A Secondary Metabolic Enzyme Functioned as an Evolutionary Seed of a Primary Metabolic Enzyme.

The antibiotic alaremycin has a structure that resembles that of 5-aminolevulinic acid (ALA), a universal precursor of porphyrins, and inhibits porphyrin biosynthesis. Genome sequencing of the alaremycin-producing bacterial strain and enzymatic analysis revealed that the first step of alaremcyin biosynthesis is catalysed by the enzyme, AlmA, which exhibits a high degree of similarity to 5-aminolevulinate synthase (ALAS) expressed by animals, protozoa, fungi, and α-proteobacteria. Site-directed mutagenesis of AlmA revealed that the substitution of two amino acids residues around the substrate binding pocket transformed its substrate specificity from that of alaremycin precursor synthesis to ALA synthesis. To estimate the evolutionary trajectory of AlmA and ALAS, we performed an ancestral sequence reconstitution analysis based on a phylogenetic tree of AlmA and ALAS. The reconstructed common ancestral enzyme of AlmA and ALAS exhibited alaremycin precursor synthetic activity, rather than ALA synthetic activity. These results suggest that ALAS evolved from an AlmA-like enzyme. We propose a new evolutionary hypothesis in which a non-essential secondary metabolic enzyme acts as an 'evolutionary seed' to generate an essential primary metabolic enzyme.

Mining RNA-seq data reveals the massive regulon of GcvB small RNA and its physiological significance in maintaining amino acid homeostasis in Escherichia coli.

Bacterial small RNAs regulate the expression of multiple genes through imperfect base-pairing with target mRNAs mediated by RNA chaperone proteins such as Hfq. GcvB is the master sRNA regulator of amino acid metabolism and transport in a wide range of Gram-negative bacteria. Recently, independent RNA-seq approaches identified a plethora of transcripts interacting with GcvB in Escherichia coli. In this study, the compilation of RIL-seq, CLASH, and MAPS data sets allowed us to identify GcvB targets with high accuracy. We validated 21 new GcvB targets repressed at the posttranscriptional level, raising the number of direct targets to >50 genes in E. coli. Among its multiple seed sequences, GcvB utilizes either R1 or R3 to regulate most of these targets. Furthermore, we demonstrated that both R1 and R3 seed sequences are required to fully repress the expression of gdhA, cstA, and sucC genes. In contrast, the ilvLXGMEDA polycistronic mRNA is targeted by GcvB through at least four individual binding sites in the mRNA. Finally, we revealed that GcvB is involved in the susceptibility of peptidase-deficient E. coli strain (Δpeps) to Ala-Gln dipeptide by regulating both Dpp dipeptide importer and YdeE dipeptide exporter via R1 and R3 seed sequences, respectively.

Requirement of the LtsA Protein for Formation of the Mycolic Acid-Containing Layer on the Cell Surface of Corynebacterium glutamicum.

The ltsA gene of Corynebacterium glutamicum encodes a purF-type glutamine-dependent amidotransferase, and mutations in this gene result in increased susceptibility to lysozyme. Recently, it was shown that the LtsA protein catalyzes the amidation of diaminopimelate residues in the lipid intermediates of peptidoglycan biosynthesis. In this study, intracellular localization of wild-type and mutant LtsA proteins fused with green fluorescent protein (GFP) was investigated. The GFP-fused wild-type LtsA protein showed a peripheral localization pattern characteristic of membrane-associated proteins. The GFP-fusions with a mutation in the N-terminal domain of LtsA, which is necessary for the glutamine amido transfer reaction, exhibited a similar localization to the wild type, whereas those with a mutation or a truncation in the C-terminal domain, which is not conserved among the purF-type glutamine-dependent amidotransferases, did not. These results suggest that the C-terminal domain is required for peripheral localization. Differential staining of cell wall structures with fluorescent dyes revealed that formation of the mycolic acid-containing layer at the cell division planes was affected in the ltsA mutant cells. This was also confirmed by observation that bulge formation was induced at the cell division planes in the ltsA mutant cells upon lysozyme treatment. These results suggest that the LtsA protein function is required for the formation of a mycolic acid-containing layer at the cell division planes and that this impairment results in increased susceptibility to lysozyme.

RNase E-dependent degradation of tnaA mRNA encoding tryptophanase is prerequisite for the induction of acid resistance in Escherichia coli.

Acid-resistance systems are essential for pathogenic Escherichia coli to survive in the strongly acidic environment of the human stomach (pH < 2.5). Among these, the glutamic acid decarboxylase (GAD) system is the most effective. However, the precise mechanism of GAD induction is unknown. We previously reported that a tolC mutant lacking the TolC outer membrane channel was defective in GAD induction. Here, we show that indole, a substrate of TolC-dependent efflux pumps and produced by the tryptophanase encoded by the tnaA gene, negatively regulates GAD expression. GAD expression was restored by deleting tnaA in the tolC mutant; in wild-type E. coli, it was suppressed by adding indole to the growth medium. RNA-sequencing revealed that tnaA mRNA levels drastically decreased upon exposure to moderately acidic conditions (pH 5.5). This decrease was suppressed by RNase E deficiency. Collectively, our results demonstrate that the RNase E-dependent degradation of tnaA mRNA is accelerated upon acid exposure, which decreases intracellular indole concentrations and triggers GAD induction.

Growth promotion in Corynebacterium glutamicum by overexpression of the NCgl2986 gene encoding a protein homologous to peptidoglycan amidases.

We previously reported the extracellular production of antibody fragment Fab by Corynebacterium glutamicum. In the course of searching for genes which improve the secretion efficiency of Fab, we coincidentally found that the final growth increased significantly when the NCgl2986 gene encoding an amidase-like protein was overexpressed. This effect was observed when cells were grown on the production medium MMTG, which contains high concentrations of glucose and neutralizing agent CaCO3, but not on MMTG without CaCO3 or Lennox medium. Not only turbidity but also dry cell weight was increased by NCgl2986 overexpression, although the growth rate was not affected. It was recently reported that the Mycobacterium tuberculosis homolog Rv3915 functions as an activator of MurA protein, which catalyzes the initial step of peptidoglycan synthesis. Growth promotion was also observed when the MurA protein was overproduced. His-tagged NCgl2986 protein was purified, but its peptidoglycan hydrolyzing activity could not be detected. These results suggest that NCgl2986 promotes cell growth by activating the peptidoglycan synthetic pathway.

RNase E/G-dependent degradation of metE mRNA, encoding methionine synthase, in Corynebacterium glutamicum.

Corynebacterium glutamicum is used for the industrial production of various metabolites, including L-glutamic acid and L-lysine. With the aim of understanding the post-transcriptional regulation of amino acid biosynthesis in this bacterium, we investigated the role of RNase E/G in the degradation of mRNAs encoding metabolic enzymes. In this study, we found that the cobalamin-independent methionine synthase MetE was overexpressed in ΔrneG mutant cells grown on various carbon sources. The level of metE mRNA was also approximately 6- to 10-fold higher in the ΔrneG mutant strain than in the wild-type strain. A rifampicin chase experiment showed that the half-life of metE mRNA was approximately 4.2 times longer in the ΔrneG mutant than in the wild-type strain. These results showed that RNase E/G is involved in the degradation of metE mRNA in C. glutamicum.

Effects of EGTA on cell surface structures of Corynebacterium glutamicum.

The mycolic acid layer and S-layer of Corynebacterium glutamicum have been considered as permeability barriers against lytic agents. EGTA, a calcium chelator, inhibited C. glutamicum growth at relatively lower concentrations compared with other Gram-positive bacteria. We investigated the effect of EGTA on C. glutamicum cell surface structures. Simultaneous addition of EGTA and lysozyme resulted in cell lysis, whereas addition of these reagents separately had no such effect. Analysis of cell surface proteins showed that CspB, an S-layer protein, was released into the culture media and degraded to several sizes upon EGTA treatment. These findings suggest that EGTA treatment causes release and proteolysis of the CspB protein, resulting in increased cell surface permeability. FE-SEM visualization further confirmed alteration of cell surface structures in EGTA-treated cells. This is the first report suggesting the importance of calcium ions in cell surface integrity of C. glutamicum.

Glutamate Fermentation-2: Mechanism of L-Glutamate Overproduction in Corynebacterium glutamicum.

The nonpathogenic coryneform bacterium, Corynebacterium glutamicum, was isolated as an L-glutamate-overproducing microorganism by Japanese researchers and is currently utilized in various amino acid fermentation processes. L-Glutamate production by C. glutamicum is induced by limitation of biotin and addition of fatty acid ester surfactants and ß-lactam antibiotics. These treatments affect the cell surface structures of C. glutamicum. After the discovery of C. glutamicum, many researchers have investigated the underlying mechanism of L-glutamate overproduction with respect to the cell surface structures of this organism. Furthermore, metabolic regulation during L-glutamate overproduction by C. glutamicum, particularly, the relationship between central carbon metabolism and L-glutamate biosynthesis, has been investigated. Recently, the role of a mechanosensitive channel protein in L-glutamate overproduction has been reported. In this chapter, mechanisms of L-glutamate overproduction by C. glutamicum have been reviewed.

Polynucleotide Phosphorylase, RNase E/G, and YbeY Are Involved in the Maturation of 4.5S RNA in Corynebacterium glutamicum.

Corynebacterium glutamicum has been applied for the industrial production of various metabolites, such as amino acids. To understand the biosynthesis of the membrane protein in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP is found in all three domains of life and plays an important role in the membrane insertion of proteins. SRP RNA is initially transcribed as precursor molecules; however, relatively little is known about its maturation. In C. glutamicum, SRP consists of the Ffh protein and 4.5S RNA lacking an Alu domain. In this study, we found that 3'-to-5' exoribonuclease, polynucleotide phosphorylase (PNPase), and two endo-type RNases, RNase E/G and YbeY, are involved in the 3' maturation of 4.5S RNA in C. glutamicum The mature form of 4.5S RNA was inefficiently formed in ΔrneG Δpnp mutant cells, suggesting the existence of an alternative pathway for the 3' maturation of 4.5S RNA. Primer extension analysis also revealed that the 5' mature end of 4.5S RNA corresponds to that of the transcriptional start site. Immunoprecipitated Ffh protein contained immature 4.5S RNA in Δpnp, ΔrneG, and ΔybeY mutants, suggesting that 4.5S RNA precursors can interact with Ffh. These results imply that the maturation of 4.5S RNA can be performed in the 4.5S RNA-Ffh complex.IMPORTANCE Overproduction of a membrane protein, such as a transporter, is useful for engineering of strains of Corynebacterium glutamicum, which is a workhorse of amino acid production. To understand membrane protein biogenesis in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP contains the Ffh protein and SRP RNA and plays an important role in the membrane insertion of proteins. Although SRP RNA is highly conserved among the three domains of life, relatively little is known about its maturation. We show that PNPase, RNase E/G, and YbeY are involved in the 3' maturation of the SRP RNA (4.5S RNA) in this bacterium. This indicates that 3' end processing in this organism is different from that in other bacteria, such as Escherichia coli.

High crude violacein production from glucose by Escherichia coli engineered with interactive control of tryptophan pathway and violacein biosynthetic pathway.

BACKGROUND: As bacteria-originated crude violacein, a natural indolocarbazole product, consists of violacein and deoxyviolacein, and can potentially be a new type of natural antibiotics, the reconstruction of an effective metabolic pathway for crude violacein (violacein and deoxyviolacein mixture) synthesis directly from glucose in Escherichia coli was of importance for developing industrial production process. RESULTS: Strains with a multivariate module for varied tryptophan productivities were firstly generated by combinatorial knockout of trpR/tnaA/pheA genes and overexpression of two key genes trpEfbr /trpD from the upstream tryptophan metabolic pathway. Then, the gene cluster of violacein biosynthetic pathway was introduced downstream of the generated tryptophan pathway. After combination of these two pathways, maximum crude violacein production directly from glucose by E. coli B2/pED+pVio was realized with a titer of 0.6±0.01 g L(-1) in flask culture, which was four fold higher than that of the control without the tryptophan pathway up-regulation. In a 5-L bioreactor batch fermentation with glucose as the carbon source, the recombinant E. coli B2/pED+pVio exhibited a crude violacein titer of 1.75 g L(-1) and a productivity of 36 mg L(-1) h(-1), which was the highest titer and productivity reported so far under the similar culture conditions without tryptophan addition. CONCLUSION: Metabolic pathway analysis using 13C labeling illustrated that the up-regulated tryptophan supply enhanced tryptophan metabolism from glucose, whereas the introduction of violacein pathway drew more carbon flux from glucose to tryptophan, thereby contributing to the effective production of crude violacein in the engineered E. coli cell factory.

Degradation of benzotrifluoride via the dioxygenase pathway in Rhodococcus sp. 065240.

We previously isolated Rhodococcus sp. 065240, which catalyzes the defluorination of benzotrifluoride (BTF). In order to investigate the mechanism of this degradation of BTF, we performed proteomic analysis of cells grown with or without BTF. Three proteins, which resemble dioxygenase pathway enzymes responsible for isopropylbenzene degradation from Rhodococcus erythropolis BD2, were induced by BTF. Genomic PCR and DNA sequence analysis revealed that the Rhodococcus sp. 065240 carries the gene cluster, btf, which is highly homologous to the ipb gene cluster from R. erythropolis BD2. A mutant strain, which could not catalyze BTF defluorination, was isolated from 065240 strain by UV mutagenesis. The mutant strain had one mutation in the btfT gene, which encodes a response regulator of the two component system. The defluorinating ability of the mutant strain was recovered by complementation of btfT. These results suggest that the btf gene cluster is responsible for degradation of BTF.

Characterization of a Corynebacterium glutamicum dnaB mutant that shows temperature-sensitive growth and mini-cell formation.

Corynebacterium glutamicum is known to perform a unique form of cell division called post-fission snapping division. In order to investigate the mechanism of cell division of this bacterium, we isolated temperature-sensitive mutants from C. glutamicum wild-type strain ATCC 31831, and found that one of them, M45, produced high frequencies of mini-cells with no nucleoids. Cell pairs composed of an elongated cell, with one nucleoid, connected to a mini-cell, with no nucleoids, were occasionally observed. The temperature sensitivity and mini-cell formation of M45 was complemented by a 2-kb DraI-EcoRI fragment derived from the ATCC 31831 chromosomal DNA, which carried a dnaB homolog encoding a replicative DNA helicase. DNA sequence analysis revealed that M45 carried a missense mutation in the dnaB gene, which caused a substitution of Thr364 to Ile. Microscopic observation after 4',6-diamidino-2-phenylindole staining revealed that the DNA content of single cells was decreased by culturing at the restrictive temperature, suggesting that the mutation affects chromosomal replication. These results suggest that the C. glutamicum dnaB mutant performs an asymmetric cell division even after DNA replication is inhibited, which results in the production of mini-cells.

Isolation of oleaginous yeast (Rhodosporidium toruloides) mutants tolerant of sugarcane bagasse hydrolysate.

Rhodosporidium toruloides is a lipid-producing yeast, the growth of which is severely suppressed when hydrolysates of lignocellulosic biomass are used as carbon source. This is probably due to the toxic substances, such as organic acids, furans, and phenolic compounds produced during the preparation of the hydrolysates. In order to solve this problem, R. toruloides cultures were subjected to atmospheric room-temperature plasma mutagenesis, resulting in the isolation of mutants showing tolerance to sugarcane bagasse hydrolysate (SBH). Three mutant strains, M11, M13, and M18, were found to grow with producing lipids with SBH as carbon source. M11 in particular appeared to accumulate higher levels (up to 60% of dry cell weight) of intracellular lipids. Further, all three mutant strains showed tolerance of vanillin, furfural, and acetic acid, with different spectra, suggesting that different genetic determinants are involved in SBH tolerance.

Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum.

BACKGROUND: Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production. RESULTS: The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion. CONCLUSION: There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system.

A secondary structure in the 5' untranslated region of adhE mRNA required for RNase G-dependent regulation.

Escherichia coli RNase G is involved in the degradation of several mRNAs, including adhE and eno, which encode alcohol dehydrogenase and enolase respectively. Previous research indicates that the 5' untranslated region (5'-UTR) of adhE mRNA gives RNase G-dependency to lacZ mRNA when tagged at the 5'-end, but it has not been elucidated yet how RNase G recognizes adhE mRNA. Primer extension analysis revealed that RNase G cleaved a phosphodiester bond between -19A and -18C in the 5'-UTR (the A of the start codon was defined as +1). Site-directed mutagenesis indicated that RNase G did not recognize the nucleotides at -19 and -18. Random deletion analysis indicated that the sequence from -145 to -125 was required for RNase G-dependent degradation. Secondary structure prediction and further site-directed deletion suggested that the stem-loop structure, with a bubble in the stem, is required for RNaseG-dependent degradation of adhE mRNA.

A role of the transcriptional regulator LldR (NCgl2814) in glutamate metabolism under biotin-limited conditions in Corynebacterium glutamicum.

Corynebacterium glutamicum is a Gram-positive, rod-shaped, aerobic bacterium used for the fermentative production of L-glutamate. LldR (NCgl2814) is known as a repressor for ldhA and lldD encoding lactate dehydrogenases. LdhA is responsible for production of L-lactate, while LldD is for its assimilation. Since L-lactate production was observed as a by-product of glutamate production under biotin-limited conditions, LldR might play a regulatory role in the glutamate metabolism. Here for the first time, we investigated effects of overproduction or deletion of LldR on the glutamate metabolism under biotin-limited conditions in C. glutamicum. It was found that glutamate production under biotin-limited conditions was decreased by overproduction of LldR. In the wild-type cells, L-lactate was produced in the first 24 h and it was re-consumed thereafter. On the other hand, in the overproduced cells, L-lactate was produced like the wild type, but it was not re-consumed. This means that L-lactate assimilation, which is catalyzed by LldD, was suppressed by the overproduction of LldR, but L-lactate production, which is catalyzed by LdhA, was not affected, indicating that LldR mainly controls the expression of lldD but not of ldhA under biotin-limited conditions. This was confirmed by quantitative real-time RT-PCR. From these results, it is suggested that L-lactate metabolism, which is controlled by LldR, has a buffering function of the pyruvate pool for glutamate production.

L-Glutamate secretion by the N-terminal domain of the Corynebacterium glutamicum NCgl1221 mechanosensitive channel.

The Corynebacterium glutamicum NCgl1221 mechanosensitive channel mediates L-glutamate secretion by sensing changes in membrane tension caused by treatments such as biotin limitation and penicillin. The NCgl1221 protein has an N-terminal domain (1-286 a.a.) homologous to the Escherichia coli MscS and a long C-terminal domain (287-533 a.a.) of unknown function. In order to investigate the role of the C-terminal domain in L-glutamate secretion, we constructed a series of C-terminally truncated mutants of NCgl1221. We found that the N-terminal domain, homologous to E. coli MscS, retained the ability to cause L-glutamate secretion in response to the treatment. Electrophysiological analysis confirmed that the N-terminal domain mediated L-glutamate secretion. 3D homology modeling has suggested that the N-terminal domain of NCgl1221 has an extra loop structure (221-232 a.a.) that is not found in most other MscS proteins. The mutant NCgl1221, deleted for this loop structure, lost the ability to secrete L-glutamate. In addition, we found that mutant NCgl1221 lacking the C-terminal extracytoplasmic domain (420-533 a.a.) produced L-glutamate without any inducing treatment. These results suggest that the N-terminal domain is necessary and sufficient for the excretion of L-glutamate in response to inducing treatment, and that the C-terminal extracytoplasmic domain has a negative regulatory role in L-glutamate production.

3' Untranslated region-dependent degradation of the aceA mRNA, encoding the glyoxylate cycle enzyme isocitrate lyase, by RNase E/G in Corynebacterium glutamicum.

We previously reported that the Corynebacterium glutamicum RNase E/G encoded by the rneG gene (NCgl2281) is required for the 5' maturation of 5S rRNA. In the search for the intracellular target RNAs of RNase E/G other than the 5S rRNA precursor, we detected that the amount of isocitrate lyase, an enzyme of the glyoxylate cycle, increased in rneG knockout mutant cells grown on sodium acetate as the sole carbon source. Rifampin chase experiments showed that the half-life of the aceA mRNA was about 4 times longer in the rneG knockout mutant than in the wild type. Quantitative real-time PCR analysis also confirmed that the level of aceA mRNA was approximately 3-fold higher in the rneG knockout mutant strain than in the wild type. Such differences were not observed in other mRNAs encoding enzymes involved in acetate metabolism. Analysis by 3' rapid amplification of cDNA ends suggested that RNase E/G cleaves the aceA mRNA at a single-stranded AU-rich region in the 3' untranslated region (3'-UTR). The lacZ fusion assay showed that the 3'-UTR rendered lacZ mRNA RNase E/G dependent. These findings indicate that RNase E/G is a novel regulator of the glyoxylate cycle in C. glutamicum.

Corynebacterium glutamicum RNase E/G-type endoribonuclease encoded by NCgl2281 is involved in the 5' maturation of 5S rRNA.

Corynebacterium glutamicum has one RNase E/G ortholog and one RNase J ortholog but no RNase Y. We previously reported that the C. glutamicum NCgl2281 gene encoding the RNase E/G ortholog complemented the rng::cat mutation in Escherichia coli but not the rne-1 mutation. In this study, we constructed an NCgl2281 knockout mutant and found that the mutant cells accumulated 5S rRNA precursor molecules. The processing of 16S and 23S rRNA, tRNA, and tmRNA was normal. Primer extension analysis revealed that the RNase E/G ortholog cleaved at the -1 site of the 5' end of 5S rRNA. However, 3' maturation was essentially unaffected. These findings showed that C. glutamicum NCgl2281 endoribonuclease is involved in the 5' maturation of 5S rRNA. This is the first report showing the physiological function of the RNase E/G ortholog in bacteria having one RNase E/G and one RNase J but no RNase Y.

A requirement of TolC and MDR efflux pumps for acid adaptation and GadAB induction in Escherichia coli.

BACKGROUND: The TolC outer membrane channel is a key component of several multidrug resistance (MDR) efflux pumps driven by H(+) transport in Escherichia coli. While tolC expression is under the regulation of the EvgA-Gad acid resistance regulon, the role of TolC in growth at low pH and extreme-acid survival is unknown. METHODS AND PRINCIPAL FINDINGS: TolC was required for extreme-acid survival (pH 2) of strain W3110 grown aerobically to stationary phase. A tolC deletion decreased extreme-acid survival (acid resistance) of aerated pH 7.0-grown cells by 10(5)-fold and of pH 5.5-grown cells by 10-fold. The requirement was specific for acid resistance since a tolC defect had no effect on aerobic survival in extreme base (pH 10). TolC was required for expression of glutamate decarboxylase (GadA, GadB), a key component of glutamate-dependent acid resistance (Gad). TolC was also required for maximal exponential growth of E. coli K-12 W3110, in LBK medium buffered at pH 4.5-6.0, but not at pH 6.5-8.5. The TolC growth requirement in moderate acid was independent of Gad. TolC-associated pump components EmrB and MdtB contributed to survival in extreme acid (pH 2), but were not required for growth at pH 5. A mutant lacking the known TolC-associated efflux pumps (acrB, acrD, emrB, emrY, macB, mdtC, mdtF, acrEF) showed no growth defect at acidic pH and a relatively small decrease in extreme-acid survival when pre-grown at pH 5.5. CONCLUSIONS: TolC and proton-driven MDR efflux pump components EmrB and MdtB contribute to E. coli survival in extreme acid and TolC is required for maximal growth rates below pH 6.5. The TolC enhancement of extreme-acid survival includes Gad induction, but TolC-dependent growth rates below pH 6.5 do not involve Gad. That MDR resistance can enhance growth and survival in acid is an important consideration for enteric organisms passing through the acidic stomach.