phz1 contributes much more to phenazine-1-carboxylic acid biosynthesis than phz2 in Pseudomonas aeruginosa rpoS mutant.

Pseudomonas aeruginosa PAO1, a common opportunistic bacterial pathogen, contains two phenazine-biosynthetic operons, phz1 (phzA1 B1 C1 D1 E1 F1 G1 ) and phz2 (phzA2 B2 C2 D2 E2 F2 G2 ). Each of two operons can independently encode a set of enzymes involving in the biosynthesis of phenazine-1-carboxylic acid. As a global transcriptional regulator, RpoS mediates a lot of genes involving secondary metabolites biosynthesis in many bacteria. In an other previous study, it was reported that RpoS deficiency caused overproduction of pyocyanin, a derivative of phenazine-1-carboxylic acid in P. aeruginosa PAO1. But it is not known how RpoS mediates the expression of each of two phz operons and modulates phenazine-1-carboxylic acid biosynthesis in detail. In this study, by deleting the rpoS gene in the mutant PNΔphz1 and the mutant PNΔphz2, we found that the phz1 operon contributes much more to phenazine-1-carboxylic acid biosynthesis than the phz2 operon in the absence of RpoS. With the construction of the translational and transcriptional fusion vectors with the truncated lacZ reporter gene, we demonstrated that RpoS negatively regulates the expression of phz1 and positively controls the expression of phz2, and the regulation of phenazine-1-carboxylic acid biosynthesis mediated by RopS occurs at the posttranscriptional level, not at the transcriptional level. Obviously, two copies of phz operons and their differential expression mediated by RpoS might help P. aeruginosa adapt to its diverse environments and establish infection in its hosts.

Zinc as a potential regulator of the BCR-ABL oncogene in chronic myelocytic leukemia cells.

BACKGROUND: Generally, decreased zinc in the serum of tumor patients but increased zinc in tumor cells can be observed. However, the role of zinc homeostasis in myeloid leukemia remains elusive. BCR-ABL is essential for the initiation, maintenance, and progression of chronic myelocytic leukemia (CML). We are currently investigating the association between zinc homeostasis and CML. METHODS: Genes involved in zinc homeostasis were examined using three GEO datasets. Western blotting and qPCR were used to investigate the effects of zinc depletion on BCR-ABL expression. Furthermore, the effect of TPEN on BCR-ABL promoter activity was determined using the dual-luciferase reporter assay. MRNA stability and protein stability of BCR-ABL were assessed using actinomycin D and cycloheximide. RESULTS: Transcriptome data mining revealed that zinc homeostasis-related genes were associated with CML progression and drug resistance. Several zinc homeostasis genes were affected by TPEN. Additionally, we found that zinc depletion by TPEN decreased BCR-ABL mRNA stability and transcriptional activity in K562 CML cells. Zinc supplementation and sodium nitroprusside treatment reversed BCR-ABL downregulation by TPEN, suggesting zinc- and nitric oxide-dependent mechanisms. CONCLUSION: Our in vitro findings may help to understand the role of zinc homeostasis in BCR-ABL regulation and thus highlight the importance of zinc homeostasis in CML.

Expression pattern and prognostic implication of zinc homeostasis-related genes in acute myeloid leukemia.

Zinc homeostasis is regulated by the SLC39A/ZIP, SLC30A/ZnT, and metallothionein (MT) protein families. The association of zinc homeostasis with acute myeloid leukemia (AML) is unclear. We previously demonstrated that zinc depletion by TPEN triggers apoptosis in NB4 AML cells with the degradation of PML-RARα oncoprotein, suggesting that zinc homeostasis may be associated with AML. The primary aim of this study was to explore the expression pattern and prognostic roles of zinc homeostasis-related genes in AML. Bioinformatics analyses were performed using integrated datasets from the TCGA and GTEx projects. The GEPIA tool was used to analyze the differential expression of zinc homeostasis-related genes. Correlations between zinc homeostasis-related genes were assessed with Spearman's correlation coefficient. OncoLnc was used to evaluate the prognostic roles of zinc homeostasis-related genes with Kaplan-Meier and Cox regression models. In both NB4 and U937 cells, the transcriptional regulation of zinc homeostasis-related genes by zinc depletion was detected through qPCR. We found that multiple ZIPs, ZnTs, and MTs were differentially expressed and correlated in AML tumors. In AML patients, higher expression of ZIP4 and lower expression of ZnT5 and ZnT7 predicted poorer survival. We further found that zinc depletion by TPEN upregulated ZIP7, ZIP9, ZIP10, ZIP13, and ZnT7 and downregulated ZIP14, ZnT1, ZnT6, and most of the positively expressed MTs in both NB4 and U937 AML cells. Our findings suggest high expression of ZIP4 and low expression of ZnT5 and ZnT7 as potential risk factors for the prognosis of AML. Zinc homeostasis may be a potential therapeutic target for AML, deserving further exploration.

vfr, A Global Regulatory Gene, is Required for Pyrrolnitrin but not for Phenazine-1-carboxylic Acid Biosynthesis in Pseudomonas chlororaphis G05.

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant G05ΔphzΔprn::lacZ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant G05Δvfr and G05ΔphzΔprn::lacZΔvfr. By quantifying ß-galactosidase activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant G05Δvfr, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.