Aerobic Respiration Control Research Articles

ArcA positively regulates the expression of virulence genes and contributes to virulence of porcine Shiga toxin-producing enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) cause severe diarrhea in humans and animals. These bacteria encode two major classes of virulence factors, adhesins that promote colonization of the small intestine and enterotoxins that induce fluid secretion and thus diarrhea. Oxygen and redox sensors are regulators of virulence expression in multiple bacterial pathogens. In this study, we show that microaerobic conditions enhance the expression of virulence factors in a porcine Shiga toxin-producing ETEC (STEC/ETEC) strain. We then investigated the role of the aerobic respiration control regulator ArcA in the pathogenicity and virulence regulation in STEC/ETEC. In a mouse model, deletion of arcA caused less severe pathology, and the arcA mutant displayed lower levels of colonization and obvious weight gain of the mice compared to the wild type (WT). In a cell culture model, a lack of arcA reduced the adherence of STEC/ETEC to porcine intestinal epithelial cells. Furthermore, ArcA positively regulated the expression of several key virulence factors, including F18 fimbriae (fed), heat-labile (eltA) and heat-stable (estB) toxins, Shiga toxin 2e (stx2e), and hemolysin (hlyC), under microaerobic conditions and in vivo conditions. We then found that ArcA positively regulated the expression of eltA, estB, and hlyC by competing with a global repressor H-NS (histone-like nucleoid structuring). Mechanistically, we show that ArcA protein directly binds to the promoters of target genes, displaces H-NS silencing from the promoter and counteracts H-NS's repression. Collectively, our data established a key role for ArcA in the pathogenicity and virulence expression of porcine STEC/ETEC. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) cause severe diarrhea in humans and animals, leading to death and huge economic loss worldwide. Thus, elucidation of ETEC's pathogenic mechanisms will provide powerful data for the discovery of drugs serving as prevention or therapeutics against ETEC-caused diarrheal diseases. Here, we report that ArcA plays an essential role in the pathogenicity and virulence regulation in ETEC by positively regulating the expression of several key virulence factors including F18 fimbriae, heat-labile and heat-stable toxins, Shiga toxin 2e, and hemolysin, under microaerobic conditions and in vivo. Moreover, we found that positive regulation of several virulence genes by ArcA requires a global repressor H-NS (histone-like nucleoid structuring), implying that ArcA may exert positive effects by antagonizing H-NS. Collectively, our data established a key role for ArcA in the pathogenicity of porcine ETEC and ETEC strains isolated from human infections. Moreover, our work reveals another layer of regulation in relation to oxygen control of virulence factors in ETEC.

Oxygen concentration modulates colibactin production

ABSTRACT Up to 25% of the E. coli strains isolated from the feces of healthy humans harbor the pks genomic island encoding the synthesis of colibactin, a genotoxic metabolite. Evidence is accumulating for an etiologic role of colibactin in colorectal cancer. Little is known about the conditions of expression of colibactin in the gut. The intestine is characterized by a unique oxygenation profile, with a steep gradient between the physiological hypoxic epithelial surface and the anaerobic lumen, which favors the dominance of obligate anaerobes. Here, we report that colibactin production is maximal under anoxic conditions and decreases with increased oxygen concentration. We show that the aerobic respiration control (ArcA) positively regulates colibactin production and genotoxicity of pks+ E. coli in response to oxygen availability. Thus, colibactin synthesis is inhibited by oxygen, indicating that the pks biosynthetic pathway is adapted to the anoxic intestinal lumen and to the hypoxic infected or tumor tissue.

Enhancing bacterial cellulose production with hypoxia-inducible factors.

Komagataeibacter xylinus is an aerobic strain that produces bacterial cellulose (BC). Oxygen levels play a critical role in regulating BC synthesis in K. xylinus, and an increase in oxygen tension generally means a decrease in BC production. Fumarate nitrate reduction protein (FNR) and aerobic respiration control protein A (ArcA) are hypoxia-inducible factors, which can signal whether oxygen is present in the environment. In this study, FNR and ArcA were used to enhance the efficiency of oxygen signaling in K. xylinus, and globally regulate the transcription of the genome to cope with hypoxic conditions, with the goal of improving growth and BC production. FNR and ArcA were individually overexpressed in K. xylinus, and the engineered strains were cultivated under different oxygen tensions to explore how their overexpression affects cellular metabolism and regulation. Although FNR overexpression did not improve BC production, ArcA overexpression increased BC production by 24.0% and 37.5% as compared to the control under oxygen tensions of 15% and 40%, respectively. Transcriptome analysis showed that FNR and ArcA overexpression changed the way K. xylinus coped with oxygen tension changes, and that both FNR and ArcA overexpression enhanced the BC synthesis pathway. The results of this study provide a new perspective on the effect of oxygen signaling on growth and BC production in K. xylinus and suggest a promising strategy for enhancing BC production through metabolic engineering. KEY POINTS: • K. xylinus BC production increased after overexpression of ArcA • The young's modulus is enhanced by the ArcA overexpression • ArcA and FNR overexpression changed how cells coped with changes in oxygen tension.

Piggybacking on Niche Adaptation Improves the Maintenance of Multidrug-Resistance Plasmids.

The persistence of plasmids in bacterial populations represents a puzzling evolutionary problem with serious clinical implications due to their role in the ongoing antibiotic resistance crisis. Recently, major advancements have been made toward resolving this “plasmid paradox” but mainly in a nonclinical context. Here, we propose an additional explanation for the maintenance of multidrug‐resistance plasmids in clinical Escherichia coli strains. After coevolving two multidrug‐resistance plasmids encoding resistance to last resort carbapenems with an extraintestinal pathogenic E. coli strain, we observed that chromosomal media adaptive mutations in the global regulatory systems CCR (carbon catabolite repression) and ArcAB (aerobic respiration control) pleiotropically improved the maintenance of both plasmids. Mechanistically, a net downregulation of plasmid gene expression reduced the fitness cost. Our results suggest that global chromosomal transcriptional rewiring during bacterial niche adaptation may facilitate plasmid maintenance.

Structure and reactivity of a siderophore-interacting protein from the marine bacterium Shewanella reveals unanticipated functional versatility

Siderophores make iron accessible under iron-limited conditions and play a crucial role in the survival of microorganisms. Because of their remarkable metal-scavenging properties and ease in crossing cellular envelopes, siderophores hold great potential in biotechnological applications, raising the need for a deeper knowledge of the molecular mechanisms underpinning the siderophore pathway. Here, we report the structural and functional characterization of a siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIBM400 (SfSIP). SfSIP is a flavin-containing ferric-siderophore reductase with FAD- and NAD(P)H-binding domains that have high homology with other characterized SIPs. However, we found here that it mechanistically departs from what has been described for this family of proteins. Unlike other FAD-containing SIPs, SfSIP did not discriminate between NADH and NADPH. Furthermore, SfSIP required the presence of the Fe2+-scavenger, ferrozine, to use NAD(P)H to drive the reduction of Shewanella-produced hydroxamate ferric-siderophores. Additionally, this is the first SIP reported that also uses a ferredoxin as electron donor, and in contrast to NAD(P)H, its utilization did not require the mediation of ferrozine, and electron transfer occurred at fast rates. Finally, FAD oxidation was thermodynamically coupled to deprotonation at physiological pH values, enhancing the solubility of ferrous iron. On the basis of these results and the location of the SfSIP gene downstream of a sequence for putative binding of aerobic respiration control protein A (ArcA), we propose that SfSIP contributes an additional layer of regulation that maintains cellular iron homeostasis according to environmental cues of oxygen availability and cellular iron demand.

Comparative analyses of proteins from Haemophilus influenzae biofilm and planktonic populations using metabolic labeling and mass spectrometry.

BackgroundNon-typeable H. influenzae (NTHi) is a nasopharyngeal commensal that can become an opportunistic pathogen causing infections such as otitis media, pneumonia, and bronchitis. NTHi is known to form biofilms. Resistance of bacterial biofilms to clearance by host defense mechanisms and antibiotic treatments is well-established. In the current study, we used stable isotope labeling by amino acids in cell culture (SILAC) to compare the proteomic profiles of NTHi biofilm and planktonic organisms. Duplicate continuous-flow growth chambers containing defined media with either “light” (L) isoleucine or “heavy” (H) 13C6-labeled isoleucine were used to grow planktonic (L) and biofilm (H) samples, respectively. Bacteria were removed from the chambers, mixed based on weight, and protein extracts were generated. Liquid chromatography-mass spectrometry (LC-MS) was performed on the tryptic peptides and 814 unique proteins were identified with 99% confidence.ResultsComparisons of the NTHi biofilm to planktonic samples demonstrated that 127 proteins showed differential expression with p-values ≤0.05. Pathway analysis demonstrated that proteins involved in energy metabolism, protein synthesis, and purine, pyrimidine, nucleoside, and nucleotide processes showed a general trend of downregulation in the biofilm compared to planktonic organisms. Conversely, proteins involved in transcription, DNA metabolism, and fatty acid and phospholipid metabolism showed a general trend of upregulation under biofilm conditions. Selected reaction monitoring (SRM)-MS was used to validate a subset of these proteins; among these were aerobic respiration control protein ArcA, NAD nucleotidase and heme-binding protein A.ConclusionsThe present proteomic study indicates that the NTHi biofilm exists in a semi-dormant state with decreased energy metabolism and protein synthesis yet is still capable of managing oxidative stress and in acquiring necessary cofactors important for biofilm survival.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0329-9) contains supplementary material, which is available to authorized users.

Construction of an l-serine producing Escherichia coli via metabolic engineering

L-Serine is a nonessential amino acid, but plays a crucial role as a building block for cell growth. Currently, L-serine production is mainly dependent on enzymatic or cellular conversion. In this study, we constructed a recombinant Escherichia coli that can fermentatively produce L-serine from glucose. To accumulate L-serine, sdaA encoding the L-serine dehydratase, iclR encoding the isocitrate lyase regulator, and arcA encoding the aerobic respiration control protein were deleted in turn. In batch fermentation, the engineered E. coli strain YF-5 exhibited obvious L-serine accumulation but poor cell growth. To restore cell growth, aceB encoding the malate synthase was knocked out, and the engineered strain was then transformed with plasmid that overexpressed serA (FR) , serB, and serC genes. The resulting strain YF-7 produced 4.5g/L L-serine in batch cultivation and 8.34g/L L-serine in fed-batch cultivation.

Proteomics of the dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1, using a matrix-assisted laser desorption/ionization-tandem-time of flight mass spectrometer.

Shewanella oneidensis MR-1 is a gram-negative facultative aerobic bacterium living at oxic-anoxic interfaces in nature. The plasticity of terminal electron-acceptors used under anaerobic conditions is huge, but the adaptation to these different environmental conditions remains unclear. In this work, we used a proteomic approach to study the protein content when the organism is grown under anaerobic respiration conditions on insoluble ferric oxide. By analysis of two-dimensional gel patterns of soluble protein extracts, we discovered 20 differentially displayed proteins. The protein spots were further analyzed by mass spectrometry for which we used, in addition to nano-high-performance liquid chromatography coupled to an electrospray ionization-quadrupole-time of flight instrument, a recently introduced matrix-assisted laser desorption/ionization (MALDI) tandem-time of flight mass spectrometer. The instrument allows the acquisition of high quality spectra, in both the mass spectrometry and tandem mass spectrometry mode, and is therefore able to identify protein spots unambiguously. Advantageous to electrospray ionization is a minimised sample handling, inherent to MALDI ionization, and the presence of high energy fragmentation ions, generating sequence information that also can differentiate isobaric amino acids. With this strategy, we could point out a regulatory protein that is up-regulated under iron(III) respiration. This protein, the aerobic respiration control protein (ArcA), has been reported as being a regulator during anaerobiosis in other species. To our knowledge, this is the first report of the possible involvement of ArcA from S. oneidensis MR-1 in the reduction of ferric oxide.

Overlapping promoters modulate Fnr- and ArcA-dependent anaerobic transcriptional activation of the focApfl operon in Escherichia coli.

The recently identified P6A promoter of the anaerobically inducible focApfl operon of Escherichia coll overlaps the Fnr (fumarate-nitrate reduction regulator)-dependent P6 promoter. The Fnr-binding site of P6 and the -35 hexamer sequence of P6A are shared between the promoters. Inactivation of P6A, through introduction of a -10 hexamer mutation, resulted in enhanced anaerobic induction of operon expression. The dependence on the ArcA (aerobic respiration control regulator) and Fnr transcription factors for anaerobic induction was tested for several focA-lacZ and pfl-lacZ gene fusions. Anaerobic induction became more dependent on Fnr in derivatives lacking a functional P6A promoter compared with wild-type constructs. Moreover, aerobic expression of the focA gene was reduced by the p6A mutation, as was the dependence on ArcA for anaerobic induction. Inactivation of P6 severely reduced Fnr-dependent anaerobic induction, in accord with previous findings. Transcription analyses demonstrated that a mutation in the -10 hexamer sequence of either P6A or P6 did not adversely affect transcription from the remaining promoter. Taken together, these results indicate that the P6A promoter moderates the Fnr-dependent activation of P6 through competition for RNA polymerase binding.

Roles of manganese and iron in the regulation of the biosynthesis of manganese-superoxide dismutase inEscherichia coli

Aerobic life-style offers both benefits and risks to living cells. The major risk comes from the formation of reactive oxygen intermediates (i.e. superoxide radical, O2-; hydrogen peroxide, H2O2; and hydroxyl radical, OH.) during normal oxygen metabolism. However, living cells are able to cope with oxygen toxicity by virtue of a unique set of antioxidant enzymes that scavenge O2- and H2O2, and prevent the formation OH.. Superoxide dismutases (SODs; EC 1.15.1.1) are metalloenzymes essential for aerobic survival. Escherichia coli contains two forms of this enzyme: an iron-containing enzyme (FeSOD) and a manganese-containing enzyme (MnSOD). In E. coli, MnSOD biosynthesis is under rigorous control. The enzyme is induced in response to a variety of environmental stress conditions including exposure to oxygen, redox cycling compounds such as paraquat which exacerbate the level of intracellular superoxide radicals, iron chelation (i.e. iron deprivation), and oxidants. A model for the regulation of the MnSOD has been proposed in which the MnSOD gene (sodA) is negatively regulated at the level of transcription by an iron-containing redox-sensitive repressor protein. The effect of iron-chelation most probably results in removal of the iron necessary for repressor activity. Recent studies have shown that sodA expression is regulated by three iron-dependent regulatory proteins, Fur (ferric uptake regulation), Fnr (fumarate nitrate regulation) and SoxR (superoxide regulon), and by the ArcA/ArcB (aerobic respiration control) system. The potential Fur-, Fnr- and ArcA-binding sites in the sodA promoter region have been identified by using different cis-acting regulatory mutations that caused anaerobic derepression of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Iron and oxygen regulation of Escherichia coli MnSOD expression: competition between the global regulators Fur and ArcA for binding to DNA

Manganese superoxide dismutase (the sodA gene product) in Escherichia coli, is negatively regulated by two global regulators, ArcA (aerobic respiration control) and Fur (ferric uptake regulation), coupling its expression to aerobic metabolism and the intracellular iron pool. Footprinting analyses were carried out on the sodA promoter region with purified Fur protein and with ArcA protein overproduced in crude extracts. ArcA is able to bind in vitro in the absence of the in vivo triggering signal. The binding occurs in one step and study of contacts within the operator sequence reveals binding on one side of the double helix. The DNA protection extends to a much larger region (about 65 bp) than would be expected for a 27 kDa protein, suggesting polymerization. Both Fur and ArcA footprints encompass the -35 and -10 promoter region and there is considerable overlap of their binding sequences, in agreement with in vivo results suggesting that either regulator alone can block sodA transcription. Furthermore, competition experiments show that Fur and ArcA binding to the sodA promoter are mutually exclusive and that ArcA can easily displace Fur, but not vice versa. The biological significance of this in vitro behaviour is discussed.

Arc and Sfr functions of the Escherichia coli K-12 arcA gene product are genetically and physiologically separable

The Escherichia coli arcA gene product regulates chromosomal gene expression in response to deprivation of oxygen (Arc function; Arc stands for aerobic respiration control) and is required for expression of the F plasmid DNA transfer (tra) genes (Sfr function; Sfr stands for sex factor regulation). Using appropriate lacZ fusions, we have examined the relationship between these two genetic regulatory functions. Arc function in vivo was measured by anaerobic repression of a chromosomal sdh-lacZ operon fusion (sdh stands for succinate dehydrogenase). Sfr function was measured by activation of a plasmid traY-lacZ gene fusion. An eight-codon insertion near the 5' terminus of arcA, designated arcA1, abolished Arc function, as previously reported by S. Iuchi and E.C.C. Lin (Proc. Natl. Acad. Sci. USA 85:1888-1892, 1988), but left Sfr function largely (greater than or equal to 60%) intact. Similarly, the arcB1 mutation, which depressed sdh expression and is thought to act by abolishing the signal input that elicits ArcA function, had little effect (less than or equal to 20%) on the Sfr function of the arcA+ gene product. Conversely, a valine-to-methionine mutation at codon 203 (the sfrA5 allele) essentially abolished Sfr activity without detectably altering Arc activity. These data indicate that Sfr and Arc functions are separately expressed and regulated properties of the same protein.

Two global regulators repress the anaerobic expression of MnSOD in Escherichia coli: Fur (ferric uptake regulation) and Arc (aerobic respiration control)

The expression of sodA, the Escherichia coli gene encoding manganese superoxide dismutase (MnSOD) is induced by aerobiosis and superoxide generators such as paraquat. Analysis of variants expressing sodA in the absence of oxygen has revealed that mutations in genes for two global regulatory systems, Fur (ferric uptake regulation) and Arc (aerobic respiration control), are simultaneously required for the expression of sodA in anaerobiosis. The Fur protein still represses sodA in an iron-dependent fashion in aerobiosis. Moreover, all mutants remain inducible by paraquat, indicating that the positive control of SoxR, which mediates the response to superoxide in E. coli, is still operative. Thus, in addition to the response to the superoxide-mediated oxidative stress which depends on SoxR, two global controls regulate MnSOD expression: ArcA couples MnSOD expression to respiration, and Fur couples it to the intracellular concentration of iron.