scholarly journals Bacterial Two-Hybrid Analysis of Interactions between Region 4 of the ς70 Subunit of RNA Polymerase and the Transcriptional Regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa

2001 ◽  
Vol 183 (21) ◽  
pp. 6413-6421 ◽  
Author(s):  
Simon L. Dove ◽  
Ann Hochschild
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Amino Acid Substitution ◽  
Transcriptional Regulators ◽  
Binding Motif ◽  
E Coli ◽  
Two Hybrid

ABSTRACT A number of transcriptional regulators mediate their effects through direct contact with the ς70 subunit ofEscherichia coli RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of ς70 that harbors conserved region 4. This region of ς contains a putative helix-turn-helix DNA-binding motif that contacts the −35 element of ς70-dependent promoters directly. Here we report the use of a recently developed bacterial two-hybrid system to study the interaction between the putative anti-ς factor Rsd and the ς70 subunit of E. coli RNAP. Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of ς70 and also that amino acid substitution R596H, within region 4 of ς70, weakens this interaction. We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between ς and two other regulators shown previously to contact region 4 of ς70. We also demonstrated that AlgQ, a homolog of Rsd that positively regulates virulence gene expression inPseudomonas aeruginosa, can contact the C-terminal region of the ς70 subunit of RNAP from this organism. We found that amino acid substitution R600H in ς70 fromP. aeruginosa, corresponding to the R596H substitution in E. coli ς70, specifically weakens the interaction between AlgQ and ς70. Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of ς70 and probably regulate gene expression through this contact.

scholarly journals Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain

2001 ◽  
Vol 183 (2) ◽  
pp. 570-579 ◽  
Author(s):  
Michal Gropp ◽  
Yael Strausz ◽  
Miriam Gross ◽  
Gad Glaser
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Amino Acid Starvation ◽  
E Coli ◽  
Terminal Domain ◽  
Negative Effect ◽  
And Control ◽  
Two Hybrid

ABSTRACT The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD inrelA + cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

scholarly journals The Bacteriophage T4 Inhibitor and Coactivator AsiA Inhibits Escherichia coli RNA Polymerase More Rapidly in the Absence of σ70 Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between σ70 and Core

2006 ◽  
Vol 188 (4) ◽  
pp. 1279-1285 ◽  
Author(s):  
Deborah M. Hinton ◽  
Srilatha Vuthoori ◽  
Rebecca Mulamba
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Dynamic Equilibrium ◽  
Bacteriophage T4 ◽  
Direct Interaction ◽  
Binding Properties ◽  
E Coli ◽  
Dna Binding Properties ◽  
Two Hybrid

ABSTRACT The N-terminal region (region 1.1) of σ70, the primary σ subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of σ70 regions 2 and 4. Region 1.1 prevents the interaction of free σ70 with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of σ70-dependent transcription from promoters that require an interaction between σ70 region 4 and the −35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with σ70 region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that σ70 region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter P uvsX . However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with σ70 that lacks region 1.1 is less stable than polymerase with full-length σ70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free σ70 and then the AsiA/σ70 complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free σ70 plus core, yielding more free σ70 at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free σ70. Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using an E. coli two-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free σ70 arises through an indirect effect.

scholarly journals A Single Amino Acid Substitution in a Mannosyltransferase, WbdA, Converts the Escherichia coli O9 Polysaccharide into O9a: Generation of a New O-Serotype Group

2000 ◽  
Vol 182 (9) ◽  
pp. 2567-2573 ◽  
Author(s):  
Nobuo Kido ◽  
Hidemitsu Kobayashi
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Chimeric Genes ◽  
Functional Studies

ABSTRACT wbdA is a mannosyltransferase gene that is involved in synthesis of the Escherichia coli O9a polysaccharide, a mannose homopolymer with a repeating unit of 2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1. The equivalent structural O polysaccharide in the E. coli O9 andKlebsiella O3 strains is 2-αMan-1,2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1, with an excess of one mannose in the 1,2 linkage. We have cloned wbdAgenes from these O9 and O3 strains and shown by genetic and functional studies that wbdA is the only gene determining the O-polysaccharide structure of O9 or O9a. Based on functional analysis of chimeric genes and site-directed mutagenesis, we showed that a single amino acid substitution, C55R, in WbdA of E. coli O9 converts the O9 polysaccharide into O9a. DNA sequencing revealed the substitution to be conserved in other E. coli O9a strains. The reverse substitution, R55C, in WbdA of E. coli O9a resulted in lipopolysaccharide synthesis showing no ladder profile instead of the conversion of O9a to O9. This suggests that more than one amino acid substitution in WbdA is required for conversion from O9a to O9.

scholarly journals ThePseudomonas aeruginosaInitiation Factor IF-2 Is Responsible for Formylation-independent Protein Initiation inP. aeruginosa

2004 ◽  
Vol 279 (50) ◽  
pp. 52262-52269 ◽  
Author(s):  
Marta Steiner-Mosonyi ◽  
Carole Creuzenet ◽  
Robert A. B. Keates ◽  
Benjamin R. Strub ◽  
Dev Mangroo
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Positive Charge ◽  
Initiation Factor ◽  
E Coli ◽  
Charge Potential ◽  
Dependent Protein ◽  
Independent Protein

Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) was generally thought to be essential for initiation of protein synthesis in all eubacteria based on studies conducted primarily inEscherichia coli. However, this view of eubacterial protein initiation has changed because some bacteria have been demonstrated to have the capacity to initiate protein synthesis with the unformylated Met-tRNAfMet. Here we show that thePseudomonas aeruginosainitiation factor IF-2 is required for formylation-independent protein initiation inP. aeruginosa, the first bacterium shown to have the ability to initiate protein synthesis with both the initiator formyl-methionyl-tRNA (fMet-tRNAfMet) and Met-tRNAfMet. TheE. coliIF-2, which participates exclusively in formylation-dependent protein initiation inE. coli, was unable to facilitate utilization of Met-tRNAfMetin initiation inP. aeruginosa. However, theE. coliIF-2 was made to function in formylation-independent protein initiation inP. aeruginosaby decreasing the positive charge potential of the cleft that binds the amino end of the amino acid attached to the tRNA. Furthermore increasing the positive charge potential of this cleft in theP. aeruginosaIF-2 prevented the protein from participating in formylation-independent protein initiation. Thus, this is the first demonstration of a eubacterial IF-2 with an inherent capacity to facilitate utilization of Met-tRNAfMetin protein initiation, discounting the dogma that eubacterial IF-2 can only allow the use of fMet-tRNAfMetin protein initiation. Furthermore these findings give important clues to the basis for discriminating the initiator Met-tRNA by IF-2 and for the evolution of alternative mechanisms for discrimination.

scholarly journals Functional Substitution of the TibC Protein of Enterotoxigenic Escherichia coli Strains for the Autotransporter Adhesin Heptosyltransferase of the AIDA System

2002 ◽  
Vol 70 (5) ◽  
pp. 2264-2270 ◽  
Author(s):  
Corinna Moormann ◽  
Inga Benz ◽  
M. Alexander Schmidt
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Consensus Sequence ◽  
Single Amino Acid Substitution ◽  
Enterotoxigenic Escherichia Coli ◽  
Single Amino Acid ◽  
E Coli ◽  
Encoding Gene ◽  
Sequence Similarities

ABSTRACT The plasmid-encoded AIDA (adhesin involved in diffuse adherence) autotransporter protein derived from diffuse-adhering clinical Escherichia coli isolate 2787 and the TibA (enterotoxigenic invasion locus B) protein encoded by the chromosomal tib locus of enterotoxigenic E. coli (ETEC) strain H10407 are posttranslationally modified by carbohydrate substituents. Analysis of the AIDA-I adhesin showed that the modification involved heptose residues. AIDA-I is modified by the heptosyltransferase activity of the product of the aah gene, which is located directly upstream of adhesin-encoding gene aidA. The carbohydrate modification of the TibA adhesin/invasin is mediated by the TibC protein but has not been elucidated. Based on the sequence similarities between TibC and AAH (autotransporter adhesin heptosyltransferase) and between the TibA and the AIDA proteins we hypothesized that the AIDA system and the Tib system encoded by the tib locus are structurally and functionally related. Here we show that (i) TibC proteins derived from different ETEC strains appear to be highly conserved, (ii) recombinant TibC proteins can substitute for the AAH heptosyltransferase in introducing the heptosyl modification to AIDA-I, (iii) this modification is functional in restoring the adhesive function of AIDA-I, (iv) a single amino acid substitution at position 358 completely abolishes this activity, and (v) antibodies directed at the functionally active AIDA-I recognize a protein resembling modified TibA in ETEC strains. In summary, we conclude that, like AAH, TibC represents an example of a novel class of heptosyltransferases specifically transferring heptose residues onto multiple sites of a protein backbone. A potential consensus sequence for the modification site is suggested.

scholarly journals Simultaneous gain and loss of functions caused by a single amino acid substitution in the beta subunit of Escherichia coli RNA polymerase: suppression of nusA and rho mutations and conditional lethality.

Genetics ◽  
1992 ◽  
Vol 130 (3) ◽  
pp. 411-428 ◽  
Author(s):  
J Sparkowski ◽  
A Das
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Amino Acid Substitution ◽  
Beta Subunit ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
N Gene ◽  
Allele Specific ◽  
Gain And Loss

Abstract Transcript elongation and termination in Escherichia coli is modulated, in part, by the nusA gene product, an acidic protein that interacts not only with RNA polymerase itself but also with ancillary factors, namely the host termination protein Rho and phage lambda antitermination protein, N. The E. coli nusA1 mutant fails to support lambda development due to a specific defect in N-mediated antitermination. Certain rifampicin-resistant (rifR) variants of the nusA1 host support lambda growth. We report here the isolation and pleiotropic properties of one such rifR mutant, ts8, resulting from a single amino acid substitution mutation in rpoB, the structural gene for polymerase beta subunit. ts8 is a recessive lethal mutation that blocks cell growth at 42 degrees. Pulse-labeling and analysis of newly synthesized proteins indicate that the mutant cell is proficient in RNA synthesis at high temperature. Apparently, ts8 causes a loss of some specialized function of RNA polymerase without a gross defect in general transcription activities. ts8 is an allele-specific suppressor of nusA1. It does not suppress nusAsal, nusB5 and nusE71 mutations nor does it bypass the requirement for a functional N gene and the nut site for antitermination and lambda growth. A mutation in the N gene, punA1, that restores lambda growth in the nusA1 mutant host but not in the nusAsal host, compensates for the nusAsal allele in the ts8 mutant. This combined effect of two allele-specific suppressors suggests that they enhance some aspect of polymerase-NusA-N interaction and function. ts8 suppresses the rho15 mutation, but not the rho112 mutation, indicating that it might render RNA polymerase susceptible to the action of a defective Rho protein. Marker rescue analysis has localized ts8 to a 910-bp internal segment of rpoB that encodes the Rif domain. By amplification, cloning and sequencing of this segment of the mutant chromosome we have determined that ts8 contains Phe in place of Ser522, caused by a C to T transition. By gene conversion, we have established that the simultaneous gain and loss of three functions of polymerase is caused by this single amino acid substitution. Clearly, a site in the beta subunit critical for the functioning of both termination and antitermination factors is altered by ts8. The alteration, we imagine, might make this site on polymerase receptive to some factors but repulsive to others.

scholarly journals Pseudomonas aeruginosa TfpW is a multifunctional D-Araf glycosyltransferase and oligosaccharyltransferase

2020 ◽  
Author(s):  
Anne D. Villela ◽  
Hanjeong Harvey ◽  
Katherine Graham ◽  
Lori L. Burrows
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Fluorescence Microscopy ◽  
Group Iv ◽  
Type Iv Pili ◽  
Central Portion ◽  
E Coli ◽  
Type Iv ◽  
C Terminus ◽  
Two Hybrid

ABSTRACTTfpW is an oligosaccharyltransferase that modifies the subunits of type IV pili from group IV strains of Pseudomonas aeruginosa with oligomers of α-1,5-linked-D-arabinofuranose (D-Araf). Besides its oligosaccharyltransferase activity, TfpW may be responsible for periplasmic translocation and polymerization of D-Araf. Here we investigated these potential roles of TfpW in Pa5196 pilin glycosylation. Topology studies confirmed the periplasmic location of loop 1 and the large C-terminus domain, however the central portion of TfpW had an indeterminate configuration. Reconstitution of the Pa5196 pilin glycosylation system by providing pilA, tfpW +/- tfpX and the D-Araf biosynthesis genes PsPA7_6246-6249 showed that TfpW is sufficient for glycan polymerization and transfer to pilins in P. aeruginosa PAO1, while TfpX is also necessary in Escherichia coli. In addition to PsPA7_6246, DprE1 (PsPA7_6248) and DprE2 (PsPA7_6249), the GtrA-like component PsPA7_6247 was required for pilin glycosylation in E. coli versus PAO1. In a PAO1 ΔarnE/F mutant, loss of PsPA7_6247 expression decreased the level of pilin glycosylation, suggesting that arnE/F may play a role in pilin glycosylation when PsPA7_6247 is absent. Bacterial two-hybrid studies showed interactions of TfpW with itself, TfpX, PsPA7_6247 and DprE2, suggesting the formation of a complex that enables efficient pilin glycosylation. Fluorescence microscopy of E. coli and Pa5196ΔdprE1 expressing a DprE1-sGFP fusion showed that the protein is expressed in the cytoplasm, supporting our model that includes cytoplasmic biosynthesis of the lipid carrier-linked D-Araf precursor prior to its periplasmic translocation. Together these data suggest that TfpW may be the first example of a trifunctional flippase, glycosyltransferase, and oligosaccharyltransferase.

scholarly journals The Escherichia colirhaSR-PrhaBADInducible Promoter System Allows Tightly Controlled Gene Expression over a Wide Range in Pseudomonas aeruginosa

2016 ◽  
Vol 82 (22) ◽  
pp. 6715-6727 ◽  
Author(s):  
Jeffrey Meisner ◽  
Joanna B. Goldberg
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Catabolite Repression ◽  
Inducible Promoter ◽  
Translational Initiation ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Promoter System

ABSTRACTThearaC-ParaBADinducible promoter system is tightly controlled and allows gene expression to be modulated over a wide range inEscherichia coli, which has led to its widespread use in other bacteria. Although anecdotal evidence suggests thataraC-ParaBADis leaky inPseudomonas aeruginosa, neither a thorough analysis of this inducible promoter system inP. aeruginosanor a concerted effort to identify alternatives with improved functionality has been reported. Here, we evaluated the functionality of thearaC-ParaBADsystem inP. aeruginosa. Using transcriptional fusions to alacZreporter gene, we determined that the noninduced expression fromaraC-ParaBADis high and cannot be reduced by carbon catabolite repression as it can inE. coli. Modulating translational initiation by altering ribosome-binding site strength reduced the noninduced activity but also decreased the maximal induced activity and narrowed the induction range. Integrating the inducible promoter system into the posttranscriptional regulatory network that controls catabolite repression inP. aeruginosasignificantly decreased the noninduced activity and increased the induction range. In addition to these improvements in the functionality of thearaC-ParaBADsystem, we found that thelacIq-PtacandrhaSR-PrhaBADinducible promoter systems had significantly lower noninduced expression and were inducible over a broader range thanaraC-ParaBAD. We demonstrated that noninduced expression from thearaC-ParaBADsystem supported the function of genes involved in antibiotic resistance and tryptophan biosynthesis inP. aeruginosa, problems that were avoided withrhaSR-PrhaBAD. rhaSR-PrhaBADis tightly controlled, allows gene expression over a wide range, and represents a significant improvement overaraC-ParaBADinP. aeruginosa.IMPORTANCEWe report the shortcomings of the commonly usedEscherichia coli araC-ParaBADinducible promoter system inPseudomonas aeruginosa, successfully reengineered it to improve its functionality, and show that theE. colirhaSR-PrhaBADsystem is tightly controlled and allows inducible gene expression over a wide range inP. aeruginosa.

scholarly journals Different Rifampin Sensitivities of Escherichia coli and Mycobacterium tuberculosis RNA Polymerases Are Not Explained by the Difference in the β-Subunit Rifampin Regions I and II

2005 ◽  
Vol 49 (4) ◽  
pp. 1587-1590 ◽  
Author(s):  
N. Zenkin ◽  
A. Kulbachinskiy ◽  
I. Bass ◽  
V. Nikiforov
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rna Polymerase ◽  
Β Subunit ◽  
E Coli ◽  
Increased Sensitivity ◽  
The Difference

ABSTRACT Mycobacterium tuberculosis RNA polymerase is 1,000-fold more sensitive to rifampin than Escherichia coli RNA polymerase. Chimeric E. coli RNA polymerase in which the β-subunit segment encompassing rifampin regions I and II (amino acids [aa] 463 through 590) was replaced with the corresponding region from M. tuberculosis (aa 382 through 509) did not show an increased sensitivity to the antibiotic. Thus, the difference in amino acid sequence between the rifampin regions I and II of the two species does not account for the difference in rifampin sensitivity of the two polymerases.

scholarly journals IMP-51, a Novel IMP-Type Metallo-β-Lactamase with Increased Doripenem- and Meropenem-Hydrolyzing Activities, in a Carbapenem-Resistant Pseudomonas aeruginosa Clinical Isolate

2015 ◽  
Vol 59 (11) ◽  
pp. 7090-7093 ◽  
Author(s):  
Tatsuya Tada ◽  
Pham Hong Nhung ◽  
Tohru Miyoshi-Akiyama ◽  
Kayo Shimada ◽  
Doan Mai Phuong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Clinical Isolate ◽  
Amino Acid Residue ◽  
Active Site ◽  
Medical Setting ◽  
Content Type ◽  
E Coli ◽  
Carbapenem Resistant

ABSTRACTA meropenem-resistantPseudomonas aeruginosaisolate was obtained from a patient in a medical setting in Hanoi, Vietnam. The isolate was found to have a novel IMP-type metallo-β-lactamase, IMP-51, which differed from IMP-7 by an amino acid substitution (Ser262Gly).Escherichia coliexpressingblaIMP-51showed greater resistance to cefoxitin, meropenem, and moxalactam thanE. coliexpressingblaIMP-7. The amino acid residue at position 262 was located near the active site, proximal to the H263 Zn(II) ligand.

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