Energetics of Saccharomyces cerevisiae CBS 426: Comparison of anaerobic and aerobic glucose limitation

The estimation of algal yield parameters associated with mixotrophic and photoheterotrophic growth under batch cultivation

While many organisms synthesize delta-aminolevulinate, the precursor of heme, by condensing succinyl-coenzyme A and glycine, others use a glutamate-dependent pathway in which glutamyl-tRNA dehydrogenase catalyzes the rate-determining step. The hemeA gene that encodes this latter enzyme in Escherichia coli has been cloned and sequenced. To examine how its expression is regulated, we constructed hemA-lacZ operon and gene fusions and inserted them into the chromosome in single copy. The effect of aerobic and anaerobic growth conditions and the availability of electron acceptors and various carbon substrates were documented. Use of different types of cell culture medium resulted in a fivefold variation in hemA-lacZ expression during aerobic cell growth. Anaerobic growth resulted in 2.5-fold-higher hemA-lacZ expression than aerobic growth. This control is mediated by the fnr and arcA gene products. Fnr functions as a repressor of hemA transcription during anaerobic cell growth only, whereas the arcA gene product activates hemA gene expression under both aerobic and anaerobic conditions. Integration host factor protein was also shown to be required for control of hemA gene regulation. To determine whether an intermediate or a product of the heme biosynthetic pathway is involved in hemA regulation, hemA-lacZ expression was analyzed in a hemA mutant. Expression was elevated by 20-fold compared with that in a wild-type strain, while the addition of the heme pathway intermediate delta-aminolevulinate to the culture medium restored expression to wild-type levels. These results suggest that the heme pathway is feedback regulated at the level of hemA gene expression, to supply heme as it is required during different modes of cell growth.

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.

Quinolone activity against Escherichia coli was examined during aerobic growth, aerobic treatment with chloramphenicol, and anaerobic growth. Nalidixic acid, norfloxacin, ciprofloxacin, and PD161144 were lethal for cultures growing aerobically, and the bacteriostatic activity of each quinolone was unaffected by anaerobic growth. However, lethal activity was distinct for each quinolone with cells treated aerobically with chloramphenicol or grown anaerobically. Nalidixic acid failed to kill cells under both conditions; norfloxacin killed cells when they were grown anaerobically but not when they were treated with chloramphenicol; ciprofloxacin killed cells under both conditions but required higher concentrations than those required with cells grown aerobically; and PD161144, a C-8-methoxy fluoroquinolone, was equally lethal under all conditions. Following pretreatment with nalidixic acid, a shift to anaerobic conditions or the addition of chloramphenicol rapidly blocked further cell death. Formation of quinolone-gyrase-DNA complexes, observed as a sodium dodecyl sulfate (SDS)-dependent drop in cell lysate viscosity, occurred during aerobic and anaerobic growth and in the presence and in the absence of chloramphenicol. However, lethal chromosome fragmentation, detected as a drop in viscosity in the absence of SDS, occurred with nalidixic acid treatment only under aerobic conditions in the absence of chloramphenicol. With PD161144, chromosome fragmentation was detected when the cells were grown aerobically and anaerobically and in the presence and in the absence of chloramphenicol. Thus, all quinolones tested appear to form reversible bacteriostatic complexes containing broken DNA during aerobic growth, during anaerobic growth, and when protein synthesis is blocked; however, the ability to fragment chromosomes and to rapidly kill cells under these conditions depends on quinolone structure.

The 74-min region of the Escherichia coli chromosome includes five open reading frames of known sequence. The first and last of these genes, nirB and cysG, are transcribed in the same direction and both are essential for NADH-dependent nitrite reductase activity. The functions of the other genes, nirD, nirE and nirC, which are located between nirB and cysG, are unknown. The nirB gene is transcribed from a promoter which is anaerobically induced, expression being dependent on the transcription activator protein, Fnr. Here we show that the nirD, nirE, nirC and cysG genes are also expressed from the nirB promoter. After subcloning cysG, a second promoter was located less than 100 bases upstream of cysG. Two groups of transcription start points separated by 40 bases were detected in this region by S1 mapping. Rates of transcription from the isolated cysG promoter were the same during aerobic growth and anaerobic growth in the presence or absence of nitrite. However, when the nirB gene and its promoter were cloned back upstream from the cysG promoter, the rate of transcription was higher during anaerobic growth than during aerobic growth and was further induced by nitrite. These increases were totally dependent on a functional fnr gene and were shown by S1 mapping experiments to be due to transcriptional read-through from the Fnr-dependent nirB promoter. No promoter activity was associated with DNA fragments between the BamHI site located within the N-terminal coding region of the nirB gene and the cysG promoter located at the C-terminus of nirC.

Streptococcus faecalis var. zymogenes was grown aerobically and anaerobically in the presence and absence of haematin, with glycerol as the carbon and energy source. Aerobic growth was stimulated by the inclusion of haematin in the medium but fumarate had no effect on growth. The bacterium was unable to grow anaerobically on glycerol unless fumarate was present; haematin had no effect on growth. NADH oxidase activity, which catalysed the oxidation of NADH + H+ to form H2O rather than H2O2, was found in the soluble fraction and was induced by aerobic growth but partially repressed when haematin was present in the medium. In contrast, a particulate NADH oxidase, which was sensitive to inhibition by antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, was induced by aerobic growth in the presence of haematin. NADH peroxidase was massively induced by aerobic growth, whereas more lactate dehydrogenase activity was found in anaerobically grown bacteria. Catalase was formed only during aerobic growth in the presence of haematin.

The sulfur of taurine can be assimilated by Klebsiella sp. during aerobic growth, but not fermentative growth. However, taurine’s N can be utilized by this bacterium as sole nitrogen source for both aerobic and anaerobic growth. Two other amino-containing sulfonates (3-aminopropanesulfonate and cysteate) were also examined for their abilities to serve as nitrogen sources for Klebsiella sp. during the different growth conditions. The result shows that 3-aminopropanesulfonate only supports aerobic growth while cysteate does not under either condition.

Anoxygenicphotosynthetic prokaryotes have simplified photosystems that represent ancient lineages that predate the more complex oxygen evolving photosystems present in cyanobacteria and chloroplasts. These organisms thrive under illuminated anaerobic photosynthetic conditions, but also have the ability to grow under dark aerobic respiratory conditions. This study provides a detailed snapshot of transcription ground states of both dark aerobic and anaerobic photosynthetic growth modes in the purple photosynthetic bacterium Rhodobactercapsulatus. Using 18 biological replicates for aerobic and photosynthetic states, we observed that 1834 genes (53 % of the genome) exhibited altered expression between aerobic and anaerobic growth. In comparison with aerobically grown cells, photosynthetically grown anaerobic cells showed decreased transcription of genes for cobalamin biosynthesis (−45 %), iron transport and homeostasis (−42 %), motility (−32 %), and glycolysis (−34 %). Conversely and more intuitively, the expression of genes involved in carbon fixation (547 %), bacteriochlorophyll biosynthesis (162 %) and carotenogenesis (114 %) were induced. We also analysed the relative contributions of known global redox transcription factors RegA, FnrL and CrtJ in regulating aerobic and anaerobic growth. Approximately 50 % of differentially expressed genes (913 of 1834) were affected by a deletion of RegA, while 33 % (598 out of 1834) were affected by FnrL, and just 7 % (136 out of 1834) by CrtJ. Numerous genes were also shown to be controlled by more than one redox responding regulator.

Lipoic acid (lip) and 2-oxoglutarate dehydrogenase (sucA) mutants of Escherichia coli K12 exhibit a requirement for exogenous succinate during aerobic growth on glucose minimal medium. Reversion studies have shown that this requirement can be suppressed by gal-linked mutations which inactivate succinate dehydrogenase. Biochemical and genetic studies confirmed that the succinate dehydrogenase gene (sdh) is affected and that suppression is mediated by the same intergenic and indirect mechanism that generates succinate independence in partial revertants of lipoamide dehydrogenase mutants (Creaghan & Guest, 1977). A series of isogenic strains containing all combinations of mutations affecting 2-oxoglutarate dehydrogenase (sucA), succinate dehydrogenase (sdh), isocitrate lyase (aceA) and fumarate reductase (frd) in a background lacking succinate semialdehyde dehydrogenase, was constructed to assess the importance of these enzymes as sources of endogenous succinate (succinyl-CoA) during aerobic and anaerobic growth on glucose. Only strains combining a deficiency in 2-oxoglutarate dehydrogenase with the presence of an active succinate dehydrogenase required succinate for aerobic growth. In all mutants, including the triple mutant (frd sucA aceA), the succinate requirement was suppressed by inactivating succinate dehydrogenase. The aerobic growth rates of succinate-independent strains were most affected by lack of isocitrate lyase but only two mutants (sdh sucA aceA and frd sdh sucA aceA) grew faster with added succinate: the growth yields were lowered by deficiencies in isocitrate lyase and also succinate dehydrogenase. It is concluded that very little succinate is needed for biosynthesis during aerobic growth on glucose and the requirement for relatively high concentrations of succinate (2 mM) by mutants lacking 2-oxoglutarate dehydrogenase or related functions stems from the presence of active succinate dehydrogenase. Anaerobically, either isocitrate lyase or fumarate reductase is essential for succinate-independent growth on glucose.

Saccharomyces cerevisiae CBS 426 was grown aerobically in continuous culture with a mixture of glucose and ethanol as the carbon source. The flows of biomass, glucose, ethanol, oxygen, and carbon dioxide were measured. A model for growth with two substrates was derived. Application of this model to the above-mentioned system yielded values for YATP and P/O. The joint confidence regions for these parameters were calculated. The relevance to industrial production of bakers' yeast is discussed.

The growth inhibitory effect of 100% carbon dioxide (CO2) at 25° C was evaluated on Aeromonas hydrophila, Bacillus cereus ATCC 14579, Brochothrix thermosphact ATCC 11509, Citrobacter freundii, Clostridium sporogenes ATCC 19404, Escherichia coli ATCC 11775, Lactobacillus sp. (homofermentative), Lactobacillus viridescens, Staphylococcus aureus, Streptococcus faecalis, Yersinia enterocolitica, and Yersinia frederiksenii. The organisms were studied in batch cultures and the maximum specific growth rate (μmax) was determined in air, 5% CO2 + nitrogen and 100% CO2; from which the relative inhibitory effect of CO2 was estimated for each organism. Using 100% CO2 reduced the growth rate for all test organisms. Compared to growth in air, the relative inhibitory effect of 100% CO2 was highest for Bac. cereus, Br. thermosphacta and A. hydrophila (> 75%), and lowest for E. coli, Str. faecalis and the Lactobacillus spp. (53%–29%). Compared to anaerobic growth, the relative inhibitory effect of 100% CO2 was generally somewhat lower and the succession of CO2-resistance was slightly altered for some organisms but drastically increased for Br. thermosphacta. The relative inhibitory effect was highest on Bac. cereus, A. hydrophila and Y. frederiksenii (67%–52%) and lowest on Y. enterocolitica, Br. thermosphacta and the Lactobacillus spp. (26%–8%). The anaerobic growth in nitrogen was generally slower than the aerobic growth. The exceptions were Streptococcus faecalis (only 2% reduction) and Clostridium sporogenes (no aerobic growth). In 100% CO2Str. faecalis, Citrobacter freundii and Escherichia coli had highest μmax and Brochothrix thermosphacta, Bacillius cereus and Staphylococcus aureus the lowest.

Influence of Modified Atmosphere Packaging on Microbiology of Broiler Drumsticks

The role of protein induction and repression in the adaptation of Escherichia coli to changes in the supply of oxygen and other electron acceptors is only poorly understood. We have studied the changes in cellular protein composition associated with this adaptation by measuring the levels of 170 individual polypeptides produced during aerobic or anaerobic growth of E. coli, with and without nitrate. Nineteen polypeptides had levels highest during aerobic growth. These proteins include the enzymes of the pyruvate dehydrogenase complex, several tricarboxylic acid cycle enzymes, superoxide dismutase, and tetrahydropteroyltriglutamate transmethylase. The other aerobiosis-induced proteins have not been identified. These polypeptides are major cellular proteins during aerobic growth and display several different patterns of regulation in response to medium composition. Induction ratios for oxygen ranged from 2.2 to 11.2, with one exceptional member, superoxide dismutase, increasing 71-fold with aeration. Most of the proteins were also induced by nitrate during anaerobic growth. The time course of induction after shifts in oxygen supply revealed similarities in response among proteins of related function or metabolic regulation class. These results are discussed in relation to previously reported information on the identified aerobiosis-induced proteins.

Fumarate dependent protein composition under aerobic and anaerobic growth conditions in Escherichia coli

The phylum Deinococcus-Thermus is a deeply-branching lineage of bacteria widely recognized as one of the most extremophilic. Members of the Thermus genus are of major interest due to both their bioremediation and biotechnology potentials. However, the molecular mechanisms associated with these key metabolic pathways remain unknown. Reverse-transcription quantitative PCR (RT-qPCR) is a high-throughput means of studying the expression of a large suite of genes over time and under different conditions. The selection of a stably-expressed reference gene is critical when using relative quantification methods, as target gene expression is normalized to expression of the reference gene. However, little information exists as to reference gene selection in extremophiles. This study evaluated 11 candidate reference genes for use with the thermophile Thermus scotoductus when grown under different culture conditions. Based on the combined stability values from BestKeeper and NormFinder software packages, the following are the most appropriate reference genes when comparing: (1) aerobic and anaerobic growth: TSC_c19900, polA2, gyrA, gyrB; (2) anaerobic growth with varied electron acceptors: TSC_c19900, infA, pfk, gyrA, gyrB; (3) aerobic growth with different heating methods: gyrA, gap, gyrB; (4) all conditions mentioned above: gap, gyrA, gyrB. The commonly-employed rpoC does not serve as a reliable reference gene in thermophiles, due to its expression instability across all culture conditions tested here. As extremophiles exhibit a tendency for polyploidy, absolute quantification was employed to determine the ratio of transcript to gene copy number in a subset of the genes. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes reflect transcript copy number, and not gene copy number, fluctuations. Even with the potential for polyploidy in extremophiles, the results obtained via absolute quantification indicate that relative quantification is appropriate for RT-qPCR studies with this thermophile.