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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]

The ferric-uptake regulator (Fur) is an Fe2+-responsive transcription factor that coordinates iron homeostasis in many bacteria. Recently, we reported that expression of the Escherichia coli Fur regulon is also impacted by O2 tension. Here, we show that for most of the Fur regulon, Fur binding and transcriptional repression increase under anaerobic conditions, suggesting that Fur is controlled by O2 availability. We found that the intracellular, labile Fe2+ pool was higher under anaerobic conditions compared with aerobic conditions, suggesting that higher Fe2+ availability drove the formation of more Fe2+-Fur and, accordingly, more DNA binding. O2 regulation of Fur activity required the anaerobically induced FeoABC Fe2+ uptake system, linking increased Fur activity to ferrous import under iron-sufficient conditions. The increased activity of Fur under anaerobic conditions led to a decrease in expression of ferric import systems. However, the combined positive regulation of the feoABC operon by ArcA and FNR partially antagonized Fur-mediated repression of feoABC under anaerobic conditions, allowing ferrous transport to increase even though Fur is more active. This design feature promotes a switch from ferric import to the more physiological relevant ferrous iron under anaerobic conditions. Taken together, we propose that the influence of O2 availability on the levels of active Fur adds a previously undescribed layer of regulation in maintaining cellular iron homeostasis. [ABSTRACT FROM AUTHOR]

Escherichia coli open reading frames ydiO and ydiQRST were identified as genes encoding components of the acyl-CoA dehydrogenase complex of anaerobic fatty acid β-oxidation. Individual or concomitant inactivation of fadE gene, encoding known aerobic acyl-CoA dehydrogenase, and ydiO and/or ydiQRST genes did not affect cellular growth on glucose as a sole carbon source. Aerobic growth on sodium oleate was observed only for the cells with intact fadE gene. With an alternative electron acceptor, the cells possessing intact fadE gene demonstrated anaerobic growth on sodium oleate irrespective of the presence or absence of ydiO and ydiQRST genes. For the fadE-deficient mutants, anaerobic growth on sodium oleate was observed only for cells with intact ydiO and ydiQRST genes, while the fadE/ ydiO and fadE/ ydiQRST mutants failed to grow under the similar conditions. [ABSTRACT FROM AUTHOR]

Using Escherichia coli for installing and maintaining anaerobiosis for hydrogen production by Clostridium acetobutylicum ATCC 824 is a cost-effective approach for industrial hydrogen production, as it does not require reducing agents or sparging with inert gases. This study was devoted for investigating the feasibility for installing and maintaining anaerobiosis of hydrogen production by C. acetobutylicum ATCC 824 when using E . coli HD701 utilizable versus non utilizable sugars as a-carbon source. Using E. coli HD701 for installing anaerobiosis showed a comparable hydrogen production yield and efficiency to the use of reducing agents and nitrogen sparging in case of hydrogen production from the E . coli HD701 non utilizable sugars. In contrast, using E . coli HD701 for installing anaerobiosis showed a lower hydrogen production yield and efficiency than the use of reducing agents and nitrogen sparging in case of using glucose as a substrate. This is possibly because E . coli HD701 when using glucose compensate for the substrate, and produce hydrogen with lower efficiency than C . acetobutylicum ATCC 824. These results indicated that the use of E . coli HD701 for installing anaerobiosis would not be economically feasible when using E . coli HD701 utilizable sugars as a carbon source. In contrast, the use of this approach for installing anaerobiosis for hydrogen production from sucrose and starch would have a high potency for industrial applications. [ABSTRACT FROM AUTHOR]

Background: The Escherichia coli response regulator NarL controls transcription of genes involved in nitrate respiration during anaerobiosis. NarL consists of two domains joined by a linker that wraps around the interdomain interface. Phosphorylation of the NarL N-terminal receiver domain (RD) releases the, otherwise sequestered, C-terminal output domain (OD) that subsequently binds specific DNA promoter sites to repress or activate gene expression. The aim of this study is to investigate the extent to which the NarL OD and RD function independently to regulate transcription, and the affect of the linker on OD function. Results: NarL OD constructs containing different linker segments were examined for their ability to repress frdA-lacZ or activate narG-lacZ reporter fusion genes. These in vivo expression assays revealed that the NarL OD, in the absence or presence of linker helix α6, constitutively repressed frdA-lacZ expression regardless of nitrate availability. However, the presence of the linker loop α5-α6 reversed this repression and also showed impaired DNA binding in vitro. The OD alone could not activate narG-lacZ expression; this activity required the presence of the NarL RD. A footprint assay demonstrated that the NarL OD only partially bound recognition sites at the narG promoter, and the binding affinity was increased by the presence of the phosphorylated RD. Analytical ultracentrifugation used to examine domain oligomerization showed that the NarL RD forms dimers in solution while the OD is monomeric. Conclusions: The NarL RD operates as an on-off switch to occlude or release the OD in a nitrate-responsive manner, but has additional roles to directly stimulate transcription at promoters for which the OD lacks independent function. One such role of the RD is to enhance the DNA binding affinity of the OD to target promoter sites. The data also imply that NarL phosphorylation results in RD dimerization and in the separation of the entire linker region from the OD. [ABSTRACT FROM AUTHOR]

The article presents a study that demonstrates the nitrate reductase' localization anaerobiosis in Escherichia coli.

The biology of Escherichia coli in its primary niche, the animal intestinal tract, is remarkably unexplored. Studies with the strep-tomycin-treated mouse model have produced important insights into the metabolic requirements for Escherichia coli to colo-nize mice. However, we still know relatively little about the physiology of this bacterium growing in the complex environment of an intestine that is permissive for the growth of competing flora. We have developed a system for studying colonization using an E. coli strain, MPI, isolated from a mouse. MPI is genetically tractable and does not require continuous antibiotic treatment for stable colonization. As an application of this system, we separately knocked out each two-component system response regulator in MPI and performed competitions against the wild-type strain. We found that only three response regulators, ArcA, CpxR, and RcsB, produce strong colonization defects, suggesting that in addition to anaerobiosis, adaptation to cell envelope stress is a crit-ical requirement for E. coli colonization of the mouse intestine. We also show that the response regulator OmpR, which had pre-viously been hypothesized to be important for adaptation between in vivo and ex vivo environments, is not required for MPI colonization due to the presence of a third major porin. [ABSTRACT FROM AUTHOR]

Abstract: Fermented or acidified vegetable foods are considered microbiologically safe although the survival of certain pathogens has occasionally been reported in these products. The aim of this research was to investigate the fate of Escherichia coli, Salmonella enterica, Listeria monocytogenes and Staphylococcus aureus when they were added to different industrial olive brines, as well as to correlate their survival with the presence of phenolic and oleosidic substances. Brines of different cultivars prepared following the Spanish-style method or preserved in acidified brine were inoculated with a cocktail of four strains of each species. The evolution of their populations was analyzed by cultural methods when the brines were kept at 4 °C or room temperature and in aerobiosis or anaerobiosis. All the pathogens investigated died off but their death rate was variable depending on the composition of the brines in phenolic compounds, temperature and oxygen availability. The time needed to reduce the inoculated pathogen populations by 5 log oscillated between less than 5 min and up to 17 days in the least deleterious conditions. [Copyright &y& Elsevier]

Adaptation to changing environments is essential to bacterial physiology. Here we report a unique role of the copper homeostasis system in adapting Escherichia coli to its host-relevant environment of anaerobiosis coupled with amino acid limitation. We found that expression of the copper/silver efflux pump CusCFBA was significantly upregulated during anaerobic amino acid limitation in E. coli without the supplement of exogenous copper. Inductively coupled plasma mass spectrometry analysis of the total intracellular copper content combined with transcriptional assay of the PcusC-lacZ reporter in the presence of specific Cu(I) chelators indicated that anaerobic amino acid limitation led to the accumulation of free Cu(I) in the periplasmic space of E. coli, resulting in Cu(I) toxicity. Cells lacking cusCFBA and another copper transporter, copA, under this condition displayed growth defects and reduced ATP production during fumarate respiration. Ectopic expression of the Fe-S cluster enzyme fumarate reductase (Frd), or supplementation with amino acids whose biosynthesis involves Fe-S cluster enzymes, rescued the poor growth of ΔcusC cells. Yet, Cu(I) treatment did not impair the Frd activity in vitro. Further studies revealed that the alternative Fe-S cluster biogenesis system Suf was induced during the anaerobic amino acid limitation, and ΔcusC enhanced this upregulation, indicating the impairment of the Fe-S cluster assembly machinery and the increased Fe-S cluster demands under this condition. Taken together, we conclude that the copper efflux system CusCFBA is induced during anaerobic amino acid limitation to protect Fe-S cluster enzymes and biogenesis from the endogenously originated Cu(I) toxicity, thus facilitating the physiological adaptation of E. coli. [ABSTRACT FROM AUTHOR]

Highlights: [•] Escherichia coli contains two l-asparaginase isozymes with distinct localization, kinetics and regulation. [•] Mutant strains were used to examine the roles of these enzymes in l-asparagine utilization. [•] We report that l-asparaginase II permits growth on asparagine and glycerol under anaerobic conditions. [•] We propose that this enzyme is the first step in a co-regulated pathway leading to fumarate. [•] The pathway is regulated by anaerobiosis and cAMP and provides a terminal elector acceptor. [ABSTRACT FROM AUTHOR]

FNR is a well-studied global regulator of anaerobiosis, which is widely conserved across bacteria. Despite the importance of FNR and anaerobiosis in microbial lifestyles, the factors that influence its function on a genome-wide scale are poorly understood. Here, we report a functional genomic analysis of FNR action. We find that FNR occupancy at many target sites is strongly influenced by nucleoid-associated proteins (NAPs) that restrict access to many FNR binding sites. At a genome-wide level, only a subset of predicted FNR binding sites were bound under anaerobic fermentative conditions and many appeared to be masked by the NAPs H-NS, IHF and Fis. Similar assays in cells lacking H-NS and its paralog StpA showed increased FNR occupancy at sites bound by H-NS in WT strains, indicating that large regions of the genome are not readily accessible for FNR binding. Genome accessibility may also explain our finding that genome-wide FNR occupancy did not correlate with the match to consensus at binding sites, suggesting that significant variation in ChIP signal was attributable to cross-linking or immunoprecipitation efficiency rather than differences in binding affinities for FNR sites. Correlation of FNR ChIP-seq peaks with transcriptomic data showed that less than half of the FNR-regulated operons could be attributed to direct FNR binding. Conversely, FNR bound some promoters without regulating expression presumably requiring changes in activity of condition-specific transcription factors. Such combinatorial regulation may allow Escherichia coli to respond rapidly to environmental changes and confer an ecological advantage in the anaerobic but nutrient-fluctuating environment of the mammalian gut. [ABSTRACT FROM AUTHOR]

Abstract: Genome-based Flux Balance Analysis (FBA) and steady-state isotopic-labeling-based Metabolic Flux Analysis (MFA) are complimentary approaches to predicting and measuring the operation and regulation of metabolic networks. Here, genome-derived models of Escherichia coli (E. coli) metabolism were used for FBA and 13C-MFA analyses of aerobic and anaerobic growths of wild-type E. coli (K-12 MG1655) cells. Validated MFA flux maps reveal that the fraction of maintenance ATP consumption in total ATP production is about 14% higher under anaerobic (51.1%) than aerobic conditions (37.2%). FBA revealed that an increased ATP utilization is consumed by ATP synthase to secrete protons from fermentation. The TCA cycle is shown to be incomplete in aerobically growing cells and submaximal growth is due to limited oxidative phosphorylation. An FBA was successful in predicting product secretion rates in aerobic culture if both glucose and oxygen uptake measurement were constrained, but the most-frequently predicted values of internal fluxes yielded from sampling the feasible space differ substantially from MFA-derived fluxes. [ABSTRACT FROM AUTHOR]

The article presents a study which seeks to confirm the assumption that Fnr[4Fe-4S] 2+, an Escherichia coli's universal anaerobiosis protein, acts as a regulator of aidB expression and an NO sensor during anaerobic growth. It says that the study used the E. coli strain MV2176 containing the [aidB1::lacZ] construct in the chromosome. The result shows that Fnr[4Fe4S] 2+ protein regulates the aidB gene's expression of the Ada regulon under anaerobic conditions.

Summary: The DNA-binding protein of starved cells (Dps) has two distinct cellular functions in Escherichia coli. The spherical Dps dodecamer can store iron and, predominantly in the stationary growth phase, high amounts of Dps protein protect the genome by binding non-specifically to DNA. In this study we investigated the role of Dps in copper homeostasis in growing cells of E. coli. Under reductive aerobic growth conditions that favor a redox shift from Cu(II) to Cu(I) or under anaerobiosis, cells deleted in dps were sensitive to low copper ion concentrations. Deletion of the DNA-binding N-terminus of Dps did not abrogate protection against copper toxicity indicating protection against copper stress is not directly related to DNA binding of Dps. The Dps protein is not a copper-storage protein in vitro or in vivo. In contrast, cells lacking Dps exhibited increased cellular copper concentrations compared to their wild-type parent. Furthermore, overproduction of Dps during growth phase resulted in decreased intracellular copper content under copper stress. [Copyright &y& Elsevier]

Bacteria have evolved different systems to tightly control both cytosolic and envelope copper concentration to fulfil their requirements and at the same time, avoid copper toxicity. We have previously demonstrated that, as in Escherichia coli, the Salmonella cue system protects the cytosol from copper excess. On the other hand, and even though Salmonella lacks the CusCFBA periplasmic copper efflux system, it can support higher copper concentrations than E. coli under anaerobic conditions. Here we show that the Salmonella cue regulon is also responsible for the control of copper toxicity in anaerobiosis. We establish that resistance in this condition requires a novel CueR-controlled gene named cueP. A Δ cueP mutant is highly susceptible to copper in the absence of oxygen, but shows a faint phenotype in aerobic conditions unless other copper-resistance genes are also deleted, resembling the E. coli CusCFBA behaviour. Species that contain a cueP homologue under CueR regulation have no functional CusR/CusS-dependent Cus-coding operon. Conversely, species that carry a CusR/CusS-regulated cus operon have no cueP homologues. Even more, we show that the CueR-controlled cueP expression increases copper resistance of a Δ cus E. coli. We posit that CueP can functionally replace the Cus complex for periplasmic copper resistance, in particular under anaerobic conditions. [ABSTRACT FROM AUTHOR]

Background: The Escherichia coli heterodimeric HU protein is a small DNA-bending protein associated with the bacterial nucleoid. It can introduce negative supercoils into closed circular DNA in the presence of topoisomerase I. Cells lacking HU grow very poorly and display many phenotypes. Methodology/Principal Findings: We analyzed the transcription profile of every Escherichia coli gene in the absence of one or both HU subunits. This genome-wide in silico transcriptomic approach, performed in parallel with in vivo genetic experimentation, defined the HU regulon. This large regulon, which comprises 8% of the genome, is composed of four biologically relevant gene classes whose regulation responds to anaerobiosis, acid stress, high osmolarity, and SOS induction. Conclusions/Significance: The regulation a large number of genes encoding enzymes involved in energy metabolism and catabolism pathways by HU explains the highly pleiotropic phenotype of HU-deficient cells. The uniform chromosomal distribution of the many operons regulated by HU strongly suggests that the transcriptional and nucleoid architectural functions of HU constitute two aspects of a unique protein-DNA interaction mechanism. [ABSTRACT FROM AUTHOR]

Transcript quantification techniques usually rely on purified mRNAs. We report here a solution-based sandwich hybridization assay for the quantification of mRNAs from Escherichia coli without the need of prior RNA isolation. This assay makes use of four DNA oligonucleotide probes adjacently hybridizing to target RNA in clarified cell extracts. Two helper probes facilitate the hybridization of a detection and a capture probe. The latter is biotin labeled, allowing binding to streptavidin-coated paramagnetic beads and the separation of the RNA-DNA hybrid from cellular constituents. Added antidigoxigenin Fab fragments conjugated to alkaline phosphatase bind to the digoxigenin-labeled detection probe, completing the sandwich of the paramagnetic bead, mRNA, probes, and alkaline phosphatase. The target transcript can be quantified by assessing phosphatase activity on a substrate that is converted into a fluorescent product. The amount of target mRNA is calculated from the fluorescence output and from a calibration curve for a known concentration of in vitro-synthesized target mRNA. This technique was used in time course experiments to investigate the expression of three genes responsible for the copper resistance of E. coli. The induction of gene expression by copper cations was rapid, but under aerobic conditions, the levels of expression returned to low, prestress levels within minutes. In anaerobiosis, high-level expression continued for at least 1 h. When cultures were shifted from anaerobiosis to aerobiosis, expression levels were diminished within minutes to prestress levels. The improved technique presented here is relatively simple, has very high degrees of sensitivity and robustness, is less laborious than other RNA quantification methods, and is not negatively affected by genomic DNA. These characteristics make it a powerful complementary application to genetic reporter fusions and to reverse transcription.PCR. [ABSTRACT FROM AUTHOR]

In Escherichia coli, Lon is an ATP-dependent protease which degrades misfolded proteins and certain rapidly-degraded regulatory proteins. Given that oxidatively damaged proteins are generally degraded rather than repaired, we anticipated that Lon deficient cells would exhibit decreased viability during aerobic, but not anaerobic, carbon starvation. We found that the opposite actually occurs. Wild-type and Lon deficient cells survived equally well under aerobic conditions, but Lon deficient cells died more rapidly than the wild-type under anaerobiosis. Aerobic induction of the Clp family of ATP-dependent proteases could explain these results, but direct quantitation of Clp protein established that its level was not affected by Lon deficiency and overexpression of Clp did not rescue the cells under anaerobic conditions. We conclude that the Lon protease supports survival during anaerobic carbon starvation by a mechanism which does not depend on Clp. [ABSTRACT FROM AUTHOR]

In the absence of oxygen, Escherichia coli can use alternative exogenous electron acceptors, including trimethylamine oxide (TMAO), to generate energy. In this study, we showed that in contrast to the other anaerobic respiratory systems, the TMAO reductase (Tor) system was expressed during both aerobiosis and anaerobiosis. By using a torA–lacZ fusion and quantitative reverse transcription polymerase chain reaction, we established that the torCAD operon encoding the Tor system was induced in the presence of TMAO mainly during exponential phase, and that optimal induction required a certain level of DNA supercoiling. We also showed that the presence of oxygen prevented neither the biogenesis of the Tor system nor the reduction of TMAO. The physiological role of TMAO reduction during aerobiosis has not been yet established, but our experiments suggest that alkaline TMA production could enhance the growth conditions by increasing the pH of the culture. [ABSTRACT FROM AUTHOR]

In anaerobiosis, Escherichia coli can use trimethylamine N-oxide (TMAO) as a terminal electron acceptor. Reduction of TMAO in trimethylamine (TMA) is mainly performed by the respiratory TMAO reductase. This system is encoded by the torCAD operon, which is induced in the presence of TMAO. This regulation involves a two-component system comprising TorS, an unorthodox histidine kinase, and TorR, a response regulator. A third protein, TorT, sharing homologies with periplasmic binding proteins, plays a key role in this regulation because disruption of the torT gene abolishes tor expression. In this study we showed that TMAO protects TorT against degradation by the GluC endoproteinase and modifies its temperature-induced CD spectrum. We also isolated a TorT negative mutant that is no longer protected by TMAO from degradation by GIuC. Isothermal titration calorimetry confirmed that TorT binds TMAO with a binding constant of 150 μM. Therefore, we conclude that TorT binds TMAO and that this binding promotes a conformational change of TorT. We also showed that TorT interacts with the periplasmic domain of TorS in both the presence and absence of TMAO but the TorT-TMAO complex induces a higher GluC protection of TorS than TorT alone. These results support the idea that TMAO binding to TorT induces a cascade of conformational changes from TorT to TorS, leading to TorS activation. We identified several homologues of the TorT protein that define a new family of periplasmic binding proteins. We thus propose that the members of this family bind TMAO or related compounds and that they are involved in signal transduction or even substrate transport. [ABSTRACT FROM AUTHOR]

Abstract: Afa/Dr diffusely adhering Escherichia coli have been shown to cause urinary tract infections and enteric infections. Virulence of Dr-positive IH11128 bacteria is associated with the presence of Dr fimbriae. In this report, we show for the first time that the Dr fimbriae are released in the extracellular medium in response to multiple environmental signals. Production and secretion of Dr fimbriae are clearly thermoregulated. A comparison of the amounts of secreted fimbriae showed that the secretion is drastically increased during anaerobic growth in minimal medium. The effect of anaerobiosis on secretion seemed to depend on both the growth phase and the culture medium. The secretion was maximal during the logarithmic-phase growth and corresponded to 27 and 57% of total Dr fimbriae produced by bacteria grown in mineral medium+glucose and LB broth, respectively. Thus, the anaerobic environment of the colon would favour the secretion of Dr fimbriae during bacterial multiplication. The controlled release of the Dr fimbriae, which is carried out in the absence of cellular lysis, appears independent of the action of proteases or a process of maturation. The mechanism employed in the liberation of Dr fimbriae thus seems different from that described for the adhesins FHA and Hap of Bordetella pertussis and Haemophilus influenzae. [Copyright &y& Elsevier]

Polymorphisms in rpoS are common in Escherichia coli. rpoS status influences a trade-off between nutrition and stress resistance and hence fitness across different environments. To analyze the selective pressures acting on rpoS, measurement of glucose transport rates in rpoS+ and rpoS bacteria was used to estimate the role of Fnc, the fitness gain due to improved nutrient uptake, in the emergence of rpoS mutations in nutrient-limited chemostat cultures. Chemostats with set atmospheres, temperatures, pH's, antibiotics, and levels of osmotic stress were followed. Fnc was reduced under anaerobiosis, high osmolarity, and with chloramphenicol, consistent with a reduced rate of rpoS enrichment in these conditions. Fnc remained high, however, with alkaline pH and low temperature but rpoS sweeps were diminished. Under these conditions, Fsp, the fitness reduction due to lowered stress protection, became significant. We also estimated whether the fitness need for the gene was related to its regulation. No consistent pattern emerged between the level of RpoS and the loss of rpoS function in particular environments. This dissection allows an unprecedented view of the genotype-by-environment interactions controlling a mutational sweep and shows that both Fnc and Fsp are influenced by individual stresses and that additional factors contribute to selection pressure in some environments. [ABSTRACT FROM AUTHOR]

Expression of the anaerobically inducible focA-pfl operon in Escherichia coli was activated nearly sevenfold relative to wild-type under aerobic growth conditions by increasing the dosage of the fnr gene on a pBR322-based plasmid (pCH21). No effect on anaerobic expression levels was observed, suggesting that operon expression under these conditions is maximal. Examination of the complex transcript pattern of the focA-pfl operon confirmed that in strains bearing pCH21 all transcripts, with the exception of the promoter 7 transcript, were up-regulated aerobically. Western analysis of strains bearing pCH21 revealed that the fumarate nitrate regulator (FNR) level was increased approximately ninefold relative to the level in strains bearing a single copy of the fnr gene aerobically, but was only overproduced threefold anaerobically. Analysis of an fnr-lacZ fusion indicated that fnr expression was more strongly negatively autoregulated in anaerobic cells compared with aerobic cells when pCH21 was present. Taken together, these findings suggest that high-level overproduction of FNR is prevented anaerobically by active FNR repressing expression of the fnr gene. Furthermore, transcription from promoter 7 of the focA-pfl operon, which depends on both ArcA-P and FNR, cannot be activated aerobically by overproduction of FNR alone, while promoter 6, which is less dependent on ArcA-P, can be activated under these conditions. [ABSTRACT FROM AUTHOR]

Aims: To establish the influence of oxygen on Escherichia coil before, during and after exposure to UVA or simulated sunlight. Methods and Results: Bacterial suspensions were exposed either to UVA or simulated sunlight. Conventional aerobic plate counts of illuminated cell suspensions were consistently lower than those obtained under conditions where reactive oxygen species (ROS) were neutralized, either (i) by the addition of the peroxide scavenger sodium pyruvate (0·05% w/v) to the medium with subsequent incubation in an anaerobic jar or (ii) by culturing on a prereduced medium within an anaerobic cabinet, indicating that a substantial proportion of such cells are sublethally injured. While the presence of oxygen during the growth period resulted in a greater resistance of aerobically grown cells to simulated sunlight compared with their anaerobic counterparts, the extent of inactivation during illumination was directly related to the dissolved oxygen content of the water. Conclusions: The results show that, at each stage, oxygen has a marked influence on the observed colony count. Significance and Impact of the Study: Overall, the results indicate that future studies of bacteria exposed to UVA or sunlight should consider the effects of oxygen at every stage in the procedure, and especially during enumeration, where the inhibitory effects of ROS must be neutralized in order to obtain a valid count. An investigation of the effects of ROS neutralization on the counts of faecal bacteria under field conditions in natural waters is now required to establish the significance of these finding to solar water treatment. [ABSTRACT FROM AUTHOR]

Shewanella oneidensisstrain MR-1 is well known for its respiratory versatility, yet little is understood about how it regulates genes involved in anaerobic respiration. The Arc two-component system plays an important role in this process inEscherichia coli; therefore, we determined its function inS. oneidensis.arcAfromS. oneidensiscomplements anE. coli arcAmutant, but the Arc regulon inS. oneidensisconstitutes a different suite of genes. For example, one of the strongest ArcA-regulated gene clusters inE. coli,sdh, is not regulated by the Arc system inS. oneidensis, and thecydlocus, which is induced by ArcA inE. coliunder microaerobic conditions, is repressed by ArcA inS. oneidensisunder anaerobic conditions.One locus that we identified as being potentially regulated by ArcA inS. oneidensiscontains genes predicted to encode subunits of a dimethyl sulphoxide (DMSO) reductase. We demonstrate that these genes encode a functional DMSO reductase, and that anarcAmutant cannot fully induce their expression and is defective in growing on DMSO under anaerobic conditions. WhileS. oneidensislacks a highly conserved full-length ArcB homologue, ArcA is partially activated by a small protein homologous to the histidine phosphotransfer domain of ArcB fromE. coli, HptA. This protein alone is unable to compensate for the lack ofarcBinE. coli, indicating that another protein is required in addition to HptA to activate ArcA inS. oneidensis. [ABSTRACT FROM AUTHOR]

Transcription of the nrdDG operon, which encodes the class III nucleotide reductase, which is only active under anaerobic conditions, was strongly induced after a shift to anaerobiosis. The induction was completely dependent on the transcriptional activator FNR and was independent of the ArcA-ArcB two-component response regulator system. The nrdD transcript start site was mapped to a position immediately downstream of two FNR binding sites. Transcription of the other two nucleotide reductase operons, nrdAB and nrdEF, did not respond to oxygen conditions in a wild-type background, but nrdAB expression was increased in the fnr mutant under anaerobic conditions. [ABSTRACT FROM AUTHOR]

Aims: In order to clarify the involvement of an energy-yielding system in the antibacterial action of surfactants, the effects of carbon source and anaerobiosis during the growth period on the surfactant sensitivity of Escherichia coli cells were investigated. Methods and Results: Cetyltrimethylammonium bromide (CTAB) and N -dodecyl-N ,N -dimethylglycine, at relatively low concentrations, caused a delay in growth of E. coli cells. Cells grown in M9 medium supplemented with glycerol, succinate or acetate as a carbon source were more sensitive to surfactants and had a higher respiratory activity than those grown with glucose. Cultivation under anaerobiosis made cells resistant to CTAB. Conclusions: Bacterial sensitivity to surfactants was affected by carbon source and anaerobiosis. Significance and Impact of the Study: The results obtained should be helpful in determining suitable conditions of treatment in the practical use of surfactants for bacterial decontamination. [ABSTRACT FROM AUTHOR]

Examines the transcription regulation of expression of the colicin K structural gene in Escherichia coli. Implication of the depletion of nutrients in cka expression; Effect of anaerobiosis, pH and short-chain acids on cka expression; Impact of the regulation of expression of colicin genes on the natural environment.

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes: it was purified 219 times with a yield of about 11%. It is an Mr-230000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis. [ABSTRACT FROM AUTHOR]

Examines the expression of the aerobic and anaerobic respiratory pathway genes in Escherichia coli during cell growth at different oxygen levels. Effect of oxygen levels on dmsA-lacZ expression; Effect of varying the oxygen levels on frdA-lacZ expression; Effect of oxygen on narG-lacZ expression.

Studies the role of the transcriptional activator appA in the regulation of the cyx appA operon of Escherichia coli by anaerobiosis, phosphate starvation and growth phase. Construction of appA operon promoter fusions; Protein overproduction; Construction of phenotypic characterization of an appY insertion mutant.

Background: Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. Results: Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential O 2 -dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. Conclusions: We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions. [ABSTRACT FROM AUTHOR]