L-Alanine Exporter, AlaE, of Escherichia coli Functions as a Safety Valve to Enhance Survival under Feast Conditions

The intracellular level of amino acids is determined by the balance between their anabolic and catabolic pathways. L-alanine is anabolized by three L-alanine synthesizing enzymes and catabolized by two racemases and D-amino acid dehydrogenase (DadA). In addition, its level is regulated by L-alanine movement across the inner membrane. We identified the novel gene alaE, encoding an L-alanine exporter. To elucidate the physiological function of L-Alanine exporter, AlaE, we determined the susceptibility of alaE-, dadA-, and alaE/dadA-deficient mutants, derived from the wild-type strain MG1655, to L-alanyl-L-alanine (Ala-Ala), which shows toxicity to the L-alanine-nonmetabolizing variant lacking alaE. The dadA-deficient mutant has a similar minimum inhibitory concentration (MIC) (>1.25 mg/mL) to that observed in MG1655. However, alaE- and alaE/dadA-deficient mutants had MICs of 0.04 and 0.0025 mg/mL, respectively. The results suggested that the efficacy of AlaE to relieve stress caused by toxic intracellular accumulation of L-alanine was higher than that of DadA. Consistent with this, the intracellular level of alanine in the alaE-mutant was much higher than that in MG1655 and the dadA-mutant. We, therefore, conclude that AlaE functions as a ‘safety-valve’ to prevent the toxic level accumulation of intracellular L-alanine under a peptide-rich environment, such as within the animal intestine.

Lessons in Membrane Engineering for Octanoic Acid Production from EnvironmentalEscherichia coliIsolates

ABSTRACTFermentative production of many attractive biorenewable fuels and chemicals is limited by product toxicity in the form of damage to the microbial cell membrane. Metabolic engineering of the production organism can help mitigate this problem, but there is a need for identification and prioritization of the most effective engineering targets. Here, we use a set of previously characterized environmentalEscherichia coliisolates with high tolerance and production of octanoic acid, a model membrane-damaging biorenewable product, as a case study for identifying and prioritizing membrane engineering strategies. This characterization identified differences in the membrane lipid composition, fluidity, integrity, and cell surface hydrophobicity from those of the lab strain MG1655. Consistent with previous publications, decreased membrane fluidity was associated with increased fatty acid production ability. Maintenance of high membrane integrity or longer membrane lipids seemed to be of less importance than fluidity. Cell surface hydrophobicity was also directly associated with fatty acid production titers, with the strength of this association demonstrated by plasmid-based expression of the multiple stress resistance outer membrane protein BhsA. This expression ofbhsAwas effective in altering hydrophobicity, but the direction and magnitude of the change differed between strains. Thus, additional strategies are needed to reliably engineer cell surface hydrophobicity. This work demonstrates the ability of environmental microbiological studies to impact the metabolic engineering design-build-test-learn cycle and possibly increase the economic viability of fermentative bioprocesses.IMPORTANCEThe production of bulk fuels and chemicals in a bio-based fermentation process requires high product titers. This is often difficult to achieve, because many of the target molecules damage the membrane of the microbial cell factory. Engineering the composition of the membrane in order to decrease its vulnerability to this damage has proven to be an effective strategy for improving bioproduction, but additional strategies and engineering targets are needed. Here, we studied a small set of environmentalEscherichia coliisolates that have higher production titers of octanoic acid, a model biorenewable chemical, than those of the lab strain MG1655. We found that membrane fluidity and cell surface hydrophobicity are strongly associated with improved octanoic acid production. Fewer genetic modification strategies have been demonstrated for tuning hydrophobicity relative to fluidity, leading to the conclusion that there is a need for expanding hydrophobicity engineering strategies inE. coli.

Subtractive hybridization and optical mapping of the enterotoxigenic Escherichia coli H10407 chromosome: isolation of unique sequences and demonstration of significant similarity to the chromosome of E. coli K-12

Enterotoxigenic Escherichia coli (ETEC) is a primary cause of diarrhoea in infants in developing countries and in travellers to endemic regions. While several virulence genes have been identified on ETEC plasmids, little is known about the ETEC chromosome, although it is expected to share significant homology in backbone sequences with E. coli K-12. In the absence of genomic sequence information, the subtractive hybridization method and the more recently described optical mapping technique were carried out to determine the degree of genomic variation between virulent ETEC strain H10407 and the non-pathogenic E. coli K-12 strain MG1655. In one round of PCR-based suppression subtractive hybridization, 153 fragments representing sequences unique to strain H10407 were identified. blast searches indicated that few unique sequences showed homology to known pathogenicity island genes identified in related E. coli pathogens. A total of 65 fragments contained sequences that were either linked to hypothetical proteins or showed no homology to any known sequence in the database. The remaining sequences were either phage or prophage related or displayed homology to classifiable genes that function in various aspects of bacterial metabolism. The 153 unique sequences showed variable distribution across different ETEC strains including ETEC strain B7A, which is attenuated in virulence and lacked several H10407-specific sequences. Restriction-enzyme-based optical maps of strain H10407 were compared to in silico restriction maps of strain MG1655 and related E. coli pathogens. The 5·1 Mb ETEC chromosome was ∼500 kb greater in length than the chromosome of E. coli K-12, collinear with it and indicated several discrete regions where insertions and/or deletions had occurred relative to the chromosome of strain MG1655. No major inversions, transpositions or gross rearrangements were observed on the ETEC chromosome. Based on comparisons with known genomic sequences and related optical-map-based restriction site similarity, the sequence of the H10407 chromosome is expected to demonstrate ∼96 % identity with that of E. coli K-12.

The Transcriptional Antiterminator RfaH Represses Biofilm Formation in Escherichia coli

ABSTRACT We investigated the influence of regulatory and pathogenicity island-associated factors (Hha, RpoS, LuxS, EvgA, RfaH, and tRNA5 Leu) on biofilm formation by uropathogenic Escherichia coli (UPEC) strain 536. Only inactivation of rfaH, which encodes a transcriptional antiterminator, resulted in increased initial adhesion and biofilm formation by E. coli 536. rfaH inactivation in nonpathogenic E. coli K-12 isolate MG1655 resulted in the same phenotype. Transcriptome analysis of wild-type strain 536 and an rfaH mutant of this strain revealed that deletion of rfaH correlated with increased expression of flu orthologs. flu encodes antigen 43 (Ag43), which mediates autoaggregation and biofilm formation. We confirmed that deletion of rfaH leads to increased levels of flu and flu-like transcripts in E. coli K-12 and UPEC. Supporting the hypothesis that RfaH represses biofilm formation through reduction of the Ag43 level, the increased-biofilm phenotype of E. coli MG1655rfaH was reversed upon inactivation of flu. Deletion of the two flu orthologs, however, did not modify the behavior of mutant 536rfaH. Our results demonstrate that the strong initial adhesion and biofilm formation capacities of strain MG1655rfaH are mediated by both increased steady-state production of Ag43 and likely increased Ag43 presentation due to null rfaH-dependent lipopolysaccharide depletion. Although the roles of rfaH in the biofilm phenotype are different in UPEC strain 536 and K-12 strain MG1655, this study shows that RfaH, in addition to affecting the expression of bacterial virulence factors, also negatively controls expression and surface presentation of Ag43 and possibly another Ag43-independent factor(s) that mediates cell-cell interactions and biofilm formation.

Microarray analysis of gene regulation by oxygen, nitrate, nitrite, FNR, NarL and NarP during anaerobic growth of Escherichia coli: new insights into microbial physiology

RNA was isolated from cultures of Escherichia coli strain MG1655 and derivatives defective in fnr, narXL, or narXL with narP, during aerobic growth, or anaerobic growth in the presence or absence of nitrate or nitrite, in non-repressing media in which both strain MG1655 and an fnr deletion mutant grew at similar rates. Glycerol was used as the non-repressing carbon source and both trimethylamine-N-oxide and fumarate were added as terminal electron acceptors. Microarray data supplemented with bioinformatic data revealed that the FNR (fumarate and nitrate reductase regulator) regulon includes at least 104, and possibly as many as 115, operons, 68 of which are activated and 36 are repressed during anaerobic growth. A total of 51 operons were directly or indirectly activated by NarL in response to nitrate; a further 41 operons were repressed. Four subgroups of genes implicated in management of reactive nitrogen compounds, NO and products of NO metabolism, were identified; they included proteins of previously unknown function. Global repression by the nitrate- and nitrite-responsive two-component system, NarQ-NarP, was shown for the first time. In contrast with the frdABCD, aspA and ansB operons that are repressed only by NarL, the dcuB-fumB operon was among 37 operons that are repressed by NarP.

Expression of fnr Is Constrained by an Upstream IS5 Insertion in Certain Escherichia coli K-12 Strains

ABSTRACT FNR is a global transcriptional regulator that controls anaerobic gene expression in Escherichia coli. Through the use of a number of approaches it was shown that fnr gene expression is reduced approximately three- to fourfold in E. coli strain MC4100 compared with the results seen with strain MG1655. This reduction in fnr expression is due to the insertion of IS5 (is5F) in the regulatory region of the gene at position −41 relative to the transcription initiation site. Transcription of the fnr gene nevertheless occurs from its own promoter in strain MC4100, but transcript levels are reduced approximately fourfold compared with those seen with strain MG1655. Remarkably, in strains bearing is5F the presence of Hfq prevents IS5-dependent transcriptional silencing of fnr expression. Thus, an hfq mutant of MC4100 is devoid of FNR protein and has the phenotype of an fnr mutant. In strain MG1655, or a derivative of MC4100 lacking is5F, mutation of hfq had no effect on fnr transcript levels. This finding indicates that IS5 mediates the effect of Hfq on fnr expression in MC4100. Western blot analysis revealed that cellular levels of FNR were reduced threefold in strain MC4100 compared with strain MG1655 results. A selection of FNR-dependent genes fused to lacZ were analyzed for the effects of reduced FNR levels on anaerobic gene expression. Expression of some operons, e.g., focA-pfl and fdnGHJI, was unaffected by reduction in the level of FNR, while the expression of other genes such as ndh and nikA was clearly affected.

Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression

ABSTRACT Escherichia coli strain MG1655 was chosen for sequencing because the few mutations it carries (ilvG rfb-50 rph-1) were considered innocuous. However, it has a number of growth defects. Internal pyrimidine starvation due to polarity of the rph-1 allele on pyrE was problematic in continuous culture. Moreover, the isolate of MG1655 obtained from the E. coli Genetic Stock Center also carries a large deletion around the fnr (fumarate-nitrate respiration) regulatory gene. Although studies on DNA microarrays revealed apparent cross-regulation of gene expression between galactose and lactose metabolism in the Stock Center isolate of MG1655, this was due to the occurrence of mutations that increased lacY expression and suppressed slow growth on galactose. The explanation for apparent cross-regulation between galactose and N-acetylglucosamine metabolism was similar. By contrast, cross-regulation between lactose and maltose metabolism appeared to be due to generation of internal maltosaccharides in lactose-grown cells and may be physiologically significant. Lactose is of restricted distribution: it is normally found together with maltosaccharides, which are starch degradation products, in the mammalian intestine. Strains designated MG1655 and obtained from other sources differed from the Stock Center isolate and each other in several respects. We confirmed that use of other E. coli strains with MG1655-based DNA microarrays works well, and hence these arrays can be used to study any strain of interest. The responses to nitrogen limitation of two urinary tract isolates and an intestinal commensal strain isolated recently from humans were remarkably similar to those of MG1655.

Definition of the Escherichia coli MC4100 Genome by Use of a DNA Array

ABSTRACT We have used an Escherichia coli K-12 whole-genome array based on the DNA sequence of strain MG1655 as a tool to identify deletions in another E. coli K-12 strain, MC4100, by probing the array with labeled chromosomal DNA. Despite the continued widespread use of MC4100 as an experimental system, the specific genetic relationship of this strain to the sequenced K-12 derivative MG1655 has not been resolved. MC4100 was found to contain four deletions, ranging from 1 to 97 kb in size. The exact nature of three of the deletions was previously unresolved, and the fourth deletion was altogether unknown.