Efflux and motility are two key biological functions in bacteria. Recent findings have shown that efflux impacts flagellum biosynthesis and motility in
Efflux and motility play a major role in bacterial growth, colonization, and survival. In
The
- Award ID(s):
- 1810599
- NSF-PAR ID:
- 10206827
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Bradford, Patricia A. (Ed.)more » « less
ABSTRACT Escherichia coli and other bacteria. AcrR is known to be the major transcriptional repressor of AcrAB-TolC, the main multidrug efflux pump inE. coli and otherEnterobacteriaceae . However, the underlying molecular mechanisms of how efflux and motility are co-regulated remain poorly understood. Here, we have studied the role of AcrR in direct regulation of motility inE. coli . By combining bioinformatics, electrophoretic mobility shift assays (EMSAs), gene expression, and motility experiments, we have found that AcrR represses motility inE. coli by directly repressing transcription of theflhDC operon, but not the other flagellum genes/operons tested.flhDC encodes the master regulator of flagellum biosynthesis and motility genes. We found that such regulation primarily occurs by direct binding of AcrR to theflhDC promoter region containing the first of the two predicted AcrR-binding sites identified in this promoter. This is the first report of direct regulation by AcrR of genes unrelated to efflux or detoxification. Moreover, we report that overexpression of AcrR restores to parental levels the increased swimming motility previously observed inE. coli strains without a functional AcrAB-TolC pump, and that such effect by AcrR is prevented by the AcrR ligand and AcrAB-TolC substrate ethidium bromide. Based on these and prior findings, we provide a novel model in which AcrR senses efflux and then co-regulates efflux and motility inE. coli to maintain homeostasis and escape hazards.IMPORTANCE Escherichia coli , the transcriptional repressor AcrR is known to directly repress efflux and was later found to also repress flagellum biosynthesis and motility by Kim et al. (J Microbiol Biotechnol 26:1824–1828, 2016, doi: 10.4014/jmb.1607.07058). However, it remained unknown whether AcrR represses flagellum biosynthesis and motility directly and through which target genes, or indirectly because of altering the amount of efflux. This study reveals that AcrR represses flagellum biosynthesis and motility by directly repressing the expression of theflhDC master regulator of flagellum biosynthesis and motility genes, but not the other flagellum genes tested. We also show that the antimicrobial, efflux pump substrate, and AcrR ligand ethidium bromide regulates motility via AcrR. Overall, these findings support a novel model of direct co-regulation of efflux and motility mediated by AcrR in response to stress inE. coli . -
The tnaC regulatory gene from the tna operon of Escherichia coli controls the transcription of its own operon through an attenuation mechanism relying on the accumulation of arrested ribosomes during inhibition of its own translation termination. This free L-Trp dependent mechanism of inhibition of translation termination remains unclear. Here, we analyzed the inhibitory effects of L-Trp on the function of two known E. coli translation termination factors, RF1 and RF2. Using a series of reporter genes, we found that the in vivo L-Trp sensitivity of tnaC gene expression is influenced by the identity of its stop codon, with the UGA stop codon producing higher expression efficiency of the tnaA-lacZ gene construct than the UAG stop codon. in vitro TnaC-peptidyl-tRNA accumulation and toeprinting assays confirmed that in the presence of L-Trp, the UGA stop codon generates higher accumulation of both TnaC-peptidyl-tRNA and arrested ribosomes than does the UAG stop codon. RF-mediated hydrolysis assays corroborated that L-Trp blocks RF2 function more than that of RF1. Mutational analyses disclosed that amino acids substitutions at the 246 and 256 residue positions surrounding the RF2-GGQ functional motif reduce L-Trp dependent expression of the tnaC(UGA) tnaA-lacZ construct and the ability of L-Trp to inhibit RF2-mediated cleavage of the TnaC-peptidyl-tRNA. Altogether, our results indicate that L-Trp preferentially blocks RF2 activity during translation termination of the tnaC gene. This inhibition depends on the identities of amino acid residues surrounding the RF2-GGQ functional motif.more » « less
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Summary Mercury (Hg) is a highly toxic and widely distributed heavy metal, which some Bacteria and Archaea detoxify by the reduction of ionic Hg (Hg[II]) to the elemental volatile form, Hg(0). This activity is specified by the
mer operon. Themer operon of the deeply branching thermophileThermus thermophilus HB27 encodes for, anO ‐acetyl‐l ‐homoacetylserine sulfhydrylase (Oah2), a transcriptional regulator (MerR), a hypothetical protein (hp) and a mercuric reductase (MerA). Here, we show that this operon has two convergently expressed and differentially regulated promoters. An upstream promoter, Poah , controls the constitutive transcription of the entire operon and a second promoter (Pmer ), located withinmerR , is responsive to Hg(II). In the absence of Hg(II), the transcription ofmerA is basal and when Hg(II) is present,merA transcription is induced. This response to Hg(II) is controlled by MerR and genetic evidence suggests that MerR acts as a repressor and activator of Pmer . When the wholemerR , including Pmer , is removed,merA is transcribed from Poah independently of Hg(II). These results suggest that the transcriptional regulation ofmer inT .thermophilus mer systems, possibly representing an early step in the evolution ofmer ‐operon regulation. -
Babitzke, Paul (Ed.)ABSTRACT Oxidative stress causes cellular damage, including DNA mutations, protein dysfunction, and loss of membrane integrity. Here, we discovered that a TrmB (transcription regulator of mal operon) family protein (Pfam PF01978) composed of a single winged-helix DNA binding domain (InterPro IPR002831) can function as thiol-based transcriptional regulator of oxidative stress response. Using the archaeon Haloferax volcanii as a model system, we demonstrate that the TrmB-like OxsR is important for recovery of cells from hypochlorite stress. OxsR is shown to bind specific regions of genomic DNA, particularly during hypochlorite stress. OxsR-bound intergenic regions were found proximal to oxidative stress operons, including genes associated with thiol relay and low molecular weight thiol biosynthesis. Further analysis of a subset of these sites revealed OxsR to function during hypochlorite stress as a transcriptional activator and repressor. OxsR was shown to require a conserved cysteine (C24) for function and to use a CG-rich motif upstream of conserved BRE/TATA box promoter elements for transcriptional activation. Protein modeling suggested the C24 is located at a homodimer interface formed by antiparallel α helices, and that oxidation of this cysteine would result in the formation of an intersubunit disulfide bond. This covalent linkage may promote stabilization of an OxsR homodimer with the enhanced DNA binding properties observed in the presence of hypochlorite stress. The phylogenetic distribution TrmB family proteins, like OxsR, that have a single winged-helix DNA binding domain and conserved cysteine residue suggests this type of redox signaling mechanism is widespread in Archaea. IMPORTANCE TrmB-like proteins, while not yet associated with redox stress, are found in bacteria and widespread in archaea. Here, we expand annotation of a large group of TrmB-like single winged-helix DNA binding domain proteins from diverse archaea to function as thiol-based transcriptional regulators of oxidative stress response. Using Haloferax volcanii as a model, we reveal that the TrmB-like OxsR functions during hypochlorite stress as a transcriptional activator and repressor of an extensive gene coexpression network associated with thiol relay and other related activities. A conserved cysteine residue of OxsR serves as the thiol-based sensor for this function and likely forms an intersubunit disulfide bond during hypochlorite stress that stabilizes a homodimeric configuration with enhanced DNA binding properties. A CG-rich DNA motif in the promoter region of a subset of sites identified to be OxsR-bound is required for regulation; however, not all sites have this motif, suggesting added complexity to the regulatory network.more » « less
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Abstract Populations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that cause constitutive expression of the lac operon. These mutations occur at a high rate and provide a significant benefit. Despite this, eight of 24 populations evolved for 8,000 generations in environments containing lactose contained no detectable repressor mutations. We report here on the basis of this observation. We find that, given relevant mutation rates, repressor mutations are expected to have fixed in all evolved populations if they had maintained the same fitness effect they confer when introduced to the ancestor. In fact, reconstruction experiments demonstrate that repressor mutations have become neutral or deleterious in those populations in which they were not detectable. Populations not fixing repressor mutations nevertheless reached the same fitness as those that did fix them, indicating that they followed an alternative evolutionary path that made redundant the potential benefit of the repressor mutation, but involved unique mutations of equivalent benefit. We identify a mutation occurring in the promoter region of the uspB gene as a candidate for influencing the selective choice between these paths. Our results detail an example of historical contingency leading to divergent evolutionary outcomes.
