Expression and characterization of the Escherichia coli fdo locus and a possible physiological role for aerobic formate dehydrogenase.

In the presence of nitrate, the major anaerobic respiratory pathway includes formate dehydrogenase (FDH-N) and nitrate reductase (NAR-A), which catalyze formate oxidation coupled to nitrate reduction. Two aerobically expressed isoenzymes, FDH-Z and NAR-Z, have been recently characterized. Enzymatic analysis of plasmid subclones carrying min 88 of the Escherichia coli chromosome was consistent with the location of the fdo locus encoding FDH-Z between the fdhD and fdhE genes which are necessary for the formation of both formate dehydrogenases. The fdo locus produced three proteins (107, 34, and 22 kDa) with sizes similar to those of the subunits of the purified FDH-N. In support to their structural role, these polypeptides were recognized by antibodies specific to FDH-N. Expression of a chromosomal fdo-uidA operon fusion was induced threefold by aerobic growth and about twofold by anaerobic growth in the presence of nitrate. However, it was independent of the two global regulatory proteins FNR and ArcA, which control genes for anaerobic and aerobic functions, respectively, and of the nitrate response regulator protein NARL. In contrast, a mutation affecting either the nucleoid-associated H-NS protein or the CRP protein abolished the aerobic expression. A possible role for FDH-Z during the transition from aerobic to anaerobic conditions was examined. Synthesis of FDH-Z was maximal at the end of the aerobic growth and remained stable after a shift to anaerobiosis, whereas FDH-N production developed only under anaerobiosis. Furthermore, in an fnr strain deprived of both FDH-N and NAR-A activities, aerobically expressed FDH-Z and NAR-Z enzymes were shown to reduce nitrate at the expense of formate under anaerobic conditions, suggesting that this pathway would allow the cell to respond quickly to anaerobiosis.     

Reversible interconversion of the functional state of the gene regulator FNR from Escherichia coli in vivo by O2 and iron availability

FNR, the gene regulator of anaerobic respiratory genes of Escherichia coli is converted in vivo by O₂ and by chelating agents to an inactive state. The interconversion process was studied in vivo in a strain with temperature controlled synthesis of FNR by measuring the expression of the frd (fumarate reductase) operon and the reactivity of FNR with the alkylating agent iodoacetic acid. FNR from aerobic bacteria is, after arresting FNR synthesis and shifting to anaerobic conditions, able to activate frd expression and behaves in the alkylation assay like anaerobic FNR. After shift from anaerobic to aerobic conditions, FNR no longer activates the expression of frd and reacts similar to aerobic FNR in the alkylation assay. The conversion of aerobic (inactive) to anaerobic (active) FNR occurs in the presence of chloramphenicol, an inhibitor of protein synthesis. Anaerobic FNR can also be converted post-translationally to inactive, metal-depleted FNR by growing the bacteria in the presence of chelating agents. The reverse is also possible by incubating metal-depleted bacteria with Fe²⁺. From the experiments it is concluded that the aerobic and the metal-depleted form of FNR can be transferred post-translationally and reversibly to the anaerobic (active) form. The response of FNR to changes in O₂ supply therefore occurs at the FNR protein level in a reversible mode.Abbreviation BV_red = reduced benzyl viologen     

Genome-wide expression analysis indicates that FNR of Escherichia coli K-12 regulates a large number of genes of unknown function

The major regulator controlling the physiological switch between aerobic and anaerobic growth conditions in Escherichia coli is the DNA binding protein FNR. To identify genes controlled by FNR, we used Affymetrix Antisense GeneChips to compare global gene expression profiles from isogenic MG1655 wild-type and Deltafnr strains grown in glucose minimal media under aerobic or anaerobic conditions. We found that 297 genes contained within 184 operons were regulated by FNR and/or by O2 levels. The expression of many genes known to be involved in anaerobic respiration and fermentation was increased under anaerobic growth conditions, while that of genes involved in aerobic respiration and the tricarboxylic acid cycle were repressed as expected. The expression of nine operons associated with acid resistance was also increased under anaerobic growth conditions, which may reflect the production of acidic fermentation products. Ninety-one genes with no presently defined function were also altered in expression, including seven of the most highly anaerobically induced genes, six of which we found to be directly regulated by FNR. Classification of the 297 genes into eight groups by k-means clustering analysis indicated that genes with common gene expression patterns also had a strong functional relationship, providing clues for studying the function of unknown genes in each group. Six of the eight groups showed regulation by FNR; while some expression groups represent genes that are simply activated or repressed by FNR, others, such as those encoding functions for chemotaxis and motility, showed a more complex pattern of regulation. A computer search for FNR DNA binding sites within predicted promoter regions identified 63 new sites for 54 genes. We suggest that E. coli MG1655 has a larger metabolic potential under anaerobic conditions than has been previously recognized.     

Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control.

In Escherichia coli, sn-glycerol-3-phosphate can be oxidized by two different flavo-dehydrogenases, an anaerobic enzyme encoded by the glpACB operon and an aerobic enzyme encoded by the glpD operon. These two operons belong to the glp regulon specifying the utilization of glycerol, sn-glycerol-3-phosphate, and glycerophosphodiesters. In glpR mutant cells grown under conditions of low catabolite repression, the glpA operon is best expressed anaerobically with fumarate as the exogenous electron acceptor, whereas the glpD operon is best expressed aerobically. Increased anaerobic expression of glpA is dependent on the fnr product, a pleiotropic activator of genes involved in anaerobic respiration. In this study we found that the expression of a glpA1(Oxr) (oxygen-resistant) mutant operon, selected for increased aerobic expression, became less dependent on the FNR protein but more dependent on the cyclic AMP-catabolite gene activator protein complex mediating catabolite repression. Despite the increased aerobic expression of glpA1(Oxr), a twofold aerobic repressibility persisted. Moreover, anaerobic repression by nitrate respiration remained normal. Thus, there seems to exist a redox control apart from the FNR-mediated one. We also showed that the anaerobic repression of the glpD operon was fully relieved by mutations in either arcA (encoding a presumptive DNA recognition protein) or arcB (encoding a presumptive redox sensor protein). The arc system is known to mediate pleiotropic control of genes of aerobic function.     

The ArcA regulon and oxidative stress resistance in Haemophilus influenzae

Haemophilus influenzae transits between niches within the human host that are predicted to differ in oxygen levels. The ArcAB two-component signal transduction system controls gene expression in response to respiratory conditions of growth and has been implicated in bacterial pathogenesis, yet the mechanism is not understood. We undertook a genome-scale study to identify genes of the H. influenzae ArcA regulon. Deletion of arcA resulted in increased anaerobic expression of genes of the respiratory chain and of H. influenzae's partial tricarboxylic acid cycle, and decreased anaerobic expression levels of genes of polyamine metabolism, and iron sequestration. Deletion of arcA also conferred a susceptibility to transient exposure to hydrogen peroxide that was greater following anaerobic growth than after aerobic growth. Array data revealed that the dps gene, not previously assigned to the ArcA modulon in bacteria, exhibited decreased expression in the arcA mutant. Deletion of dps resulted in hydrogen peroxide sensitivity and complementation restored resistance, providing insight into the previously uncharacterized mechanism of arcA-mediated H(2)O(2) resistance. The results indicate a role for H. influenzae arcA and dps in pre-emptive defence against transitions from growth in low oxygen environments to aerobic exposure to hydrogen peroxide, an antibacterial oxidant produced by phagocytes during infection.     

Anaerobic growth on elemental sulfur using dissimilar iron reduction by autotrophic Thiobacillus ferrooxidans

Abstract Anaerobic growth on elemental sulfur using dissimilar iron reduction by Thiobacillus ferrooxidans has been demonstrated. The ferric ion reducing activity (FIR) of the anaerobic cells was double that of the aerobic cells. Significant differences in inhibition of FIR by respiratory inhibitors were observed between aerobic and anaerobic cells. A higher amount of cytochrome was detected in anaerobic cells compared to aerobic cells. Absorption minima developed with the addition of ferric sulfate in the dithionite reduced cell suspension demonstrated that the ferric ion could accept electrons from the cytochrome system of this bacterium. The possibility of two different electron transport chains in ferric ion reduction is discussed.     

Proteomics of Shewanella oneidensis MR-1 biofilm reveals differentially expressed proteins, including AggA and RibB

Shewanella oneidensis MR-1 is a Gram-negative, facultative aerobic bacterium, able to respire a variety of electron acceptors. Due to its capability to reduce solid ferric iron, S. oneidensis plays an important role in microbially induced corrosion of metal surfaces. Since this requires cellular adhesion to the metal surface, biofilm growth is an essential feature of this process. The goal of this work was to compare the global protein expression patterns of sessile and planktonic grown S. oneidensis cells by two-dimensional (2-D) gel electrophoresis. Mass spectrometry was used as an identification tool of the differentially expressed proteins. An IPG strip of pH 3-10 as well as pH 4-7 was applied for iso-electrofocusing. Analysis of the 2-D patterns pointed out a total of 59 relevant spots. Among these proteins, we highlight the involvement of a protein annotated as an agglutination protein (AggA). AggA is a TolC-like protein which is presumably part of an ABC transporter. Another differentially expressed protein is RibB, an enzyme of the riboflavin biosynthesis pathway. Riboflavin is the precursor molecule of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and may be necessary for the altered respiratory properties of the biofilm cells versus planktonic cells. Some proteins that were identified indicate an anaerobic state of the biofilm. This anaerobic way of living affects the energy gaining pathways of the cell and is reflected by the presence of several proteins, including those of a heme-utilization system.     

Genetic regulation of nitrate assimilation in Klebsiella pneumoniae M5al.

We isolated Mu dI1734 insertion mutants of Klebsiella pneumoniae that were unable to assimilate nitrate or nitrite as the sole nitrogen source during aerobic growth (Nas- phenotype). The mutants were not altered in respiratory (anaerobic) nitrate and nitrite reduction or in general nitrogen control. The mutations were linked and thus defined a single locus (nas) containing genes required for nitrate assimilation. beta-Galactosidase synthesis in nas+/phi(nas-lacZ) merodiploid strains was induced by nitrate or nitrite and was inhibited by exogenous ammonia or by anaerobiosis. beta-Galactosidase synthesis in phi(nas-lacZ) haploid (Nas-) strains was nearly constitutive during nitrogen-limited aerobic growth and uninducible during anaerobic growth. A general nitrogen control regulatory mutation (ntrB4) allowed nitrate induction of phi(nas-lacZ) expression during anaerobic growth. This and other results suggest that the apparent anaerobic inhibition of phi(nas-lacZ) expression was due to general nitrogen control, exerted in response to ammonia generated by anaerobic (respiratory) nitrate reduction.     

Regulation of Klebsiella pneumoniae hut operons by oxygen.

We investigated the regulation of genes concerned with nitrogen metabolism by oxygen in the facultative anaerobe Klebsiella pneumoniae. We found oxygen to be required for the expression of the hut operons; the effect of O2 on the glutamine synthetase and urease was less pronounced than on the hut operons. Glutamine synthetase was transiently repressed during the transition from an aerobic to an anaerobic environment. Regulation of hut by O2 suppressed the effect of nitrogen limitation on the expression of these genes.