Requirement for terminal cytochromes in generation of the aerobic signal for the arc regulatory system in Escherichia coli: study utilizing deletions and lac fusions of cyo and cyd.

Escherichia coli has two terminal oxidases for its respiratory chain: cytochrome o (low O2 affinity) and cytochrome d (high O2 affinity). Expression of the cyo operon, encoding cytochrome o, is decreased by anaerobic growth, whereas expression of the cyd operon, encoding cytochrome d, is increased by anaerobic growth. We show by the use of lac gene fusion that the expressions of cyo and cyd are under the control of the two-component arc system. In a cyo+ cyd+ background, expression of phi(cyo-lac) is higher when the organism is grown aerobically than when it is grown anaerobically. A mutation in either the sensor gene arcB or the pleiotropic regulator gene arcA almost abolishes the anaerobic repression. In the same background, expression of phi(cyd-lac) is higher under anaerobic growth conditions than under aerobic growth conditions. A mutation in arcA or arcB lowers both the aerobic and anaerobic expressions, suggesting that ArcA plays an activating role instead of the typical repressing role. Under aerobic growth conditions, double deletions of cyo and cyd lower phi(cyo-lac) expression but enhance phi(cyd-lac) expression. The double deletions also prevent elevated aerobic induction of the lct operon (encoding L-lactate dehydrogenase), another target operon of the arc system. In contrast, these deletions do not circumvent aerobic repression of the nar operon (encoding the anaerobic respiratory enzyme nitrate reductase) under the control of the pleiotropic fnr gene product. It thus appears that ArcB senses the presence of O2 by level of an electron transport component in reduced form or that of an nonautoxidizable compound linked to the process by a redox reaction, whereas Fnr senses O2 by a different mechanism.     

Identification of the cydC locus required for expression of the functional form of the cytochrome d terminal oxidase complex in Escherichia coli.

The aerobic respiratory chain of Escherichia coli contains two terminal oxidases which are differentially regulated. The cytochrome o complex predominates under growth conditions of high aeration, whereas the cytochrome d complex predominates when the oxygen tension is low. Either terminal oxidase will support aerobic growth. The goal of the work presented in this paper was to identify genes required for the expression of the functional form of the cytochrome d complex, other than the genes encoding the polypeptide components of the oxidase complex (cyd locus). A strain lacking the cytochrome o complex (cyo mutant strain) was mutagenized by using a lambda-Mu hybrid hopper bacteriophage, lambda placMu53, which inserts randomly into the chromosome and carries a kanamycin resistance marker. Strains were isolated and examined which were unable to grow aerobically, i.e., which lacked functional cytochrome d complex, and which could not be complemented by introduction of the cyd gene on F-prime episomes. One strain was selected for characterization. The phage insert was mapped to min 18.9 on the genetic linkage map, defining a new genetic locus, cydC. Evidence described in the text suggests that the gene product is probably required for the synthesis of the unique heme d component of the cytochrome d complex.     

Regulation of the citrate synthase (gltA) gene of Escherichia coli in response to anaerobiosis and carbon supply: role of the arcA gene product.

As an enzyme of the tricarboxylic acid cycle pathway, citrate synthase participates in the generation of a variety of cellular biosynthetic intermediates and in that of reduced purine nucleotides that are used in energy generation via electron transport-linked phosphorylation reactions. It catalyzes the condensation of oxaloacetate and acetyl coenzyme A to produce citrate plus coenzyme A. In Escherichia coli this enzyme is encoded by the gltA gene. To investigate how gltA expression is regulated, a gltA-lacZ operon fusion was constructed and analyzed following aerobic and anaerobic cell growth on various types of culture media. Under aerobic culture conditions, expression was elevated to a level twofold higher than that reached under anaerobic culture conditions. ArcA functions as a repressor of gltA expression under each set of conditions: in a delta arcA strain, gltA-lacZ expression was elevated to levels two- and eightfold higher than those seen in a wild-type strain under aerobic and anaerobic conditions, respectively. This control is independent of the fnr gene product, an alternative anaerobic gene regulator in E. coli. When the richness or type of carbon compound used for cell growth was varied, gltA-lacZ expression varied by 10- to 14-fold during aerobic and anaerobic growth. This regulation was independent of both the crp and fruR gene products, suggesting that another regulatory element in E. coli is responsible for the observed control. Finally, gltA-lacZ expression was shown to be inversely proportional to the cell growth rate. These findings indicate that the regulation of gltA gene expression is complex in meeting the differential needs of the cell for biosynthesis and energy generation under various cell culture conditions.     

arc-dependent thermal regulation and extragenic suppression of the Escherichia coli cytochrome d operon.

In a screen for Escherichia coli genes whose products are required for high-temperature growth, we identified and characterized a mini-Tn10 insertion that allows the formation of wild-type-size colonies at 30 degrees C but results in microcolony formation at 36 degrees C and above (Ts- phenotype). Mapping, molecular cloning, and DNA sequencing analyses showed that the mini-Tn10 insertion resides in the cydB gene, the distal gene of the cydAB operon (cytochrome d). The Ts- growth phenotype was also shown to be associated with previously described cyd alleles. In addition, all cyd mutants were found to be extremely sensitive to hydrogen peroxide. Northern (RNA) blot analysis showed that cyd-specific mRNA levels accumulate following a shift to high temperature. Interestingly, this heat shock induction of the cyd operon was not affected in an rpoH delta background but was totally absent in an arcA or arcB mutant background. Extragenic suppressors of the Cyd Ts- phenotype are found at approximately 10(-3). Two extragenic suppressors were shown to be null alleles in either arcA or arcB. One interpretation of our results is that in the absence of ArcA or ArcB, which are required for the repression of the cyo operon (cytochrome o), elevated levels of Cyo are produced, thus compensating for the missing cytochrome d function. Consistent with this interpretation, the presence of the cyo gene on a multicopy plasmid suppressed the Ts- and hydrogen peroxide-sensitive phenotypes of cyd mutants.