scholarly journals H-NS Is a Repressor of the Proteus mirabilis Urease Transcriptional Activator GeneureR

2000 ◽  
Vol 182 (9) ◽  
pp. 2649-2653 ◽  
Author(s):  
Christopher Coker ◽  
Olubunmi O. Bakare ◽  
Harry L. T. Mobley
Keyword(s):  
Nucleotide Sequence ◽  
Proteus Mirabilis ◽  
Binding Sites ◽  
Recombinant Plasmid ◽  
Urease Activity ◽  
Transcriptional Activator ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Urease Gene ◽  
In Trans

ABSTRACT Expression of Proteus mirabilis urease is governed by UreR, an AraC-like positive transcriptional activator. A poly(A) tract nucleotide sequence, consisting of A6TA2CA2TGGTA5GA6TGA5, is located 16 bp upstream of the ς70-likeureR promoter P2. Since poly(A) tracts of DNA serve as binding sites for the gene repressor histone-like nucleoid structuring protein (H-NS), we measured β-galactosidase activity of wild-typeEscherichia coli MC4100 (H-NS+) and its isogenic derivative ATM121 (hns::Tn10) (H-NS−) harboring a ureR-lacZ operon fusion plasmid (pLC9801). β-Galactosidase activity in the H-NS− host strain was constitutive and sevenfold greater (P < 0.0001) than that in the H-NS+ host. A recombinant plasmid containing cloned P. mirabilis hns was able to complement and restore repression of the ureR promoter in the H-NS−host when provided in trans. Deletion of the poly(A) tract nucleotide sequence from pLC9801 resulted in an increase in β-galactosidase activity in the H-NS+ host to nearly the same levels as that observed for wild-type pLC9801 harbored by the H-NS−host. Urease activity in strains harboring the recombinant plasmid pMID1010 (encoding the entire urease gene cluster of P. mirabilis) was equivalent in both the H-NS−background and the H-NS+ background in the presence of urea but was eightfold greater (P = 0.0001) in the H-NS− background in the absence of urea. We conclude that H-NS represses ureR expression in the absence of urea induction.

scholarly journals UreR, the Transcriptional Activator of the Proteus mirabilis Urease Gene Cluster, Is Required for Urease Activity and Virulence in Experimental Urinary Tract Infections

2003 ◽  
Vol 71 (2) ◽  
pp. 1026-1030 ◽  
Author(s):  
Jonathan D. Dattelbaum ◽  
C. Virginia Lockatell ◽  
David E. Johnson ◽  
Harry L. T. Mobley
Keyword(s):  
Urinary Tract ◽  
Gene Cluster ◽  
Proteus Mirabilis ◽  
Urease Activity ◽  
Complicated Urinary Tract Infection ◽  
Wild Type ◽  
Urease Gene ◽  
Low Copy Number ◽  
Mutant Strains ◽  
Tract Infections

ABSTRACT Proteus mirabilis, a cause of complicated urinary tract infection, produces urease, an essential virulence factor for this species. UreR, a member of the AraC/XylS family of transcriptional regulators, positively activates expression of the ure gene cluster in the presence of urea. To specifically evaluate the contribution of UreR to urease activity and virulence in the urinary tract, a ureR mutation was introduced into P. mirabilis HI4320 by homologous recombination. The isogenic ureR::aphA mutant, deficient in UreR production, lacked measurable urease activity. Expression was not detected in the UreR-deficient strain by Western blotting with monoclonal antibodies raised against UreD. Urease activity and UreD expression were restored by complementation of the mutant strain with ureR expressed from a low-copy-number plasmid. Virulence was assessed by transurethral cochallenge of CBA mice with wild-type and mutant strains. The isogenic ureR::aphA mutant of HI4320 was outcompeted in the urine (P = 0.004), bladder (P = 0.016), and kidneys (P ≤ 0.001) 7 days after inoculation. Thus, UreR is required for basal urease activity in the absence of urea, for induction of urease by urea, and for virulence of P. mirabilis in the urinary tract.

scholarly journals MexR Repressor of the mexAB-oprMMultidrug Efflux Operon of Pseudomonas aeruginosa: Identification of MexR Binding Sites in the mexA-mexRIntergenic Region

2001 ◽  
Vol 183 (3) ◽  
pp. 807-812 ◽  
Author(s):  
Kelly Evans ◽  
Lateef Adewoye ◽  
Keith Poole
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Nucleotide Sequence ◽  
Binding Sites ◽  
Metal Chelate ◽  
Negative Regulation ◽  
Proximal Promoter ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Distal Promoter ◽  
Metal Chelate Affinity Chromatography

ABSTRACT The MexR repressor of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa was purified as a C-terminal histidine-tagged protein by metal chelate affinity chromatography. The purified protein was shown to bind ca. 200 bp upstream of mexA, at two sites, each of which contains a repeat of the nucleotide sequence GTTGA in inverse orientation. DNA sequence analysis identified mexA and mexRpromoters within the MexR binding regions, consistent with the previously observed negative regulation of mexR andmexAB-oprM expression by MexR. Transcription ofmexA from the promoter originating within the MexR binding site II was confirmed and shown to be markedly enhanced in analB (i.e., mexR) mutant of P. aeruginosa. A second mexA promoter was also identified, ca. 70 bp upstream of mexAB-oprM, and transcription from this promoter appeared to occur in both the wild type and a nalB mutant. Production of MexAB-OprM in wild-type cells may be due to expression from a constitutively expressed proximal promoter, while MexAB-OprM hyperexpression innalB mutants is due to the additional expression from a MexR-regulated distal promoter.

scholarly journals FlrA, a σ54-Dependent Transcriptional Activator in Vibrio fischeri, Is Required for Motility and Symbiotic Light-Organ Colonization

2003 ◽  
Vol 185 (12) ◽  
pp. 3547-3557 ◽  
Author(s):  
Deborah S. Millikan ◽  
Edward G. Ruby
Keyword(s):  
Vibrio Fischeri ◽  
Transcriptional Activator ◽  
Pcr Analysis ◽  
Euprymna Scolopes ◽  
Wild Type ◽  
Flagellar Regulon ◽  
Colonization Factors ◽  
Recognition Motifs ◽  
Arbitrarily Primed Pcr ◽  
Organ Colonization

ABSTRACT Flagellum-mediated motility of Vibrio fischeri is an essential factor in the bacterium's ability to colonize its host, the Hawaiian squid Euprymna scolopes. To begin characterizing the nature of the flagellar regulon, we have cloned a gene, designated flrA, from V. fischeri that encodes a putative σ54-dependent transcriptional activator. Genetic arrangement of the flrA locus in V. fischeri is similar to motility master-regulator operons of Vibrio cholerae and Vibrio parahaemolyticus. In addition, examination of regulatory regions of a number of flagellar operons in V. fischeri revealed apparent σ54 recognition motifs, suggesting that the flagellar regulatory hierarchy is controlled by a similar mechanism to that described in V. cholerae. However, in contrast to its closest known relatives, flrA mutant strains of V. fischeri ES114 were completely abolished in swimming capability. Although flrA provided in trans restored motility to the flrA mutant, the complemented strain was unable to reach wild-type levels of symbiotic colonization in juvenile squid, suggesting a possible role for the proper expression of FlrA in regulating symbiotic colonization factors in addition to those required for motility. Comparative RNA arbitrarily primed PCR analysis of the flrA mutant and its wild-type parent revealed several differentially expressed transcripts. These results define a regulon that includes both flagellar structural genes and other genes apparently not involved in flagellum elaboration or function. Thus, the transcriptional activator FlrA plays an essential role in regulating motility, and apparently in modulating other symbiotic functions, in V. fischeri.

scholarly journals CaNdt80 Is Involved in Drug Resistance in Candida albicans by Regulating CDR1

2004 ◽  
Vol 48 (12) ◽  
pp. 4505-4512 ◽  
Author(s):  
Chia-Geun Chen ◽  
Yun-Liang Yang ◽  
Hsin-I Shih ◽  
Chia-Li Su ◽  
Hsiu-Jung Lo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Candida Albicans ◽  
Type Strain ◽  
Antifungal Agents ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Antifungal Drugs ◽  
Galactosidase Activity ◽  
Wild Type

ABSTRACT Overexpression of CDR1, an efflux pump, is one of the major mechanisms contributing to drug resistance in Candida albicans. CDR1 p-lacZ was constructed and transformed into a Saccharomyces cerevisiae strain so that the lacZ gene could be used as the reporter to monitor the activity of the CDR1 promoter. Overexpression of CaNDT80, the C. albicans homolog of S. cerevisiae NDT80, increases the β-galactosidase activity of the CDR1 p-lacZ construct in S. cerevisiae. Furthermore, mutations in CaNDT80 abolish the induction of CDR1 expression by antifungal agents in C. albicans. Consistently, the Candt80/Candt80 mutant is also more susceptible to antifungal drugs than the wild-type strain. Thus, the gene for CaNdt80 may be the first gene among the regulatory factors involved in drug resistance in C. albicans whose function has been identified.

scholarly journals Roles of rpoN, fliA,and flgR in Expression of Flagella inCampylobacter jejuni

2001 ◽  
Vol 183 (9) ◽  
pp. 2937-2942 ◽  
Author(s):  
Aparna Jagannathan ◽  
Chrystala Constantinidou ◽  
Charles W. Penn
Keyword(s):  
Campylobacter Jejuni ◽  
Genome Sequence ◽  
Sigma Factor ◽  
Transcriptional Activator ◽  
Alternative Sigma Factor ◽  
Wild Type ◽  
Electron Microscopic ◽  
Global Regulation ◽  
Mutant Strains ◽  
Microscopic Studies

ABSTRACT Three potential regulators of flagellar expression present in the genome sequence of Campylobacter jejuni NCTC 11168, the genes rpoN, flgR, andfliA, which encode the alternative sigma factor ς54, the ς54-associated transcriptional activator FlgR, and the flagellar sigma factor ς28, respectively, were investigated for their role in global regulation of flagellar expression. The three genes were insertionally inactivated inC. jejuni strains NCTC 11168 and NCTC 11828. Electron microscopic studies of the wild-type and mutant strains showed that therpoN and flgR mutants were nonflagellate and that the fliA mutant had truncated flagella. Immunoblotting experiments with the three mutants confirmed the roles of rpoN, flgR, and fliA in the expression of flagellin.

scholarly journals AcrAB Multidrug Efflux Pump Is Associated with Reduced Levels of Susceptibility to Tigecycline (GAR-936) in Proteus mirabilis

2003 ◽  
Vol 47 (2) ◽  
pp. 665-669 ◽  
Author(s):  
Melissa A. Visalli ◽  
Ellen Murphy ◽  
Steven J. Projan ◽  
Patricia A. Bradford
Keyword(s):  
Proteus Mirabilis ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Transposon Insertion ◽  
Spectrum Activity ◽  
Insertion Mutants ◽  
Broad Spectrum Activity ◽  
Increased Susceptibility

ABSTRACT Tigecycline has good broad-spectrum activity against many gram-positive and gram-negative pathogens with the notable exception of the Proteeae. A study was performed to identify the mechanism responsible for the reduced susceptibility to tigecycline in Proteus mirabilis. Two independent transposon insertion mutants of P. mirabilis that had 16-fold-increased susceptibility to tigecycline were mapped to the acrB gene homolog of the Escherichia coli AcrRAB efflux system. Wild-type levels of decreased susceptibility to tigecycline were restored to the insertion mutants by complementation with a clone containing a PCR-derived fragment from the parental wild-type acrRAB efflux gene cluster. The AcrAB transport system appears to be associated with the intrinsic reduced susceptibility to tigecycline in P. mirabilis.

DNA-binding and transcriptional regulatory properties of hepatic leukemia factor (HLF) and the t(17;19) acute lymphoblastic leukemia chimera E2A-HLF

1994 ◽  
Vol 14 (9) ◽  
pp. 5986-5996
Author(s):  
S P Hunger ◽  
R Brown ◽  
M L Cleary
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Lymphoblastic Leukemia ◽  
Consensus Sequence ◽  
Reporter Genes ◽  
Wild Type ◽  
Basic Leucine Zipper

The t(17;19) translocation in acute lymphoblastic leukemias results in creation of E2A-hepatic leukemia factor (HLF) chimeric proteins that contain the DNA-binding and protein dimerization domains of the basic leucine zipper (bZIP) protein HLF fused to a portion of E2A proteins with transcriptional activation properties. An in vitro binding site selection procedure was used to determine DNA sequences preferentially bound by wild-type HLF and chimeric E2A-HLF proteins isolated from various t(17;19)-bearing leukemias. All were found to selectively bind the consensus sequence 5'-GTTACGTAAT-3' with high affinity. Wild-type and chimeric HLF proteins also bound closely related sites identified previously for bZIP proteins of both the proline- and acidic amino acid-rich (PAR) and C/EBP subfamilies; however, E2A-HLF proteins were significantly less tolerant of certain deviations from the HLF consensus binding site. These differences were directly attributable to loss of an HLF ancillary DNA-binding domain in all E2A-HLF chimeras and were further exacerbated by a zipper mutation in one isolate. Both wild-type and chimeric HLF proteins displayed transcriptional activator properties in lymphoid and nonlymphoid cells on reporter genes containing HLF or C/EBP consensus binding sites. But on reporter genes with nonoptimal binding sites, their transcriptional properties diverged and E2A-HLF competitively inhibited activation by wild-type PAR proteins. These findings establish a spectrum of binding site-specific transcriptional properties for E2A-HLF which may preferentially activate expression of select subordinate genes as a homodimer and potentially antagonize expression of others through heteromeric interactions.

The number of ribosomes on simian virus 40 late 16S mRNA is determined in part by the nucleotide sequence of its leader

1984 ◽  
Vol 4 (4) ◽  
pp. 813-816
Author(s):  
A Barkan ◽  
J E Mertz
Keyword(s):  
Nucleotide Sequence ◽  
Direct Evidence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
The Body ◽  
Size Distributions ◽  
Wild Type ◽  
Leader Protein ◽  
16S Rna

The size distributions of polyribosomes containing each of three simian virus 40 late 16S mRNA species that differ in nucleotide sequence only within their leaders were determined. The two 16S RNA species with shorter leaders were incorporated into polysomes that were both larger (on average) and narrower in size distribution than was the predominant wild-type 16S RNA. Therefore, the nucleotide sequence of the leader can influence the number of ribosomes present on the body of an mRNA molecule. We propose a model in which the excision from leaders of sizeable translatable regions permits more frequent utilization of internally located translation initiation signals, thereby enabling genes encoded within the bodies of polygenic mRNAs to be translated at higher rates. In addition, the data provide the first direct evidence that VP1 can, indeed, be synthesized in vivo from the species of 16S mRNA that also encodes the 61-amino acid leader protein.

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