The Pseudomonas aeruginosa phosphodiesterase gene nbdA is transcriptionally regulated by RpoS and AmrZ

Promoter Region ◽

Transcriptional Start Site ◽

Guanosine Monophosphate ◽

Enzymatic Function ◽

Nitrite Reductase Gene ◽

Biofilm Dispersal ◽

Pseudomonas aeruginosa is an opportunistic pathogen causing serious infections in immune compromised persons. These infections are difficult to erase with antibiotics, due to the formation of biofilms. The biofilm lifecycle is regulated by the second messenger molecule c-di-GMP (bis-3,5-cyclic di-guanosine monophosphate). P. aeruginosa encodes 40 genes for enzymes presumably involved in the biosynthesis and degradation of c-di-GMP. A tight regulation of expression, subcellular localized function and protein interactions control the activity of these enzymes. In this work we elucidated the transcriptional regulation of the gene encoding the membrane-bound phosphodiesterase NbdA. We previously reported a transcriptional and posttranslational role of nitric oxide (NO) on nbdA and its involvement in biofilm dispersal. NO is released from macrophages during infections but can also be produced by P. aeruginosa itself during anaerobic denitrification. Recently however, contradictory results about the role of NbdA within NO-induced biofilm dispersal were published. Therefore, the transcriptional regulation of nbdA was reevaluated to obtain insights into this discrepancy. Determination of the transcriptional start site of nbdA by 5'-RACE and subsequent identification of the promoter region revealed a shortened open reading frame (ORF) in contrast to the annotated one. In addition, putative binding sites for RpoS and AmrZ were discovered in the newly defined promoter region. Employing chromosomally integrated transcriptional lacZ reporter gene fusions demonstrated a RpoS-dependent activation and AmrZ repression of nbdA transcription. In order to investigate the impact of NO on nbdA transcription, conditions mimicking exogenous and endogenous NO were applied. While neither exogenous nor endogenous NO had an influence on nbdA promoter activity, deletion of the nitrite reductase gene nirS strongly increased nbdA transcription independently of its enzymatic activity during denitrification. The latter supports a role of NirS in P. aeruginosa apart from its enzymatic function.

PmtA Regulates Pyocyanin Expression and Biofilm Formation in Pseudomonas aeruginosa

Frontiers in Microbiology ◽

10.3389/fmicb.2021.789765 ◽

2021 ◽

Vol 12 ◽

Author(s):

Amy V. Thees ◽

Kathryn M. Pietrosimone ◽

Clare K. Melchiorre ◽

Jeremiah N. Marden ◽

Joerg Graf ◽

...

Keyword(s):

Oxidative Stress ◽

Pseudomonas Aeruginosa ◽

Metal Binding ◽

Biofilm Formation ◽

Binding Capacity ◽

Opportunistic Pathogen ◽

Small Molecular Weight ◽

Heavy Metal Binding ◽

The opportunistic pathogen Pseudomonas aeruginosa expresses a small molecular weight, cysteine-rich protein (PmtA), identified as a metallothionein (MT) protein family member. The MT family proteins have been well-characterized in eukaryotes as essential for zinc and copper homeostasis, protection against oxidative stress, and the ability to modify a variety of immune activities. Bacterial MTs share sequence homology, antioxidant chemistry, and heavy metal-binding capacity with eukaryotic MTs, however, the impact of bacterial MTs on virulence and infection have not been well-studied. In the present study, we investigated the role of PmtA in P. aeruginosa PAO1 using a PmtA-deficient strain (ΔpmtA). Here we demonstrated the virulence factor, pyocyanin, relies on the expression of PmtA. We showed that PmtA may be protective against oxidative stress, as an alternative antioxidant, glutathione, can rescue pyocyanin expression. Furthermore, the expression of phzM, which encodes a pyocyanin precursor enzyme, was decreased in the ΔpmtA mutant during early stationary phase. Upregulated pmtA expression was previously detected in confluent biofilms, which are essential for chronic infection, and we observed that the ΔpmtA mutant was disrupted for biofilm formation. As biofilms also modulate antibiotic susceptibility, we examined the ΔpmtA mutant susceptibility to antibiotics and found that the ΔpmtA mutant is more susceptible to cefepime and ciprofloxacin than the wild-type strain. Finally, we observed that the deletion of pmtA results in decreased virulence in a waxworm model. Taken together, our results support the conclusion that PmtA is necessary for the full virulence of P. aeruginosa and may represent a potential target for therapeutic intervention.

Autophagy plays a protective role during Pseudomonas aeruginosa-induced apoptosis via ROS–MAPK pathway

Innate Immunity ◽

10.1177/1753425920952156 ◽

2020 ◽

Vol 26 (7) ◽

pp. 580-591

Author(s):

Lu Han ◽

Qinmei Ma ◽

Jialin Yu ◽

Zhaoqian Gong ◽

Chenjie Ma ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Mapk Pathway ◽

Protective Role ◽

Vital Role ◽

Raw264.7 Cells ◽

Cellular Reactive Oxygen Species ◽

Induced Apoptosis ◽

Related Proteins ◽

Pseudomonas aeruginosa infection can induce alveolar macrophage apoptosis and autophagy, which play a vital role in eliminating pathogens. These two processes are usually not independent. Recently, autophagy has been found to interact with apoptosis during pathogen infections. Nevertheless, the role of autophagy in P. aeruginosa-infected cell apoptosis is unclear. In this study, we explored the impact of P. aeruginosa infection on autophagy and apoptosis in RAW264.7 cells. The autophagy activator rapamycin was used to stimulate autophagy and explore the role of autophagy on apoptosis in P. aeruginosa-infected RAW264.7 cells. The results indicated that P. aeruginosa infection induced autophagy and apoptosis in RAW264.7 cells, and that rapamycin could suppress P. aeruginosa-induced apoptosis by regulating the expression of apoptosis-related proteins. In addition, rapamycin scavenged the cellular reactive oxygen species (ROS) and diminished p-JNK, p-ERK1/2 and p-p38 expression of MAPK pathways in RAW264.7 cells infected with P. aeruginosa. In conclusion, the promotion of autophagy decreased P. aeruginosa-induced ROS accumulation and further attenuated the apoptosis of RAW264.7 cells through MAPK pathway. These results provide novel insights into host–pathogen interactions and highlight a potential role of autophagy in eliminating P. aeruginosa.

Independent Regulation of MucD, an HtrA-Like Protease in Pseudomonas aeruginosa, and the Role of Its Proteolytic Motif in Alginate Gene Regulation

Journal of Bacteriology ◽

10.1128/jb.188.8.3134-3137.2006 ◽

2006 ◽

Vol 188 (8) ◽

pp. 3134-3137 ◽

Cited By ~ 39

Author(s):

Lynn F. Wood ◽

Dennis E. Ohman

Keyword(s):

Pseudomonas Aeruginosa ◽

Gene Regulation ◽

Transcriptional Start Site ◽

Single Copy ◽

Start Site ◽

Temperature Resistance

ABSTRACT Expression of mucD, encoding a homologue of the HtrA(DegP) family of endoserine proteases, was investigated in Pseudomonas aeruginosa. Expressed from the algT-mucABCD operon, MucD was detected in mucoid (FRD1) and nonmucoid (PAO1) parental strains and also when polar insertions were placed upstream in algT or mucB. A transcriptional start site for a mucD promoter (PmucD) was mapped within mucC. Expression of single-copy mucD217, encoding MucD altered in the protease motif (S217A), was defective in temperature resistance and alginate gene regulation.

Fucose-Mediated Transcriptional Activation of the fcs Operon by FcsR in Streptococcus pneumoniae

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000377724 ◽

2015 ◽

Vol 25 (2-3) ◽

pp. 120-128 ◽

Cited By ~ 3

Author(s):

Irfan Manzoor ◽

Sulman Shafeeq ◽

Muhammad Afzal ◽

Oscar P. Kuipers

Keyword(s):

Streptococcus Pneumoniae ◽

Transcriptional Activation ◽

Promoter Region ◽

Transcriptional Regulator ◽

Transcriptional Activator ◽

Regulatory Site ◽

Rt Pcr ◽

In this study, we explore the impact of fucose on the transcriptome of S. pneumoniae D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (fcs operon) was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the fcs operon, as a transcriptional activator of the fcs operon. We also predict a 19-bp putative FcsR regulatory site (5′-ATTTGAACATTATTCAAGT-3′) in the promoter region of the fcs operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the fcs operon.

The role of Biofilm for Pseudomonas aeruginosa to resistance ultraviolet and MIC concentration for antibiotics

The Iraqi Journal of Veterinary Medicine ◽

10.30539/iraqijvm.v36i0a.348 ◽

2012 ◽

Vol 36 (0A) ◽

pp. 13-19

Author(s):

احمد محمد تركي

Keyword(s):

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

High Resistance ◽

The Other ◽

Bacterial Isolates ◽

Staph Aureus ◽

E Coli ◽

The Impact ◽

And Staphylococcus Aureus

The present study is conducted to in restigate the bacteria Pseudomonas aeruginosa, the impact of ultraviolet on the bacterial isolates under study and the resistance of these isolates to ultraviolet are studied in comparison to two standard isolates ( E . coli and Staphylococcus aureus ) which are considered sensitive to ultraviolet . The natures of the resistance of the isolates, under study, are also being investigated against the different antibiotics. The isolates are subjected to a test to examine their sensitivity to (12) types of antibiotics used routinely in the treatment of various infection of these bacteria. They are (streptomycin , cephalothin ,Gentamycin , cefotaxime ,nitrofurantion ,ampicillin, amoxicillin, rifampin, lincomycin, tetracycline, erythromycin and ciprofloxacin ).The lowest concentration installer ( MIC ) is also testified in accordance with six types of antibiotics (streptomycin, cefotaxime , rifampin , nitrofurantion , Gentamycin , amoxicillin ).The biologic effectiveness of the overlap between the bacterial isolates , under study, is examined against four bacteria (klebseilla pneumonia , Staphylococcus aureus , Enterobacter , Proteus ) The result of using the ultraviolet with different wavelength show the ability of the five local isolates used to resistance of ultraviolet reaching (180 s.) in comparison to the isolates E.coli and staph. aureus in which the ratio of killing is %100 at a time of exposing 40 , 60 sec. respectively. The results indicated that the five local bacterial isolates have high resistance to the most tested antibiotics, It is shouted that all of them have resistance to (erythromycin , tetracycline , lincomycin , Gentamycin ) but they are sensitive towards antibiotic streptomycin . as for the other antibiotics , over can find that the isolates are varied of them for being resisting or sensitive towards them .The results of testing the inhabited effectiveness of the five bacterial isolates towards some other bacterial isolates show the efficiency of the five local isolates in the inhabitation of growth of the five studied bacterial isolates.

Role of yoaE Gene Regulated by CpxR in the Survival of Salmonella enterica Serovar Enteritidis in Antibacterial Egg White

mSphere ◽

10.1128/msphere.00638-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 1

Author(s):

Xiaozhen Huang ◽

Mengjun Hu ◽

Xiujuan Zhou ◽

Yanhong Liu ◽

Chunlei Shi ◽

...

Keyword(s):

Salmonella Enterica ◽

Promoter Region ◽

Antibacterial Properties ◽

Egg White ◽

Global Public Health ◽

Mobility Shift ◽

Content Type ◽

Egg Products

ABSTRACT The survival ability of Salmonella enterica serovar Enteritidis in antibacterial egg white is an important factor leading to Salmonella outbreaks through eggs and egg products. In this study, the role of the gene yoaE, encoding an inner membrane protein, in the survival of Salmonella Enteritidis in egg white, and its transcriptional regulation by CpxR were investigated. Quantitative reverse transcription-PCR (RT-qPCR) results showed that the yoaE gene expression was upregulated 35-fold after exposure to egg white for 4 h compared to that in M9FeS medium, and the deletion of yoaE (ΔyoaE) dramatically decreased the survival rate of bacteria in egg white to less than 1% of the wild type (WT) and the complementary strain at both 37 and 20°C, indicating that yoaE was essential for bacteria to survive in egg white. Furthermore, the ΔyoaE strain was sensitive to a 3-kDa ultrafiltration matrix of egg white because of its high pH and antimicrobial peptide components. Putative conserved binding sites for the envelope stress response regulator CpxR were found in the yoaE promoter region. In vivo, the RT-qPCR assay results showed that the upregulation of yoaE in a ΔcpxR strain in egg white was 1/5 that of the WT. In vitro, results from DNase I footprinting and electrophoretic mobility shift assays further demonstrated that CpxR could directly bind to the yoaE promoter region, and a specific CpxR binding sequence was identified. In conclusion, it was shown for the first time that CpxR positively regulated the transcription of yoaE, which was indispensable for survival of Salmonella Enteritidis in egg white. IMPORTANCE Salmonella enterica serovar Enteritidis is the predominant Salmonella serotype that causes human salmonellosis mainly through contaminated chicken eggs or egg products and has been a global public health threat. The spread and frequent outbreaks of this serotype through eggs correlate significantly with its exceptional survival in eggs, despite the antibacterial properties of egg white. Research on the survival mechanisms of S. Enteritidis in egg white will help develop effective strategies to control the contamination of eggs by this Salmonella serotype and help further elucidate the complex antibacterial mechanisms of egg white. This study revealed the importance of yoaE, a gene with unknown function, on the survival of S. Enteritidis in egg white, as well as its transcriptional regulation by CpxR. Our work provides the basis to reveal the mechanisms of survival of S. Enteritidis in egg white and the specific function of the yoaE gene.

Mechanisms of Transcriptional Regulation and Transformation by HOXA9

Blood ◽

10.1182/blood.v120.21.sci-30.sci-30 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-30-SCI-30

Author(s):

Jay L. Hess ◽

Cailin Collins ◽

Joel Bronstein ◽

Yuqing Sun ◽

Surya Nagaraja

Keyword(s):

Transcription Factors ◽

Transcriptional Activation ◽

Binding Sites ◽

Target Genes ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

A Genome ◽

Abstract Abstract SCI-30 HOXA9 plays important roles in both development and hematopoiesis and is overexpressed in more than 50 percent of acute myeloid leukemias (AML). Nearly all cases of AML with mixed lineage leukemia (MLL) translocations show increased HOXA9 expression, as well as cases with mutation of the nucleophosmin gene NPM1, overexpression of CDX2, and fusions of NUP98. In most cases, upregulation of HOXA9 is accompanied by upregulation of its homeodomain-containing cofactor MEIS1, which directly interacts with HOXA9. While HOXA9 alone is sufficient for transformation of hematopoietic stem cells in culture, the addition of MEIS1 increases the transformation efficiency and results in rapidly fatal leukemias in transplanted animals. Despite the crucial role that HOXA9 plays in development, hematopoiesis, and leukemia, its transcriptional targets and mechanisms of action are poorly understood. We have used ChIP-seq to identify Hoxa9 and Meis1 binding sites on a genome-wide level in myeloblastic cells, profiled their associated epigenetic modifications, identified the target genes regulated by HOXA9 and identified HOXA9 interacting proteins. HOXA9 and MEIS1 cobind at hundreds of promoter distal, highly evolutionarily conserved sites showing high levels of histone H3K4 monomethylation and CBP/P300 binding. These include many proleukemogenic gene loci, such as Erg, Flt3, Myb, Lmo2, and Sox4. In addition, HOXA9 binding sites overlap a subset of enhancers previously implicated in myeloid differentiation and inflammation. HOXA9 binding at enhancers stabilizes association of MEIS1 and lineage-restricted transcription factors, including C/EBPα, PU.1, and STAT5A/B thereby promoting CBP/p300 recruitment, histone acetylation, and transcriptional activation. Current efforts are focused on using both biochemical and genetic approaches to assess the role of HOXA9 “enhanceosome” components C/EBPα, PU.1, and STAT5A/B in transcriptional regulation and leukemogenesis. Studies to date suggest that C/EBPα and PU.1 binding can occur in the absence of HOXA9/MEIS1, supporting a model in which these proteins act as pioneer transcription factors for establishment of poised, but not activated, HOXA9-regulated enhancers. Work is under way to assess the impact of high-level HOXA9 and MEIS1 on enhanceosome assembly and the role of recruitment of transcriptional coactivators involved in target gene up- or downregulation, including histone acetyltransferases and chromatin remodeling complexes. Collectively, our findings suggest that HOXA9-regulated enhancers are a fundamental mechanism of HOX-mediated transcription in normal development that is deregulated in leukemia. Disclosures: No relevant conflicts of interest to declare.

Elastase LasB of Pseudomonas aeruginosa promotes biofilm formation partly through rhamnolipid-mediated regulation

Canadian Journal of Microbiology ◽

10.1139/cjm-2013-0667 ◽

2014 ◽

Vol 60 (4) ◽

pp. 227-235 ◽

Cited By ~ 29

Author(s):

Hua Yu ◽

Xiaomei He ◽

Wei Xie ◽

Junzhi Xiong ◽

Halei Sheng ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Deletion Mutant ◽

Gene Expression Analysis ◽

Bacterial Attachment ◽

Transcriptional Level ◽

Complementary Strain ◽

Host Infection ◽

Elastase LasB, an important extracellular virulence factor, is shown to play an important role in the pathogenicity of Pseudomonas aeruginosa during host infection. However, the role of LasB in the life cycle of P. aeruginosa is not completely understood. This report focuses on the impact of LasB on biofilm formation of P. aeruginosa PAO1. Here, we reported that the lasB deletion mutant (ΔlasB) displayed significantly decreased bacterial attachment, microcolony formation, and extracellular matrix linkage in biofilm associated with decreased biosynthesis of rhamnolipids compared with PAO1 and lasB complementary strain (ΔlasB+). Nevertheless, the ΔlasB developed restored biofilm formation with supplementation of exogenous rhamnolipids. Further gene expression analysis revealed that the mutant of lasB could result in the downregulation of rhamnolipid synthesis at the transcriptional level. Taken together, these results indicated that LasB could promote biofilm formation partly through the rhamnolipid-mediated regulation.

Role of MexZ and PA5471 in transcriptional regulation of mexXY in Pseudomonas aeruginosa

Microbiology ◽

10.1099/mic.0.028993-0 ◽

2009 ◽

Vol 155 (10) ◽

pp. 3312-3321 ◽

Cited By ~ 24

Author(s):

Masaki Yamamoto ◽

Atsuhisa Ueda ◽

Makoto Kudo ◽

Yasuhiro Matsuo ◽

Jun Fukushima ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Molecular Mechanisms ◽

Efflux Pump ◽

Mobility Shift ◽

Drug Induced ◽

Drug Efflux Pump ◽

Mobility Shift Assay

MexXY, a drug efflux pump in Pseudomonas aeruginosa, confers resistance to aminoglycoside antibiotics. We recently reported that MexZ binds to the promoter region of the mexXY operon. Electrophoretic mobility shift assay (EMSA) using recombinant MexZ and oligonucleotide probes prepared from the intergenic region between mexZ and mexX revealed that MexZ binds to a 20 bp palindromic sequence. Culture of P. aeruginosa in the presence of tetracycline induced higher levels of MexX and MexZ, as measured by immunoblotting and EMSA, than in the absence of antibiotics. When MexZ was expressed by a mexZ expression plasmid, the plasmid-borne MexZ repressed drug-induced MexX production, further confirming that MexZ acts as a repressor of the mexXY operon. PA5471 protein has been reported to be essential for drug-induced MexXY production. Similarly to that report, we observed that plasmid-borne PA5471 induced both MexX and MexZ production in PAO1 cells. Interestingly, interaction between MexZ and PA5471 was observed in a yeast two-hybrid assay. Furthermore, EMSA and in vitro transcription assays revealed that interaction between PA5471 and MexZ reduced MexZ DNA-binding ability, leading to mexXY transcription. These findings contribute to the understanding of the molecular mechanisms underlying the transcriptional regulation of mexZ and mexXY by drug-induced PA5471 expression.

Glucocorticoid Receptor Regulates TNFSF11 Transcription by Binding to Glucocorticoid Responsive Element in TNFSF11 Proximal Promoter Region

International Journal of Molecular Sciences ◽

10.3390/ijms22031054 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1054

Author(s):

Nika Lovšin ◽

Janja Marc

Keyword(s):

Glucocorticoid Receptor ◽

Promoter Region ◽

Promoter Activity ◽

Mutational Analysis ◽

Proximal Promoter ◽

Rankl Mrna ◽

Glucocorticoid Responsive Element ◽

Responsive Element ◽

Glucocorticoid osteoporosis is a serious side effect of long term glucocorticoid uptake and it is caused by osteoblast apoptosis and imbalance in the major bone remodeling pathway RANK/RANKL/OPG. The impact of glucocorticoid on the maintenance of RANK/RANKL/OPG is well explored; dexamethasone was shown to disturb the ratio between OPG and RANKL level by decreasing the expression level of OPG and increasing level of RANKL. Here, were aimed to decipher whether glucocorticoid receptor directly influences RANKL promoter activity and its transcriptional regulation. We demonstrate that overexpression of glucocorticoid receptor (GR) NR3C1 increased RANKL promoter activity in human osteosarcoma, cervical cancer (2-fold) and adenocarcinoma cells (4.5-fold). Mutational analysis revealed that +352 site in the RANKL promoter is functional glucocorticoid responsive element (GRE) since the effect of GR on RANKL promoter activity was diminished by mutation at this site. Overexpression of NR3C1 upregulated RANKL mRNA expression 1.5-fold in human A549 and HOS cells. On the other hand silencing of NR3C1 caused slight decrease in RANKL mRNA level, suggesting that NR3C1 directly accounts for RANKL transcriptional regulation. Using electrophoretic mobility shift assay we demonstrate that NR3C1 binds to the proximal RANKL promoter region. Our study provides evidences that NR3C1 directly upregulates RANKL transcription in human cell lines and connects the missing link in the mechanism of RANK/RANKL/OPG imbalance of glucocorticoid induced osteoporosis.