Progress on the GntR family transcription regulators in bacteria.

In bacteria, GntR family transcription regulators are the widespread family of transcription factors. Members of this family consist of two functional domains, a conserved N-terminal DNA-binding domain that contains a typical helix-turn-helix (HTH) motif and a C-terminal effector-binding or oligomerization domain. Usually, the amino acid sequences of N-terminal DNA-binding domains are highly conserved, but differ in the C-terminal effector-binding or oligomerization domains. In the past several decades, many GntR family transcription regulators have been characterized in a number of bacteria. These regulators control a variety of cellular processes such as cell motility, glucose metabolism, bacterial resistance, pathogenesis and virulence. In this review, we summarized the discovery, C-terminal domains, biological function and regulation mode of GntR family transcription regulators. This review will help researchers to obtain more knowledge about the functions and mechanisms of the GntR family transcriptional regulatory factors.

MicroRNA-329-mediated PTTG1 downregulation inactivates the MAPK signaling pathway to suppress cell proliferation and tumor growth in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a severe malignancy usually producing a poor prognosis and high mortality rate. MicroRNAs (miRNAs) have been reported in association with CCA; however, the role miR-329 plays in the CCA condition still remains unclear. Therefore, this study was conducted to explore the underlying mechanism of which miR-329 is influencing the progression of CCA. This work studied the differential analysis of the expression chips of CCA obtained from the Gene Expression Omnibus database. Next, to determine both the expression and role of pituitary tumor transforming gene-1 (PTTG1) in CCA, the miRNAs regulating PTTG1 were predicted. In the CCA cells that had been intervened with miR-329 upregulation or inhibition, along with PTTG1 silencing, expression of miR-329, PTTG1, p-p38/p38, p-ERK5/ERK5, proliferating cell nuclear antigen (PCNA), Cyclin D1, Bcl-2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and caspase-3 were determined. The effects of both miR-329 and PTTG1 on cell proliferation, cell-cycle distribution, and apoptosis were also assayed. The miR-329 was likely to affect the CCA development through regulation of the PTTG1-mediated mitogen-activated protein kinase (MAPK) signaling pathway. The miR-329 targeted PTTG1, leading to inactivation of the MAPK signaling pathway. Upregulation of miR-329 and silencing of PTTG1 inhibited the CCA cell proliferation, induced cell-cycle arrest, and subsequently promoted apoptosis with elevations in Bax, cleaved caspase-3, and total caspase-3, but showed declines in PCNA, Cyclin D1, and Bcl-2. Moreover, miR-329 was also found to suppress the tumor growth by downregulation of PTTG1. To summarize, miR-329 inhibited the expression of PTTG1 to inactivate the MAPK signaling pathway, thus suppressing the CCA progression, thereby providing a therapeutic basis for the CCA treatment.

Competitive control of endoglucanase gene engXCA expression in the plant pathogen Xanthomonas campestris by the global transcriptional regulators HpaR1 and Clp.

Transcriptional regulators are key players in pathways that allow bacteria to alter gene expression in response to environmental conditions. However, work to understand how such transcriptional regulatory networks interact in bacterial plant pathogens is limited. Here, in the phytopathogen Xanthomonas campestris, we demonstrate that the global transcriptional regulator HpaR1 influences many of the same genes as another global regulator Clp, including the engXCA gene that encodes extracellular endoglucanase. We demonstrate that HpaR1 facilitates the binding of RNA polymerase to the engXCA promoter. In addition, we show that HpaR1 binds directly to the engXCA promoter. Furthermore, our in vitro tests characterize two binding sites for Clp within the engXCA promoter. Interestingly, one of these sites overlaps with the HpaR1 binding site. Mobility shift assays reveal that HpaR1 has greater affinity for binding to the engXCA promoter. This observation is supported by promoter activity assays, which show that the engXCA expression level is lower when both HpaR1 and Clp are present together, rather than alone. The data also reveal that HpaR1 and Clp activate engXCA gene expression by binding directly to its promoter. This transcriptional activation is modulated as both regulators compete to bind to overlapping sites on the engXCA promoter. Bioinformatics analysis suggests that this mechanism may be used broadly in Xanthomonas campestris pv. campestris (Xcc) and is probably widespread in Xanthomonads and, potentially, other bacteria. Taken together, these data support a novel mechanism of competitive activation by two global regulators of virulence gene expression in Xcc which is probably widespread in Xanthomonads and, potentially, other bacteria.

Diagnostic and prognostic value of lncRNA cancer susceptibility candidate 9 in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common malignant gastrointestinal tumor. There are currently few clinical diagnostic and prognostic markers for HCC. LncRNA cancer susceptibility candidate 9 (CASC9) is a long-chain non-coding RNA discovered in recent years, and previous studies have found that lncRNA CASC9 participates in the occurrence and development of HCC, but its clinical value remains unclear. AIM: To determine the expression of lncRNA CASC9 in HCC and its diagnostic and prognostic value. METHODS: Data on CASC9 expression in patients with HCC were collected from the Cancer Genome Atlas (TCGA) database to analyze the relationship between CASC9 and patient survival. A total of 80 HCC patients treated in The First Affiliated Hospital of Guangxi Medical University from May 2012 to January 2014 were enrolled in the patient group, and 50 healthy subjects were enrolled in the control group during the same period. CASC9 expression in the two groups was determined using quantitative real-time polymerase chain reaction, and its diagnostic and prognostic value was analyzed based on the CASC9 data and pathological data in these HCC patients. The relationship between CASC9 and patient survival was assessed during the 5-year follow-up period. RESULTS: Analysis of data from TCGA database revealed that control samples showed significantly lower CASC9 expression than carcinoma tissue samples (P < 0.001); the low CASC9 expression group had a higher survival rate than the high CASC9 expression group (P = 0.011), and the patient group showed significantly increased expression of serum CASC9, with the area under the curve (AUC) of 0.933. CASC9 expression was related to tumor size, combined hepatitis, tumor, node, metastasis (TNM) staging, lymph node metastasis, differentiation and alpha fetoprotein, and the high CASC9 expression group showed lower 1-year, 3-year and 5-year survival rates than the low CASC9 expression group (all a P < 0.05). Multivariate Cox regression analysis revealed that TNM staging, lymph node metastasis, differentiation, alpha fetoprotein and CASC9 were independent factors affecting the prognosis of patients. Stage I+II patients with lymph node metastasis, low differentiation, and alpha fetoprotein > 200 ng/mL had a poor 5-year survival rate. CONCLUSION: High CASC9 expression is beneficial in the prognosis of HCC patients. CASC9 is expected to be a potential diagnostic and prognostic indicator of HCC.

Characterization of the GntR family regulator HpaR1 of the crucifer black rot pathogen Xanthomonas campestris pathovar campestris.

The GntR family transcription regulator HpaR1 identified from Xanthomonas campestris pv. campestris has been previously shown to positively regulate the genes responsible for hypersensitive reaction and pathogenicity and to autorepress its own expression. Here, we demonstrated that HpaR1 is a global regulator that positively regulates diverse biological processes, including xanthan polysaccharide production, extracellular enzyme activity, cell motility and tolerance to various stresses. To investigate the regulatory mechanisms of HpaR1, we began with xanthan polysaccharide production, which is governed by a cluster of gum genes. These are directed by the gumB promoter. Disruption of HpaR1 significantly reduced gumB transcription and an electrophoretic mobility shift assay demonstrated that HpaR1 interacts directly with gumB promoter. DNase I footprint analysis revealed that HpaR1 and RNA polymerase were bound to the sequences extending from -21 to +10 and -41 to +29 relative to the transcription initiation site of gumB, respectively. Furthermore, in vitro transcription assays showed that HpaR1 facilitated the binding of RNA polymerase to gumB promoter, leading to an enhancement of its transcription. These results suggest that HpaR1 regulates gumB transcription via a mechanism similar but different to what was found, until now, to only be used by some MerR family transcription activators.

Identification of a putative cognate sensor kinase for the two-component response regulator HrpG, a key regulator controlling the expression of the hrp genes in Xanthomonas campestris pv. campestris.

The bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc) relies on the hrp (hypersensitive response and pathogenicity) genes to cause disease and induce hypersensitive response (HR). The hrp genes of bacterial phytopathogens are divided into two groups. Xcc hrp genes belong to group II. It has long been known that the group II hrp genes are activated by an AraC-type transcriptional regulator whose expression is controlled by a two-component system (TCS) response regulator (named HrpG in Xcc). However, no cognate sensor kinase has yet been identified. Here, we present evidence showing that the Xcc open-reading frame XC_3670 encodes a TCS sensor kinase (named HpaS). Mutation of hpaS almost completely abolished the HR induction and virulence. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacted with HrpG. Phos-tag™ SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of HrpG in vivo. These data suggest that HpaS and HrpG are most likely to form a TCS. We also showed that XC_3669 (named hpaR2), which is adjacent to hpaS and encodes a putative TCS response regulator, is required for full virulence but not HR induction. HpaR2 also physically interacted with HpaS, suggesting that HpaS may also form another TCS with HpaR2.

Systematic mutagenesis of all predicted gntR genes in Xanthomonas campestris pv. campestris reveals a GntR family transcriptional regulator controlling hypersensitive response and virulence.

The GntR family is one of the most abundant and widely distributed groups of helix-turn-helix transcriptional regulators in bacteria. Six open reading frames in the genome of the plant pathogen Xanthomonas campestris pv. campestris were predicted to encode GntR regulators. All six of the predicted GntR-encoding genes were individually mutagenized and mutants from five of them were successfully obtained. Plant disease response assays revealed that one, whose product belongs to the YtrA subfamily and has been named HpaR1, is involved in the hypersensitive response (HR) and virulence. Electrophoretic mobility shift assays and in vitro transcription assays revealed that HpaR1 could repress its own transcription level through binding to its promoter sequence, indicating an autoregulatory feedback inhibition mechanism for HpaR1 expression. Promoter-gusA reporter and reverse-transcription polymerase chain reaction analyses revealed that HpaR1 positively and negatively affects the expression of HR and pathogenicity (hrp) genes in host plant and standard media, respectively. Constitutive expression of the key hrp regulator, hrpG, in the hpaR1 mutant could bypass the requirement of HpaR1 for the induction of wild-type HR, suggesting that HpaR1 regulates the expression of hrp genes that encode the type III secretion system via hrpG.

[Identification of a gene involved in the expression of the pathogenicity-related gene XC3814 in Xanthomonas campestris].

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of the black rot disease of cruciferous plants. Our previous work had demonstrated that XC3814 is required for full virulence and extracellular polysaccharide production. In this work, the reporter plasmid pL3814sac was constructed by fusing the promoter region of XC3814 to the coding region of the gene sacB, and introduced into Xcc wild-type strain 8004. The resulted strain 8004/pL3814sac was mutagenized randomly by the transposon EZ::Tn5, and 3 mutant strains insensitive to sucrose were isolated. One of the mutants was due to the disruption of the open reading frame XC3882, which was assigned to code a hypothetical protein. To verify whether XC3882 has an impact on the expression level of XC3814, the reporter plasmid pGUS3814 was constructed by fusing the promoter region of XC3814 to the coding region of the gusA gene. This construct was introduced into the wild-type strain 8004 and the XC3882 mutant strain 190A10, which was derived from the transposon Tn5gusA5 insertion. The GUS activity, produced by pGUS3814 in the XC3882 mutant background, was reduced by 81.3% compared to that in the wild type background. These results indicate that the expression of XC3814 is influenced by XC3882.

Glyceraldehyde-3-phosphate dehydrogenase of Xanthomonas campestris pv. campestris is required for extracellular polysaccharide production and full virulence.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glucose catabolism, converting glyceraldehyde 3-phosphates to 1,3-bisphosphoglycerates. Open reading frame (ORF) XC_0972 in the genome of Xanthomonas campestris pv. campestris (Xcc) strain 8004 is the only ORF in this strain annotated to encode a GAPDH. In this work, we have demonstrated genetically that this ORF encodes a unique GAPDH in Xcc strain 8004, which seems to be constitutively expressed. A GAPDH-deficient mutant could still grow in medium with glucose or other sugars as the sole carbon source, and no phosphofructokinase activity was detectable in strain 8004. These facts suggest that Xcc may employ the Entner-Doudoroff pathway, but not glycolysis, to utilize glucose. The mutant could not utilize pyruvate as sole carbon source, whereas the wild-type could, implying that the GAPDH of Xcc is involved in gluconeogenesis. Furthermore, inactivation of the Xcc GAPDH resulted in impairment of bacterial growth and virulence in the host plant, and reduction of intracellular ATP and extracellular polysaccharide (EPS). This reveals that GAPDH is required for EPS production and full pathogenicity of Xcc.