Proteogenomic evidence for β-oxidation of plant-derived 3-phenylpropanoids in "Aromatoleum aromaticum" EbN1

The betaproteobacterium "Aromatoleum aromaticum" EbN1 utilizes eight different plant-derived nonhydroxylated (e.g. cinnamate) and hydroxylated (e.g. p-coumarate) 3-phenylpropanoids with nitrate as electron acceptor. Differential protein profiling (2D-DIGE) revealed abundance increases of five proteins (EbA5316 to EbA5320) during anaerobic growth with cinnamate, hydrocinnamate, p-coumarate, and 3-(4-hydroxyphenyl)propanoate, compared to anaerobic benzoate-adapted cells serving as reference state. The predicted functions of four of these proteins (EbA5317, fatty acid-coenzyme A (CoA) ligase; EbA5318, enoyl-CoA hydratase/isomerase; EbA5319, β-ketothiolase; and EbA5320, 3-hydroxyacyl-CoA dehydrogenase) suggest β-oxidation of the above 3-phenylpropanoids to benzoyl-CoA and p-hydroxybenzoyl-CoA, respectively. The fifth protein (EbA5316, ABC-type periplasmic solute-binding protein) could be involved in 3-phenylpropanoid uptake. The detection of 3-hydroxy-3-phenylpropanoate during anaerobic growth with cinnamate and hydrocinnamate or 3-hydroxy-3-(4-hydroxyphenyl)propanoate during anaerobic growth with p-coumarate and 3-(4-hydroxyphenyl)propanoate supports the proteome-predicted β-oxidation pathway. Based on the specific formation of EbA5316-20 also during anaerobic growth with further 3-phenylpropanoid growth substrates including cinnamyl alcohol, m-coumarate, 3-(3,4-dihydroxyphenyl)propanoate and 3,4-dihydroxycinnamate (caffeate), a common β-oxidation route is proposed for 3-phenylpropanoid degradation in strain EbN1. The low amount of metabolites attributable to cometabolic transformation of nongrowth supporting 3-phenylpropanoids (e.g. o-coumarate, ferulate) may be indicative for a high substrate specificity of the involved enzymes.     

Towards determining details of anaerobic growth coupled to ferric iron reduction by the acidophilic archaeon ‘<Emphasis Type="Italic">Ferroplasma</Emphasis><Emphasis Type="Italic"> acidarmanus</Emphasis>’ Fer1

Elucidation of the different growth states of Ferroplasma species is crucial in understanding the cycling of iron in acid leaching sites. Therefore, a proteomic and biochemical study of anaerobic growth in ‘Ferroplasma acidarmanus’ Fer1 has been carried out. Anaerobic growth in Ferroplasma spp. occurred by coupling oxidation of organic carbon with the reduction of Fe³⁺; but sulfate, nitrate, sulfite, thiosulfate, and arsenate were not utilized as electron acceptors. Rates of Fe³⁺ reduction were similar to other acidophilic chemoorganotrophs. Analysis of the ‘F. acidarmanus’ Fer1 proteome by 2-dimensional polyacrylamide gel electrophoresis revealed ten key proteins linked with central metabolic pathways ≥4 fold up-regulated during anaerobic growth. These included proteins putatively identified as associated with the reductive tricarboxylic acid pathway used for anaerobic energy production, and others including a putative flavoprotein involved in electron transport. Inhibition of anaerobic growth and Fe³⁺ reduction by inhibitors suggests the involvement of electron transport in Fe³⁺ reduction. This study has increased the knowledge of anaerobic growth in this biotechnologically and environmentally important acidophilic archaeon.     

Differential proteomic analysis of Bacillus subtilis nitrate respiration and fermentation in defined medium

A comparative investigation of protein expression by two-dimensional gel electrophoresis was conducted between Bacillus subtilis cultures grown in defined medium under aerobic, anaerobic nitrate respiration, or fermentation conditions. Defined medium specific for either nitrate respiration or fermentation allowed distinction between proteins induced by each individual growth process. Our differential protein profiling analysis between aerobic and anaerobic conditions showed that anaerobic fermentation induced at least 44 proteins and nitrate respiration induced at least 19 proteins compared to aerobic controls. Certain proteins were specifically induced during nitrate respiration or fermentation, while others were induced by both anaerobic processes. Eleven proteins induced by nitrate respiration and/or fermentation were identified by peptide mass matching using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Proteins encoded by feuA, hmp, and ytkD were induced by nitrate respiration. Proteins encoded by pyrR, sucD, trpC, and ywjH were induced by fermentation. Proteins encoded by acuB, pdhC, ydjL, and yvyD were induced by nitrate respiration and fermentation. This proteomic analysis has provided a more complete characterization of B. subtilis anaerobic growth and increased our understanding of its metabolic pathways of nitrate respiration and fermentation.     

Global gene expression profiles of Bacillus subtilis grown under anaerobic conditions

Bacillus subtilis can grow under anaerobic conditions, either with nitrate or nitrite as the electron acceptor or by fermentation. A DNA microarray containing 4,020 genes from this organism was constructed to explore anaerobic gene expression patterns on a genomic scale. When mRNA levels of aerobic and anaerobic cultures during exponential growth were compared, several hundred genes were observed to be induced or repressed under anaerobic conditions. These genes are involved in a variety of cell functions, including carbon metabolism, electron transport, iron uptake, antibiotic production, and stress response. Among the highly induced genes are not only those responsible for nitrate respiration and fermentation but also those of unknown function. Certain groups of genes were specifically regulated during anaerobic growth on nitrite, while others were primarily affected during fermentative growth, indicating a complex regulatory circuitry of anaerobic metabolism.     

Effect of the medium redox potential on the growth and metabolism of anaerobic bacteria

Based on the available literature data on a decrease in the redox potential of medium to low negative values and a decrease in pH during the growth of sugar-fermenting anaerobic bacteria, it was concluded that these processes cannot be described by the theory of redox potential. A theory was developed according to which the regulation of bacterial metabolism is accomplished through changes in the redox potential. The theory considers the redox potential as a factor determining the growth of anaerobic bacteria, which is regulated by oxidizers and reducers. The assumption is put forward that, under anaerobic conditions, bacteria are sensitive to changes in the redox potential and have a redox taxis. The effect of the redox potential on the transport of protons and other substances through membranes and the activity of membrane-bound enzymes, including the proton F1-F0-ATPase, whose mechanisms of action involve changes in the proton conductance of the membrane, the generation of proton-driving force, and dithiol-disulfide transitions in proteins was studied.     

Proteins induced by anaerobiosis in Escherichia coli

The contribution of protein induction and repression to the adaptation of cells to changes in oxygen supply is only poorly understood. We assessed this contribution by measuring the levels of 170 individual polypeptides produced by Escherichia coli K-12 in cells growing aerobically or anaerobically with and without nitrate. Eighteen reached their highest levels during anaerobic growth. These 18 polypeptides include at least 4 glycolytic enzymes and pyruvate formate-lyase (beta-subunit). Most of these proteins were found at significant levels during aerobic growth and appeared to undergo metabolic regulation by stimuli other than anaerobiosis. Anaerobic induction ratios ranged from 1.8- to 11-fold, and nitrate antagonized the anaerobic induction of all of the proteins except one. The time course of synthesis of the proteins after shifts in oxygen supply revealed at least three distinct temporal patterns. These results are discussed in light of known physiological alterations associated with changes in oxygen availability.     

Characterization of anaerobic sulfite reduction by Salmonella typhimurium and purification of the anaerobically induced sulfite reductase.

Mutants of Salmonella typhimurium that lack the biosynthetic sulfite reductase (cysI and cysJ mutants) retain the ability to reduce sulfite for growth under anaerobic conditions (E. L. Barrett and G. W. Chang, J. Gen. Microbiol., 115:513-516, 1979). Here we report studies of sulfite reduction by a cysI mutant of S. typhimurium and purification of the associated anaerobic sulfite reductase. Sulfite reduction for anaerobic growth did not require a reducing atmosphere but was prevented by an argon atmosphere contaminated with air (less than 0.33%). It was also prevented by the presence of 0.1 mM nitrate, which argues against a strictly biosynthetic role for anaerobic sulfite reduction. Anaerobic growth in liquid minimal medium, but not on agar, was found to require additions of trace amounts (10(-7)M) of cysteine. Spontaneous mutants that grew under the argon contaminated with air also lost the requirement for 10(-7)M cysteine for anaerobic growth in liquid. A role for sulfite reduction in anaerobic energy generation was contraindicated by the findings that sulfite reduction did not improve cell yields, and anaerobic sulfite reductase activity was greatest during the stationary phase of growth. Sulfite reductase was purified from the cytoplasmic fraction of the anaerobically grown cysI mutant and was purified 190-fold. The most effective donor in crude extracts was NADH. NADPH and methyl viologen were, respectively, 40 and 30% as effective as NADH. Oxygen reversibly inhibited the enzyme. Two high-molecular-weight proteins separated by gel filtration (Mr 360,000 and 490,000, respectively) were required for maximal activity with NADH. Indirect evidence, including in vitro complementation experiments with a cysG mutant extract, suggested that the 360,000-Mr component contains siroheme and is the terminal reductase. This component was further purified to near homogeneity and was found to consist of a single subunit of molecular weight 67,500. The anaerobic sulfite reductase showed some resemblance to the biosynthetic sulfite reductase, but apparently it has a unique, as yet unidentified function.     

Acid- and base-induced proteins during aerobic and anaerobic growth of Escherichia coli revealed by two-dimensional gel electrophoresis

Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified in Escherichia coli. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 proteins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may reverse alkalinization by metabolizing amino acids to produce acidic products. Also induced at high pH, but only in anaerobiosis, was glutamate decarboxylase (GadA). The gad system (GadA/GadBC) neutralizes acidity and enhances survival in extreme acid; its induction during anaerobic growth may help protect alkaline-grown cells from the acidification resulting from anaerobic fermentation. To investigate possible responses to internal acidification, cultures were grown in propionate, a membrane-permeant weak acid which acidifies the cytoplasm. YfiD, a homologue of pyruvate formate lyase, was induced to high levels at pH 4.4 and induced twofold more by propionate at pH 6; both of these conditions cause internal acidification. At neutral or alkaline pH, YfiD was virtually absent. YfiD is therefore a strong candidate for response to internal acidification. Acid or propionate also increased the expression of alkyl hydroperoxide reductase (AhpC) but only during aerobic growth. At neutral or high pH, AhpC showed no significant difference between aerobic and anaerobic growth. The increase of AhpC in acid may help protect the cell from the greater concentrations of oxidizing intermediates at low pH. Isocitrate lyase (AceA) was induced by oxygen across the pH range but showed substantially greater induction in acid or in base than at pH 7. Additional responses observed included the induction of MalE at high pH and induction of several enzymes of sugar metabolism at low pH: the phosphotransferase system components ManX and PtsH and the galactitol fermentation enzyme GatY. Overall, our results indicate complex relationships between pH and oxygen and a novel permeant acid-inducible gene, YfiD.     

Application of a tetrazolium dye as an indicator of viability in anaerobic bacteria.

The use of the redox dye 5-cyano-2,3,-ditolyl tetrazolium chloride (CTC) for evaluating the metabolic activity of aerobic bacteria has gained wide application in recent years. In this study, we examined the utility of CTC in capturing the metabolic activity of anaerobic bacteria. In addition, the factors contributing to abiotic reduction of CTC were also examined. CTC was used in conjunction with the fluorochrome 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF), that targets bacterial cell wall proteins, to quantitate the active fraction of total bacterial numbers. Facultative anaerobic bacteria, including Escherichia coli grown fermentatively, and Pseudomonas chlorophis, P. fluorescens, P. stutzeri, and P. pseudoalcalegenes subsp. pseudoalcalegenes grown under nitrate-reducing conditions, actively reduced CTC during all phases of growth. Greater than 95% of these cells accumulated intracellular CTC-formazan crystals during the exponential phase. Obligate anaerobic bacteria, including Syntrophus aciditrophicus grown fermentatively, Geobacter sulfurreducens grown with fumarate as the electron acceptor, Desulfovibrio desulfuricans subsp. desulfuricans and D. halophilus grown under sulfate-reducing conditions, Methanobacterium formicicum grown on formate, H2 and CO2, and Methanobacterium thermoautotrophicum grown autotrophically on H2 and CO2 all reduced CTC to intracellular CTC-formazan crystals. The optimal CTC concentration for all organisms examined was 5 mM. Anaerobic CTC incubations were not required for quantification of anaerobically grown cells. CTC-formazan production by all cultures examined was proportional to biomass production, and CTC reduction was observed even in the absence of added nutrients. CTC was reduced by culture fluids containing ferric citrate as electron acceptor following growth of either G. metallireducens or G. sulfurreducens. Abiotic reduction of CTC was observed in the presence of ascorbic acid, cysteine hydrochloride, dithiothreitol, NADH, NADPH, Fe(II)Cl2, sodium thioglycolic acid and sodium sulfide. These results suggest that while CTC can be used to capture the metabolic activity of anaerobic bacteria, care must be taken to avoid abiotic reduction of CTC.     

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.     

Solvent stress response of the denitrifying bacterium "Aromatoleum aromaticum" strain EbN1

The denitrifying betaproteobacterium "Aromatoleum aromaticum" strain EbN1 degrades several aromatic compounds, including ethylbenzene, toluene, p-cresol, and phenol, under anoxic conditions. The hydrophobicity of these aromatic solvents determines their toxic properties. Here, we investigated the response of strain EbN1 to aromatic substrates at semi-inhibitory (about 50% growth inhibition) concentrations under two different conditions: first, during anaerobic growth with ethylbenzene (0.32 mM) or toluene (0.74 mM); and second, when anaerobic succinate-utilizing cultures were shocked with ethylbenzene (0.5 mM), toluene (1.2 mM), p-cresol (3.0 mM), and phenol (6.5 mM) as single stressors or as a mixture (total solvent concentration, 2.7 mM). Under all tested conditions impaired growth was paralleled by decelerated nitrate-nitrite consumption. Additionally, alkylbenzene-utilizing cultures accumulated poly(3-hydroxybutyrate) (PHB) up to 10% of the cell dry weight. These physiological responses were also reflected on the proteomic level (as determined by two-dimensional difference gel electrophoresis), e.g., up-regulation of PHB granule-associated phasins, cytochrome cd(1) nitrite reductase of denitrification, and several proteins involved in oxidative (e.g., SodB) and general (e.g., ClpB) stress responses.     

A metaproteomic approach gives functional insights into anaerobic digestion

Aims: The objective of this work was to provide functional evidence of key metabolic pathways important for anaerobic digestion processes through the identification of highly expressed proteins in a mixed anaerobic microbial consortium. Methods and Results: The microbial communities from an anaerobic industrial‐like wastewater treatment bioreactor were characterized using phylogenetic analyses and metaproteomics. Clone libraries indicated that the bacterial community in the bioreactor was diverse while the archaeal population was mainly composed of Methanocorpusculum‐like (76%) micro‐organisms. Three hundred and eighty‐eight reproducible protein spots were obtained on 2‐D gels, of which 70 were excised and 33 were identified. The putative functions of the proteins detected in the anaerobic bioreactor were related to cellular processes, including methanogenesis from CO₂ and acetate, glycolysis and the pentose phosphate pathway. Metaproteomics also indicated, by protein assignment, the presence of specific micro‐organisms in the bioreactor. However, only a limited overlap was observed between the phylogenetic and metaproteomic analyses. Conclusions: This study provides some direct evidence of the microbial activities taking place during anaerobic digestion. Significance and Impact of Study: This study demonstrates metaproteomics as a useful tool to uncover key biochemical pathways underpinning specific anaerobic bioprocesses.     

pH-dependent catabolic protein expression during anaerobic growth of Escherichia coli K-12

During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.     

Energy-dependent inactivation of citrate lyase in Enterobacter aerogenes.

Enterobacter aerogenes was grown in continous culture with ammonia as the growth-limiting substrate, and changes in citrate lyase and citrate synthase activities were monitored after growth shifts from anaerobic growth on citrate to aerobic growth on citrate, aerobic growth on glucose, anaerobic growth on glucose, and anaerobic growth on glucose plus nitrate. Citrate lyase was inactivated during aerobic growth on glucose and during anaerobic growth with glucose plus nitrate. Inactivation did not occur during anaerobic growth on glucose, and as a result of the simultaneous presence of citrate lyase and citrate synthase, growth difficulties were observed. Citrate lyase inactivation consisted of deacetylation of the enzyme. The corresponding deacetylase could not be demonstrated in cell extracts, and it is concluded that, as in a number of other inactivations, electron transport to oxygen or nitrate was required for inactivation.     

Biochemically Induced Carbonate Precipitation in Aerobic and Anaerobic Environments by Sporosarcina pasteurii

Microbially induced calcium carbonate precipitation (MICP) at laboratory scale for modifying the geotechnical properties of soils has been extensively investigated. The successful implementation of MICP in the field encounters many biotic and abiotic challenges. The study aimed to comprehend the role of oxygen availability on the efficacy of MICP catalyzed by S. pasteurii microbe. For this purpose, microbial growth rate, its ureolytic activities and carbonate precipitation by S. pasteurii over aerated, anoxic and anaerobic conditions were studied. The growth rate, ureolytic activity and amount of mineral precipitated were found to be insignificant under anaerobic environment compared to the remaining exposure conditions, which signifies the importance of oxygen for successful implementation of MICP process. The limited ureolytic activities and a minute amount of precipitation observed under anaerobic system were primarily attributed to the enzymes, already produced during the aerobic culture. The rise in the pH during the MICP process was not only because of ureolytic activities but also due to the breakdown of complex proteins in the stationary growth phase. As a whole, the MICP process was significantly inhibited in the absence of oxygen, or without frequent injection of S. pasteurii.     

Studies on Crystalline Growth of O2-susceptible Proteins in Space

In order to meet the requirement for crystalline growth of O2-susceptible proteins in space, crystallization conditions on the earth was optimized for the proteins using a simple and suitable device for anaerobic addition of the protein samples. Nitrogenase is susceptible to O2. ΔnifZ MoFe protein from a nifZ deleted strain and MnFe protein from mutant strain UW3 grown on a medium containing Mn were crystallized at the first time in the world using an anaerobic device equipped with plastic bags or using a small simplified box, as a replacement for the cumbersome dry box. And the proteins could be also crystallized far from laboratory by sitting-drop method using a much lighter device. It was equipped with a smaller plastic food bag and a first-aid bag filled with Ar, as a substitute for the cumbersome dry box and the Ar cylinder, respectively. The results showed that the device could meet the requirement for studies on crystal growth of the above anaerobic proteins in space.     

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.     

Reciprocal regulation of anaerobic and aerobic cell wall mannoprotein gene expression in Saccharomyces cerevisiae

The DAN/TIR genes encode nine cell wall mannoproteins in Saccharomyces cerevisiae which are expressed during anaerobiosis (DAN1, DAN2, DAN3, DAN4, TIR1, TIR2, TIR3, TIR4, and TIP1). Most are expressed within an hour of an anaerobic shift, but DAN2 and DAN3 are expressed after about 3 h. At the same time, CWP1 and CWP2, the genes encoding the major mannoproteins, are down-regulated, suggesting that there is a programmed remodeling of the cell wall in which Cwp1 and Cwp2 are replaced by nine anaerobic counterparts. TIP1, TIR1, TIR2, and TIR4 are also induced during cold shock. Correspondingly, CWP1 is down-regulated during cold shock. As reported elsewhere, Mox4 is a heme-inhibited activator, and Mot3 is a heme-induced repressor of the DAN/TIR genes (but not of TIP1). We show that CWP2 (but not CWP1) is controlled by the same factors, but in reverse fashion-primarily by Mot3 (which can function as either an activator or repressor) but also by Mox4, accounting for the reciprocal regulation of the two groups of genes. Disruptions of TIR1, TIR3, or TIR4 prevent anaerobic growth, indicating that each protein is essential for anaerobic adaptation. The Dan/Tir and Cwp proteins are homologous, with the greatest similarities shown within three subgroups: the Dan proteins, the Tip and Tir proteins, and, more distantly, the Cwp proteins. The clustering of homology corresponds to differences in expression: the Tip and Tir proteins are expressed during hypoxia and cold shock, the Dan proteins are more stringently repressed by oxygen and insensitive to cold shock, and the Cwp proteins are oppositely regulated by oxygen and temperature.