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Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs). Successful urinary tract colonization requires appropriate expression of virulence factors in response to host environmental cues, such as limited oxygen and iron availability. Hemolysin is a pore‐forming toxin, and its expression correlates with the severity of UPEC infection. Previously, we showed that hemolysin expression is enhanced under anaerobic conditions; however, the genetic basis and regulatory mechanisms involved remain undefined. Here, a transposon‐based forward screen identified bis‐molybdopterin guanine dinucleotide cofactor (bis‐MGD) biosynthesis as an important factor for a full transcription of hemolysin under anaerobiosis but not under aerobiosis. bis‐MGD positively influences hemolysin transcription via c3566‐c3568, an operon immediately upstream of and cotranscribed with hlyCABD. Furthermore, suppressor mutation analysis identified the nitrogen regulator NtrC as a direct repressor of c3566‐c3568‐hlyCABD expression, and intact bis‐MGD biosynthesis downregulated ntrC expression, thus at least partially explaining the positive role of bis‐MGD in modulating hemolysin expression. Finally, bis‐MGD is involved in hemolysin‐mediated uroepithelial cell death and contributes to the competitive fitness of UPEC in a murine model of UTI. Collectively, our data establish that bis‐MGD biosynthesis plays a crucial role in UPEC fitness in vivo, thus providing a potential target for combatting UTIs. [ABSTRACT FROM AUTHOR]

All biological hydroxylation reactions are thought to derive the oxygen atom from one of three inorganic oxygen donors, O2, H2O2 or H2O. Here, we have identified the organic compound prephenate as the oxygen donor for the three hydroxylation steps of the O2-independent biosynthetic pathway of ubiquinone, a widely distributed lipid coenzyme. Prephenate is an intermediate in the aromatic amino acid pathway and genetic experiments showed that it is essential for ubiquinone biosynthesis in Escherichia coli under anaerobic conditions. Metabolic labeling experiments with 18O-shikimate, a precursor of prephenate, demonstrated the incorporation of 18O atoms into ubiquinone. The role of specific iron--sulfur enzymes belonging to the widespread U32 protein family is discussed. Prephenate-dependent hydroxylation reactions represent a unique biochemical strategy for adaptation to anaerobic environments. [ABSTRACT FROM AUTHOR]

Promoters adjust cellular gene expression in response to internal or external signals and are key elements for implementing dynamic metabolic engineering concepts in fermentation processes. One useful signal is the dissolved oxygen content of the culture medium, since production phases often proceed in anaerobic conditions. Although several oxygen-dependent promoters have been described, a comprehensive and comparative study is missing. The goal of this work is to systematically test and characterize 15 promoter candidates that have been previously reported to be induced upon oxygen depletion in Escherichia coli. For this purpose, we developed a microtiter plate-level screening using an algal oxygen-independent flavin-based fluorescent protein and additionally employed flow cytometry analysis for verification. Various expression levels and dynamic ranges could be observed, and six promoters (nar-strong, nar-medium, nar-weak, nirB-m, yfiD-m, and fnrF8) appear particularly suited for dynamic metabolic engineering applications. We demonstrate applicability of these candidates for dynamic induction of enforced ATP wasting, a metabolic engineering approach to increase productivity of microbial strains that requires a narrow level of ATPase expression for optimal function. The selected candidates exhibited sufficient tightness under aerobic conditions while, under complete anaerobiosis, driving expression of the cytosolic F 1 -subunit of the ATPase from E. coli to levels that resulted in unprecedented specific glucose uptake rates. We finally utilized the nirB-m promoter to demonstrate the optimization of a two-stage lactate production process by dynamically enforcing ATP wasting, which is automatically turned on in the anaerobic (growth-arrested) production phase to boost the volumetric productivity. Our results are valuable for implementing metabolic control and bioprocess design concepts that use oxygen as signal for regulation and induction. [Display omitted] • 15 oxygen-dependent promoters systematically characterized. • Microtiter plate level screening using an oxygen-independent LOV fluorescent reporter. • Verification of expression levels and dynamic ranges using flow cytometry. • Six most suitable promoters for dynamic metabolic engineering applications identified. • Example study: nirB-m driven ATP wasting boosts lactate productivity in a two-stage process. [ABSTRACT FROM AUTHOR]

Aerobic Escherichia coli growth at restricted iron concentrations (≤ 1.75 ± 0.04 μM) is characterized by lower biomass yield, higher acetate accumulation and higher activation of the siderophore iron-acquisition systems. Although iron homeostasis in E. coli has been studied intensively, previous studies focused only on understanding the regulation of the iron import systems and the iron-requiring enzymes. Here, the effect of iron availability on the energy metabolism of E. coli has been investigated. It was established that aerobic cultures growing under limiting iron conditions showed lower ATP yield per glucose, lower growth rate and lower TCA cycle activity and respiration, at the same time as increased glucose consumption, acetate and pyruvate accumulation, practically mimicking microaerobic growth. However, at excess iron, independent of oxygen availability, the cultures showed high cellular energetics (5.8 ATP/mol of glucose) by using pathways requiring iron-rich complex proteins found in the TCA cycle and respiratory chain. In conditions of iron excess, some iron-requiring terminal reductases of the respiratory chain, that were thought to function only under anaerobiosis, were used by the E. coli, when in aerobic conditions, to maintain high respiratory activity. This allowed it to produce more biomass and more reactive oxygen species that were controlled by the higher activity of the antioxidant defenses (SOD, peroxidase and catalase) and the iron-sulfur cluster repair systems. • Aerobic iron limitation mimics microaerobic growth response of E. coli. • Low cellular energetics is associated to low iron availability. • Iron-rich protein complexes are the main repressed target of iron limitation conditions. [ABSTRACT FROM AUTHOR]

Repairing oxidative‐targeted macromolecules is a central mechanism necessary for living organisms to adapt to oxidative stress. Reactive oxygen and chlorine species preferentially oxidize sulfur‐containing amino acids in proteins. Among these amino acids, methionine can be converted into methionine sulfoxide. This post‐translational oxidation can be reversed by methionine sulfoxide reductases, Msr enzymes. In Gram‐negative bacteria, the antioxidant MsrPQ system is involved in the repair of periplasmic oxidized proteins. Surprisingly, in this study, we observed in Escherichia coli that msrPQ was highly expressed in the absence of oxygen. We have demonstrated that the anaerobic induction of msrPQ was due to chlorate (ClO3−) contamination of the Casamino Acids. Molecular investigation led us to determine that the reduction of chlorate to the toxic oxidizing agent chlorite (ClO2−) by the three nitrate reductases (NarA, NarZ, and Nap) led to methionine oxidation of periplasmic proteins. In response to this stress, the E. coli HprSR two‐component system was activated, leading to the over‐production of MsrPQ. This study, therefore, supports the idea that methionine oxidation in proteins is part of chlorate toxicity, and that MsrPQ can be considered as an anti‐chlorate/chlorite defense system in bacteria. Finally, this study challenges the traditional view of the absence of Met‐oxidation during anaerobiosis. [ABSTRACT FROM AUTHOR]

Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In the recent years it has become evident that the availability of Fe-S clusters play an important role for the biosynthesis of Moco. First, the MoaA protein binds two [4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional [4Fe-4S] cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is an enzyme involved in the transfer of sulfur to various acceptor proteins with a main role in the assembly of Fe-S clusters. In this review, we dissect the dependence of the production of active molybdoenzymes in detail, starting from the regulation of gene expression and further explaining sulfur delivery and Fe-S cluster insertion into target enzymes. Further, Fe-S cluster assembly is also linked to iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, we explain that the expression of the genes is dependent on an active FNR protein. FNR is a very important transcription factor that represents the master-switch for the expression of target genes in response to anaerobiosis. Moco biosynthesis is further directly dependent on the presence of ArcA and also on an active Fur protein. • While the biosynthesis of Moco and FeS cluster assembly has been well studied in E. coli , there are still some remaining unsolved questions. • Moco biosynthesis is tightly connected to the assembly of FeS clusters at several levels. • The specificity of the cluster transfer process into target proteins, their recognition and the hierarchy how metal-center insertion for Fe-S clusters and Moco occurs into target proteins is still largely unresolved. • The main questions to be solved are how iron availability and Fe-S cluster assembly is connected to Moco biosynthesis. • In this review we provide an overview on the crosstalk between FeS cluster assembly and Moco biosynthesis that lead to the production of active molybdoenzymes. [ABSTRACT FROM AUTHOR]