulating microbiota in patients with end-stage kidney disease undergoing hemodialysis.

Circulating Microbial Signatures and Cardiovascular Death in Patients With ESRD.

INTRODUCTION: Patients with end-stage renal disease (ESRD) experience disproportionately high cardiovascular morbidity and mortality. Accumulating evidence suggests a role for the circulating microbiome in the pathogenesis of cardiovascular disease; however, little is known about its association with premature cardiovascular mortality in ESRD. METHODS: In a pilot case-control study of 17 hemodialysis patients who died of a cardiovascular event and 17 matched hemodialysis controls who remained alive during a median follow-up of 2.0 years, we compared the levels and composition of circulating microbiome, including Bacteria, Archaea, and Fungi, in serum samples by quantitative polymerase chain reaction and 16S or Internal Transcribed Spacer (ITS) ribosomal RNA (rRNA) sequencing, respectively. Associations of the circulating cell-free microbial signatures with clinical parameters and cardiovascular death were examined using the Spearman rank correlation and multivariable conditional logistic regression, respectively. RESULTS: Both 16S and ITS rRNA were detectable in all (except 3 for ITS) examined patients' serum samples. Despite no significant difference in 16S rRNA levels and α diversity between cases and controls, taxonomic analysis demonstrated differential community membership between groups, with significantly greater Actinobacteria and less Proteobacteria observed in cases than in controls at the phylum level. Proportions of Actinobacteria and Proteobacteria phyla were significantly correlated with plasma nuclear factor erythroid 2-related factor 2 (Nrf2) levels (rho = -0.41 and 0.42, P = 0.015 and 0.013, respectively) and marginally associated with risk of cardiovascular death (adjusted odds ratios [95% confidence intervals] = 1.12 [0.98-1.29] and 0.88 [0.76-1.02] for 1% increase, respectively). CONCLUSION: Alterations of the circulating cell-free microbial signatures may be associated with higher premature cardiovascular mortality in ESRD.

Homocysteine-induced neural tube defects in chick embryos via oxidative stress and DNA methylation associated transcriptional down-regulation of miR-124.

Although moderate homocysteine (HCY) elevation is associated with neural tube defects (NTDs), the underlying mechanisms have not been elucidated. In this study, we aimed to investigate that whether HCY-induced NTDs were associated with oxidative stress and methyl metabolism in chick embryos. The potential role of miR-124 in neurogenesis was also investigated. In this study, increased intracellular oxidative species and alterations in DNA methylation were observed following HCY treatment. This alteration coincided with decreases of Mn superoxide dismutase and glutathione peroxidase activities, as well as the expression of anti-rabbit DNA methyltransferase (DNMT) 1 and 3a. In addition, HCY induced significant decreases of S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) (P < 0.05). N-acetyl-L-cysteine and choline ameliorated global DNA hypomethylation induced by HCY. MiR-124 levels were significantly suppressed by HCY (P < 0.05), while elevated by 5-aza-2'-deoxycytidine (5-aza-dC). MiR-124 knockdown resulted in spina bifida occulta. Our research suggests that HCY-induced NTDs were associated with oxidative stress and methyl metabolism in chick embryos. MiR-124 down-regulation may occur via epigenetic mechanisms and contribute to HCY-induced NTDs in chick embryo models.

Association between Nrf2 and CDKN2A expression in patients with end-stage renal disease: a pilot study.

Patients with end-stage renal disease (ESRD) display phenotypic features of premature biological aging, characterized by disproportionately high morbidity and mortality at a younger age. Nuclear factor erythroid 2-related factor 2 (Nrf2) activity, a master regulator of antioxidative responses, declines with age and is implicated in the pathogenesis of age-related disorders; however, little is known about the association between Nrf2 and premature biological aging in ESRD patients. In a cross-sectional pilot cohort of 34 ESRD patients receiving maintenance hemodialysis, we measured the expression of Nrf2 and cyclin-dependent kinase inhibitor 2A (CDKN2A, or p16INK4a, a biomarker of biological aging) genes in whole blood and examined the association of Nrf2 with CDKN2A expression, using Spearman's rank correlation and multivariable linear regression models with adjustment for potential confounders. There was a significant negative correlation between Nrf2 and CDKN2A expression (rho=-0.51, P=0.002); while no significant correlation was found between Nrf2 expression and chronological age (rho=-0.02, P=0.91). After multivariable adjustment, Nrf2 expression remained significantly and negatively associated with CDKN2A expression (ß coefficient=-1.51, P=0.01), independent of chronological age, gender, race, and diabetes status. These findings suggest a potential contribution of Nrf2 dysfunction to the development of premature biological aging and its related morbidities in ESRD patients.

Validation of a Novel Modified Aptamer-Based Array Proteomic Platform in Patients with End-Stage Renal Disease.

End stage renal disease (ESRD) is characterized by complex metabolic abnormalities, yet the clinical relevance of specific biomarkers remains unclear. The development of multiplex diagnostic platforms is creating opportunities to develop novel diagnostic and therapeutic approaches. SOMAscan is an innovative multiplex proteomic platform which can measure >1300 proteins. In the present study, we performed SOMAscan analysis of plasma samples and validated the measurements by comparison with selected biomarkers. We compared concentrations of SOMAscan-measured prostate specific antigen (PSA) between males and females, and validated SOMAscan concentrations of fibroblast growth factor 23 (FGF23), FGF receptor 1 (FGFR1), and FGFR4 using Enzyme-Linked immunosorbent assay (ELISA). The median (25th and 75th percentile) SOMAscan PSA level in males and females was 4304.7 (1815.4 to 7259.5) and 547.8 (521.8 to 993.4) relative fluorescence units (p = 0.002), respectively, suggesting biological plausibility. Pearson correlation between SOMAscan and ELISA was high for FGF23 (R = 0.95, p < 0.001) and FGFR4 (R = 0.69, p < 0.001), indicating significant positive correlation, while a weak correlation was found for FGFR1 (R = 0.13, p = 0.16). In conclusion, there is a good to near-perfect correlation between SOMAscan and standard immunoassays for FGF23 and FGFR4, but not for FGFR1. This technology may be useful to simultaneously measure a large number of plasma proteins in ESRD, and identify clinically important prognostic markers to predict outcomes.

Changes With Lanthanum Carbonate, Calcium Acetate, and Phosphorus Restriction in CKD: A Randomized Controlled Trial.

INTRODUCTION: Abnormal phosphorus homeostasis develops early in chronic kidney disease (CKD). It is unclear if its correction results in improved clinical outcomes in non-dialysis dependent CKD. METHODS: We conducted a randomized controlled, parallel design clinical trial in 120 patients with estimated glomerular filtration rate 15 to 59 ml/min per 1.73 m2 and abnormal phosphorus homeostasis (serum phosphorus >4.6 mg/dl, parathyroid hormone [PTH] >70 pg/ml or tubular reabsorption of phosphorus [TRP] <80%). Patients were randomized to open-label lanthanum carbonate versus calcium acetate versus dietary intervention over 1 year. The co-primary outcomes were month 12 (vs. baseline) biochemical (serum phosphorus, TRP, PTH, calcium, bone-specific alkaline phosphatase [bALP], and fibroblast growth factor 23 [FGF23]) and vascular parameters (coronary artery calcium score, pulse wave velocity, and endothelial dysfunction) in all patients. Secondary outcomes were between-treatment differences in change for each parameter between month 12 and baseline. All analyses were intention to treat. RESULTS: Baseline characteristics were similar in the 3 groups. A total of 107 patients (89%) completed 12 months of follow-up. Differences were not significant at month 12 (vs. baseline) for any of the outcomes except bALP (median [25th, 75th] percentile at month 12 versus baseline: 13.8 [10.6, 17.6] vs. 15.8 [12.1, 21.1], P < .001) and FGF23 (132 [99, 216] vs. 133 [86, 189], P = .002). Changes for all outcomes were similar in the 3 arms except for PTH, which was suppressed more effectively by calcium acetate (P < .001). CONCLUSION: A 1-year intervention to limit phosphorus absorption using dietary restriction or 2 different phosphorus binders resulted in decreased bALP suggesting improved bone turnover, but no other significant changes in biochemical or vascular parameters in patients with CKD stage 3/4. (ClinicalTrials.gov: NCT01357317).

Identification of an Epitope from Adenine Nucleotide Translocator 1 That Induces Inflammation in Heart in A/J Mice.

Heart failure, a leading cause of death in humans, can emanate from myocarditis. Although most individuals with myocarditis recover spontaneously, some develop chronic dilated cardiomyopathy. Myocarditis may result from both infectious and noninfectious causes, including autoimmune responses to cardiac antigens. In support of this notion, intracellular cardiac antigens, like cardiac myosin heavy chain-α, cardiac troponin-I, and adenine nucleotide translocator 1 (ANT1), have been identified as autoantigens in cardiac autoimmunity. Herein, we demonstrate that ANT1 can induce autoimmune myocarditis in A/J mice by generating autoreactive T cells. We show that ANT1 encompasses multiple immunodominant epitopes (namely, ANT1 21-40, ANT1 31-50, ANT1 171-190, and ANT1 181-200). Although all four peptides induce comparable T-cell responses, only ANT1 21-40 was found to be a major myocarditogenic epitope in immunized animals. The myocarditis-inducing ability of ANT1 21-40 was associated with the generation of T cells producing predominantly IL-17A, and the antigen-sensitized T cells could transfer the disease to naïve recipients. These data indicate that cardiac mitochondrial proteins can be target autoantigens in myocarditis, supporting the notion that the antigens released as a result of primary damage may contribute to the persistence of chronic inflammation through autoimmunity.

Localization of CD8 T cell epitope within cardiac myosin heavy chain-α334-352 that induces autoimmune myocarditis in A/J mice.

BACKGROUND: Cardiac myosin heavy chain-α (Myhc), an intracellular protein expressed in the cardiomyocytes, has been identified as a major autoantigen in cardiac autoimmunity. In our studies with Myhc334-352-induced experimental autoimmune myocarditis in A/J mice (H-2a), we discovered that Myhc334-352, supposedly a CD4 T cell epitope, also induced antigen-specific CD8 T cells that transfer disease to naive animals. METHODS AND RESULTS: In our efforts to identify the CD8 T cell determinants, we localized Myhc338-348 within the full length-Myhc334-352, leading to four key findings. (1) By acting as a dual epitope, Myhc338-348 induces both CD4 and CD8 T cell responses. (2) In a major histocompatibility complex (MHC) class I-stabilization assay, Myhc338-348 was found to bind H-2Dd-but not H-2Kk or H-2Ld-alleles. (3) The CD8 T cell response induced by Myhc338-348 was antigen-specific, as evaluated by MHC class I/H-2Dd dextramer staining. The antigen-sensitized T cells predominantly produced interferon-γ, the critical cytokine of effector cytotoxic T lymphocytes. (4) Myhc338-348 was found to induce myocarditis in immunized animals as determined by histology and magnetic resonance microscopy imaging. CONCLUSIONS: Our data provide new insights as to how different immune cells can recognize the same antigen and inflict damage through different mechanisms.

MR elastography for evaluating regeneration of tissue-engineered cartilage in an ectopic mouse model.

PURPOSE: The purpose of the present study was to apply noninvasive methods for monitoring regeneration and mechanical properties of tissue-engineered cartilage in vivo at different growth stages using MR elastography (MRE). METHODS: Three types of scaffolds, including silk, collagen, and gelatin seeded by human mesenchymal stem cells, were implanted subcutaneously in mice and imaged at 9.4T where the shear stiffness and transverse MR relaxation time (T2 ) were measured for the regenerating constructs for 8 wk. An MRE phase contrast spin echo-based sequence was used for collecting MRE images. At the conclusion of the in vivo study, constructs were excised and transcript levels of cartilage-specific genes were quantitated using reverse-transcription polymerase chain reaction. RESULTS: Tissue-engineered constructs showed a cartilage-like construct with progressive tissue formation characterized by increase in shear stiffness and decrease in T2 that can be correlated with increased cartilage transcript levels including aggrecan, type II collagen, and cartilage oligomeric matrix protein after 8 wk of in vivo culture. CONCLUSION: Altogether, the outcome of this research demonstrates the feasibility of MRE and MRI for noninvasive monitoring of engineered cartilage construct's growth after implantation and provides noninvasive biomarkers for regeneration, which may be translated into treatment of tissue defects.

Effects of choline on sodium arsenite-induced neural tube defects in chick embryos.

Arsenic passes through the placenta and accumulates in the neuroepithelium of embryo, whereby inducing congenital malformations such as neural tube defects (NTDs) in animals. Choline (CHO), a methyl-rich nutrient, functions as a methyl donor to participate in methyl group metabolism. Arsenic methylation has been regarded as a detoxification process and choline (CHO) is the major source of methyl-groups. However, whether CHO intake reverses the abnormal embryo development induced by sodium arsenite (SA) and the relationship between CHO intake and arsenite-induced NTDs are still unclear. In this study, we used chick embryos as animal model to investigate the effects of SA and CHO supplementation on the early development of nervous system. Our results showed that the administration of SA led to reduction in embryo viability, embryo body weight and extraembryonic vascular area, accompanied by a significantly increased incidence of the failed closure of the caudal end of the neural tube. CHO, at low dose (25 µg/µL), reversed the decrease in embryo viability and the increase in the failed closure of the caudal end of the neural tube, which were induced by SA. In addition, CHO (25 µg/µL) inhibited not only the SA-induced cell apoptosis by up-regulating Bcl-2 level, but also the global DNA methylation by increasing the expressions of DNMT1 and DNMT3a. However, less significant difference was found between the embryos co-treated with SA and CHO (50 µg/µL) and the ones treated with SA alone. Taken together, these findings suggest that low dose CHO could protect chick embryos from arsenite-induced NTDs by a possible mechanism related to the methyl metabolism.

MicroRNA-181b and microRNA-9 mediate arsenic-induced angiogenesis via NRP1.

Environmental exposure to inorganic arsenic compounds has been reported to have serious health effects on humans. Recent studies reported that arsenic targets endothelial cells lining blood vessels, and endothelial cell activation or dysfunction, may underlie the pathogenesis of arsenic-induced diseases and developmental toxicity. It has been reported that microRNAs (miRNAs) may act as an angiogenic switch by regulating related genes. The present study was designed to test the hypothesis that arsenite-regulated miRNAs play pivotal roles in arsenic-induced toxicity. Fertilized eggs were injected via the yolk sac with 100 nM sodium arsenite at Hamburger-Hamilton (HH) stages 6, 9, and 12, and harvested at HH stage 18. To identify the individual miRNAs and mRNAs that may regulate the genetic network, the expression profiles of chick embryos were analyzed by microarray analysis. Microarray analyses revealed that the expression of a set of miRNAs changed after arsenite administration, especially miRNA-9, 181b, 124, 10b, and 125b, which exhibited a massive decrease in expression. Integrative analyses of the microarray data revealed that several miRNAs, including miR-9 and miR-181b, might target several key genes involved in arsenic-induced developmental toxicity. A luciferase reporter assay confirmed neuropilin-1 (Nrp1) as a target of mir-9 and mir-181b. Data from the transwell migration assay and the tube-formation assay indicated that miR-9 and mir-181b inhibited the arsenic-induced EA.hy926 cell migration and tube formation by targeting NRP1. Our study demonstrates that the environmental toxin, sodium arsenite, induced angiogenesis by altering the expression of miRNAs and their cognate mRNA targets.

Oxidative stress is implicated in arsenic-induced neural tube defects in chick embryos.

The potential of arsenic to induce neural tube defects (NTDs) remains a topic of controversy. In our previous study, oxidative stress and altered DNA methylation were observed in arsenic-exposed animal models. However, the correlation between these conditions was not fully understood. Therefore, our present aim was to determine whether arsenic exposure results in altered reactive oxygen species levels that affect DNA methylation and may contribute to NTDs in chick embryos. We demonstrated that arsenic-induced NTDs were associated with oxidative stress. Increased intracellular oxidative species and DNA methylation changes were observed following arsenic exposure. These changes were accompanied by a decrease in manganese superoxide dismutase activity. Furthermore, a significant decrease in DNA methyltransferase (DNMT) 1 and 3a expression was observed following arsenic exposure. The known antioxidant N-acetyl-l-cysteine, a known antioxidant, ameliorated global DNA hypomethylation and the decreased DNMT 1 and 3a expression observed during arsenic exposure. In addition, arsenic caused a significant decrease in S-adenosylmethionine (SAM) and significant increase in S-adenosylhomocysteine (SAH). This effect resulted in a significant reduction of the SAM/SAH ratio, which may also contribute to DNA hypomethylation. In conclusion, oxidative stress and reduction in SAM/SAH ratio during arsenic exposure in chick embryos seem to modulate DNA methylation and contribute to arsenic-induced NTDs via epigenetic mechanisms.

WITHDRAWN: Effects of choline on sodium arsenite-induced neural tube defects in chick embryos.

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Sodium thiosulfate exposure disrupts in vitro and in vivo heart development.

It is well-known that the majority of malformations found in the human population is based on complex gene-environment interactions. As an industrial chemical sodium thiosulfate (STS) is used heavily in many industries. Nevertheless, there is little known about the effects of STS on embryo development. In the present study, we have investigated the effects of STS on cardiac development in rat cardiomyocyte H9C2 cell line and chick embryos. As determined by MTT assays, the proliferation of H9C2 cells was inhibited by STS in a dose-dependent manner. Fertilized eggs injected via the yolk sac with STS at Hamburger-Hamilton (HH) stages 6, 9 and 12 showed significantly increased cardiotoxicity at HH stage 18, including cardiomyocyte apoptosis and animal mortality. Western blot analysis showed that STS significantly affected the expression of the apoptosis-related genes bcl-2, bax, and caspase-3 in a dose-dependent manner in the H9C2 cell line and in chick embryos. Dysregulation of apoptosis was correlated with embryonic heart malformations. Thus, STS may be a potent cardiac teratogen during embryo development.

Protective effects of N-acetylcysteine on homocysteine induced injury in chick embryos.

Protective effects of N-acetylcysteine (NAC) on homocysteine (Hcy)-induced injury have been reported in vitro. However, it is not known whether NAC has a similar effect in Hcy-induced injury during embryonic development. In this study, we tested the hypothesis that exogenous NAC can attenuate Hcy-induced injury in chick embryos. Hcy-induced apoptosis and reduced embryo viability were effectively attenuated by application of exogenous NAC. NAC could also rescue Hcy-induced inhibition of extra-embryonic vascular development. 2',7'-Dichlorofluorescein diacetate, an indicator of reactive oxygen species, was detected in H9C2 cells after treatment with Hcy. The results of this study provide the first evidence that NAC can protect against the adverse effect of Hcy during chick embryo development, and suggest that these effects are at least partly mediated by oxidative stress.

Genome-scale mutagenesis and phenotypic characterization of two-component signal transduction systems in Xanthomonas campestris pv. campestris ATCC 33913.

The gram-negative bacterium Xanthomonas campestris pv. campestris is the causal agent of black rot disease of cruciferous plants. Its genome encodes a large repertoire of two-component signal transduction systems (TCSTSs), which consist of histidine kinases and response regulators (RR) to monitor and respond to environmental stimuli. To investigate the biological functions of these TCSTS genes, we aimed to inactivate all 54 RR genes in X. campestris pv. campestris ATCC 33913, and successfully generated 51 viable mutants using the insertion inactivation method. Plant inoculation identified two novel response regulator genes (XCC1958 and XCC3107) that are involved in virulence of this strain. Genetic complementation demonstrated that XCC3107, designated as vgrR (virulence and growth regulator), also affects bacterial growth and activity of extracellular proteases. In addition, we assessed the survival of these mutants under various stresses, including osmotic stress, high sodium concentration, heat shock, and sodium dodecyl sulfate exposure, and identified a number of genes that may be involved in the general stress response of X. campestris pv. campestris. Mutagenesis and phenotypic characterization of RR genes in this study will facilitate future studies on signaling networks in this important phytopathogenic bacterium.

Two-component signal transduction systems of Xanthomonas spp.: a lesson from genomics.

The two-component signal transduction systems (TCSTSs), consisting of a histidine kinase sensor (HK) and a response regulator (RR), are the dominant molecular mechanisms by which prokaryotes sense and respond to environmental stimuli. Genomes of Xanthomonas generally contain a large repertoire of TCSTS genes (approximately 92 to 121 for each genome), which encode diverse structural groups of HKs and RRs. Among them, although a core set of 70 TCSTS genes (about two-thirds in total) which accumulates point mutations with a slow rate are shared by these genomes, the other genes, especially hybrid HKs, experienced extensive genetic recombination, including genomic rearrangement, gene duplication, addition or deletion, and fusion or fission. The recombinations potentially promote the efficiency and complexity of TCSTSs in regulating gene expression. In addition, our analysis suggests that a co-evolutionary model, rather than a selfish operon model, is the major mechanism for the maintenance and microevolution of TCSTS genes in the genomes of Xanthomonas. Genomic annotation, secondary protein structure prediction, and comparative genomic analyses of TCSTS genes reviewed here provide insights into our understanding of signal networks in these important phytopathogenic bacteria.