Role of the Multidrug Resistance Regulator MarA in Global Regulation of the
            <i>hdeAB</i>
            Acid Resistance Operon in
            <i>Escherichia coli</i>

Ruiz, Cristian; McMurry, Laura M.; Levy, Stuart B.

doi:10.1128/jb.01729-07

Cited by 38 publications

(37 citation statements)

References 45 publications

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“…These genes included slp and hdeAB-yhiD, which are repressed by MarA during the transition to stationary phase (68,76) but are also affected by numerous other regulators. Increased expression of these genes was also accompanied by dramatic increases in expression of gadA, gadB, gadC, gadE, and mdtEF, with more moderate increases in expression (Ͻ5-fold) of gadX and gadW (see the supplemental material), strongly suggesting a relative lack of MarA involvement.…”

Section: Induction Of the Mara/rob/soxs Regulonmentioning

confidence: 99%

Membrane Stresses Induced by Overproduction of Free Fatty Acids in Escherichia coli

Lennen

Kruziki

Kumar

et al. 2011

Appl Environ Microbiol

137

157

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Microbially produced fatty acids are potential precursors to high-energy-density biofuels, including alkanes and alkyl ethyl esters, by either catalytic conversion of free fatty acids (FFAs) or enzymatic conversion of acyl-acyl carrier protein or acyl-coenzyme A intermediates. Metabolic engineering efforts aimed at overproducing FFAs in Escherichia coli have achieved less than 30% of the maximum theoretical yield on the supplied carbon source. In this work, the viability, morphology, transcript levels, and protein levels of a strain of E. coli that overproduces medium-chain-length FFAs was compared to an engineered control strain. By early stationary phase, an 85% reduction in viable cell counts and exacerbated loss of inner membrane integrity were observed in the FFA-overproducing strain. These effects were enhanced in strains endogenously producing FFAs compared to strains exposed to exogenously fed FFAs. Under two sets of cultivation conditions, longchain unsaturated fatty acid content greatly increased, and the expression of genes and proteins required for unsaturated fatty acid biosynthesis were significantly decreased. Membrane stresses were further implicated by increased expression of genes and proteins of the phage shock response, the MarA/Rob/SoxS regulon, and the nuo and cyo operons of aerobic respiration. Gene deletion studies confirmed the importance of the phage shock proteins and Rob for maintaining cell viability; however, little to no change in FFA titer was observed after 24 h of cultivation. The results of this study serve as a baseline for future targeted attempts to improve FFA yields and titers in E. coli.Microbially derived free fatty acids (FFAs) are attractive intermediates for producing a wide range of high-energy-density biofuels from sustainable carbon sources, such as biomass (34). FFAs can be extracted from culture medium and catalytically converted to esters or alkanes (48, 55). Alternatively, enzymatic pathways exist for intracellular conversion to esters (42, 81), olefins (10, 59, 75), alkanes (78), or fatty aldehydes and fatty alcohols (22,81,82). These pathways can either be exploited in their native host or heterologously expressed in a genetically pliable microorganism (3). The physical and chemical properties of the resulting products are dependent on chain length and hydrophobicity; however, medium-chainlength (8-to 14-carbon) methyl esters, olefins, and alkanes exhibit many properties analogous to those of diesel and jet fuel and are therefore potential drop-in replacements (44,61).Several studies have demonstrated FFA overproduction in Escherichia coli (19,48,52,81,83). In each, the key strain modifications included overexpression of one or more cytosolic acyl-acyl carrier protein (ACP) thioesterases and deletion of fadD, or both fadD and fadE, which encode an acyl-coenzyme A (CoA) synthetase and acyl-CoA dehydrogenase, respectively. Overexpression of an acyl-ACP thioesterase depletes the level of acyl-ACP intermediates, which inhibit via feedback enzymes of fatty acid bi...

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Section: Induction Of the Mara/rob/soxs Regulonmentioning

confidence: 99%

Membrane Stresses Induced by Overproduction of Free Fatty Acids in Escherichia coli

Lennen

Kruziki

Kumar

et al. 2011

Appl Environ Microbiol

137

157

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“…MarAactivated lacZ promoter fusions (Kan r ) were described previously (6). The repressed promoter regions cloned upstream of lacZ are (with respect to their transcriptional start sites in base pairs): hdeA, Ϫ349 to ϩ23 (22), purA, Ϫ97 to ϩ19 (T. M. Barbosa and L. M. McMurry, unpublished data), and rob, Ϫ88 to ϩ136 (21,24); the hdeA-lacZ and purA-lacZ lysogens are Amp r , while the rob-lacZ lysogen is Kan r and was obtained from R. G. Martin. The purA-lacZ and hdeA-lacZ fusions were transferred into host N8452 by phage P1 transduction and confirmed as single lysogens by PCR as described previously (18).…”

Section: E Coli Strains and Promoter-lacz Fusionsmentioning

confidence: 99%

“…The induction of MarA specified by pMPM-TmarA did not begin until the OD 600 reached about 0.3, irrespective of the number of generations of growth in arabinose. After growth for about 3 h (or 6 to 7 h for hdeA expression, which is maximally repressed in stationary phase [22]), the OD 600 was determined and one or, usually, two sequential LacZ assays were done 45 min apart on 50 l of cells in 0.5 ml of LacZ assay buffer (17) at room temperature with lysis by 0.005% SDS, 25 l chloroform. Each experiment was repeated one or more times.…”

Section: E Coli Strains and Promoter-lacz Fusionsmentioning

confidence: 99%

Evidence that Regulatory Protein MarA of Escherichia coli Represses rob by Steric Hindrance

McMurry¹,

Levy

2010

J Bacteriol

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The MarA protein of Escherichia coli can both activate and repress the initiation of transcription, depending on the position and orientation of its degenerate 20-bp binding site ("marbox") at the promoter. For all three known repressed genes, the marbox overlaps the promoter. It has been reported that MarA represses the rob promoter via an RNA polymerase (RNAP)-DNA-MarA ternary complex. Under similar conditions, we found a ternary complex for the repressed purA promoter also. These findings, together with the backwards orientation of repressed marboxes, suggested a unique interaction of MarA with RNAP in repression. However, no repression-specific residues of MarA could be found among 38 single-alanine replacement mutations previously shown to retain activation function or among mutants from random mutagenesis. Mutations Thr12Ala, Arg36Ala, Thr95Ile, and Pro106Ala were more damaging for activation than for repression, some up to 10-fold, so these residues may play a specific role in activation. We found that nonspecific binding of RNAP to promoterless regions of DNA was presumably responsible for the ternary complexes seen previously. When RNAP binding was promoter specific, MarA reduced RNAP access to the rob promoter; there was little or no ternary complex. These findings strongly implicate steric hindrance as the mechanism of repression of rob by MarA.

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“…In addition, many other genes which express metabolic enzymes, periplasmic proteins and regulators involved in AR in E. coli have also been examined by microarray and proteomic 2D-gel analyses (Blankenhorn et al, 1999;Tucker et al, 2002). Recently, the Lon protease, endoRNase RNase E and chaperone Hsp31 have been shown to be important in controlling AR systems in E. coli (Heuveling et al, 2008;Mujacic & Baneyx, 2007;Takada et al, 2007), and the roles of AraC-family regulators GadX and GadW and the multidrug resistance regulator MarA in AR systems have also been investigated (Ruiz et al, 2008;Sayed et al, 2007;Tramonti et al, 2006). Nevertheless, despite advances in unravelling some of the regulatory networks involved in AR systems (Foster, 2004;Masuda & Church, 2003), it remains unclear how these factors function together to promote cell survival in low pH conditions.…”

Section: Introductionmentioning

confidence: 99%

OmpR positively regulates urease expression to enhance acid survival of Yersinia pseudotuberculosis

Wang

et al. 2009

Microbiology

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Yersinia pseudotuberculosis is an enteric bacterium which must overcome the acidic stress in host organs for successful colonization, but how this bacterium survives in acidic conditions remains largely unknown. In the present study, the importance of OmpR in acid survival of Y. pseudotuberculosis YpIII was confirmed by the fact that mutation of ompR (strain ΔompR) greatly reduced cell survival at pH 4.5 or lower. To characterize the regulatory role of OmpR in this acid survival process, proteomic analysis was carried out to compare YpIII at pH 7.0 and pH 4.5 with ΔompR at pH 7.0, and urease components were revealed to be the main targets for OmpR regulation. Addition of urea to the culture medium also enhanced acid survival of YpIII but not ΔompR and urease activity was significantly induced by acid in YpIII but not in ΔompR. Each of the seven components of the YpIII urease gene cluster was fused to a lacZ reporter and their expression was dramatically decreased in a ΔompR background; this supports the notion that OmpR positively regulates urease expression. Furthermore, gel shift analysis revealed that OmpR binds to the deduced promoter regions of three polycistronic transcriptional units (ureABC, ureEF and ureGD) in the urease cluster, suggesting that the regulation of OmpR to urease components is direct. Taken together, these data strongly suggest that OmpR activates urease expression to enhance acid survival in Y. pseudotuberculosis.

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Role of the Multidrug Resistance Regulator MarA in Global Regulation of the hdeAB Acid Resistance Operon in Escherichia coli

Cited by 38 publications

References 45 publications

Membrane Stresses Induced by Overproduction of Free Fatty Acids in Escherichia coli

Membrane Stresses Induced by Overproduction of Free Fatty Acids in Escherichia coli

Evidence that Regulatory Protein MarA of Escherichia coli Represses rob by Steric Hindrance

OmpR positively regulates urease expression to enhance acid survival of Yersinia pseudotuberculosis

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