Genetic and physiological characterization of new Escherichia coli mutants impaired in hydrogenase activity

The Mu dl (ApR lac) bacteriophage was used to generate mutants of Escherichia coli which were defective in formate hydrogenlyase. Three mutants were chosen for further analysis: they lacked hydrogenase (hydrogen: benzyl viologen oxidoreductase) activity, but produced normal levels of fumarate reductase activity and two- to three-fold reduced levels of benzyl viologen (BV)-dependent formate dehydrogenase activity. Two of them (hydC) were shown to contain about 4-fold reduced amounts of formate hydrogenlyase and fumarate-dependent H2 uptake activities. The third one (hydD) was totally devoid of both activities. Their insertion sites were located at 77 min on the E. coli map. Subdivision of these mutants into two classes was subsequently based on the restoration capacity of hydrogenase activity with high concentration of nickel in the growth media. Addition of 500 microM NiCl2 led to a complete recovery of hydrogenase activity, and to the concomitant restoration of normal BV-linked formate dehydrogenase, formate hydrogenlyase and fumarate-dependent H2 uptake activities in the hydC mutants. The hydD mutant was insensitive to the effect of nickel. Expression of the lac operon in hydC and hydD mutants was induced by anaerobiosis. It was not increased by the addition of formate under anaerobic conditions. The presence of nitrate resulted in slightly reduced beta-galactosidase activities in the hydC mutants, whereas those found in the hydD mutant reached only one third of the level obtained in its absence. Fumarate had no effect on both classes. Moreover, in contrast to the hydD locus, the hydC::Mu dl fusions were found to be dependent upon the positive control exerted by the nirR gene product and were totally repressed by an excess of nickel. In addition, the low levels of overall hydrogenase-dependent activities found in a nirR strain were also relieved by the presence of nickel. Our results strongly suggest that the pleiotropic regulatory gene nirR is essential for the expression of a gene (hydC) involved in either transport or processing of nickel in the cell, whose alteration leads to a loss of hydrogenase activity.     

Cloning of hydrogenase genes and fine structure analysis of an operon essential for H2 metabolism in Escherichia coli.

Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2 (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome.     

Roles for menaquinone and the two trimethylamine oxide (TMAO) reductases in TMAO respiration in Salmonella typhimurium: Mu d(Apr lac) insertion mutations in men and tor.

Three groups of mutants defective in trimethylamine oxide (TMAO) reduction were isolated from Salmonella typhimurium LT2 subjected to transposition mutagenesis with Mu d(Apr lac). Mutants were identified by their acidic reaction on a modified MacConkey-TMAO medium. Group I consisted of pleiotropic chlorate-resistant mutants which were devoid of TMAO reductase activity. None expressed the lac operon. Group II mutants were partially defective in TMAO reductase. Electrophoretic studies revealed that they lacked the inducible TMAO reductase, but retained the constitutive activity. The genotypic designation tor was suggested for these mutants. The tor mutation in one was located between 80 and 83 U on the S. typhimurium chromosome. Expression of the lac operon in these mutants was not affected by air, TMAO, or nitrate. Group III mutants reduced little or no TMAO in vivo, but their extracts retained full capacity to reduce it with methyl viologen. These mutants also failed to produce hydrogen sulfide from thiosulfate and could not grow anaerobically on glycerol-fumarate. Two subgroups were distinguished. Vitamin K5 restored wild-type phenotype in subgroup IIIa only; vitamin K1 restored wild-type phenotype in both IIIa and IIIb isolates. The genotypic designation men (menaquinone) was suggested for group III isolates. The mutation in IIIa mutants was cotransducible with glpT, which corresponds to the menBCD site in Escherichia coli. That in IIIb mutants was cotransducible with glpK, which corresponds to the menA site in E. coli. Expression of the lac operon in IIIa, but not IIIb, mutants was repressed by air. An additional mutant group isolated on the same medium consisted of strains defective in formate hydrogenlyase.     

The phs gene and hydrogen sulfide production by Salmonella typhimurium.

Salmonella typhimurium produces H2S from thiosulfate or sulfite. The respective pathways for the two reductions must be distinct as mutants carrying motations in phs, chlA, and menB reduced sulfite, but not thiosulfate, to H2S, and glucose repressed the production of H2S from thiosulfate while it stimulated its production from sulfite. The phs and chlA mutants also lacked a methyl viologen-linked thiosulfate reductase activity present in anaerobically grown wild-type cultures. A number of hydroxylamine, transposon Tn10 insertion, and Mu d1(Apr lac) operon fusion mutants defective in phs were characterized. One of the hydroxylamine mutants was an amber mutant, as indicated by suppression of its mutation in a supD background. The temperature-sensitive phs mutants produced H2S and methyl viologen-linked thiosulfate reductase at 30 degrees C but not at 42 degrees C. The reductases in all such mutants grown at 30 degrees C were as thermostable as the wild-type enzyme and did not differ in electrophoretic relative mobility, suggesting that phs is not the structural gene for thiosulfate reductase. Expression of beta-galactosidase in phs::Mu d1(Apr lac) mutants was dependent on anaerobiosis and the presence of reduced sulfur. It was also strongly influenced by carbon source and growth stage. The results are consistent with a model in which the phs gene encodes a regulatory protein essential for the reduction of thiosulfate to hydrogen sulfide.     

Isolation and characterization of mutant strains of Escherichia coli altered in H2 metabolism

A positive selection procedure is described for the isolation of hydrogenase-defective mutant strains of Escherichia coli. Mutant strains isolated by this procedure can be divided into two major classes. Class I mutants produced hydrogenase activity (determined by using a tritium-exchange assay) and formate hydrogenlyase activity but lacked the ability to reduce benzyl viologen or fumarate with H2 as the electron donor. Class II mutants failed to produce active hydrogenase and hydrogenase-dependent activities. All the mutant strains produced detectable levels of formate dehydrogenase-1 and -2 and fumarate reductase. The mutation in class I mutants mapped near 65 min of the E. coli chromosome, whereas the mutation in class II mutants mapped between srl and cys operons (58 and 59 min, respectively) in the genome. The class II Hyd mutants can be further subdivided into two groups (hydA and hydB) based on the cotransduction characteristics with cys and srl. These results indicate that there are two hyd operons and one hup operon in the E. coli chromosome. The two hyd operons are needed for the production of active hydrogenase, and all three are essential for hydrogen-dependent growth of the cell.     

Mu dX, a derivative of Mu d1 (lac Apr) which makes stable lacZ fusions at high temperature.

We describe defective Mu phage Mu dX (Mu d1 Bx::Tn9 [lac Apr Cmr]) which is useful for insertion mutagenesis and for construction of lac operon fusions in vivo. Mu dX retains the insertion properties of Mu d1 but produces temperature-resistant lysogens and transposes at a reduced frequency. A method is described to convert existing Mu d1 insertions to Mu dX.     

Anaerobiosis induces expression of ant, a new Escherichia coli locus with a role in anaerobic electron transport

Escherichia coli has a formate hydrogenlyase system which allows it to maintain an electron balance during anaerobic growth by passing electrons from formate to H+ ions, thus generating H2. The Mu d1(Ap lac) bacteriophage was used to generate mutants that were defective in passing electrons from formate to benzyl viologen, an artificial electron acceptor. A subset of these mutants was studied in which beta-galactosidase was expressed at much higher levels under anaerobic conditions than under aerobic conditions. If nitrate was present during anaerobic growth, the same levels of beta-galactosidase were seen in these fusion strains as were seen under aerobic conditions. The Mu d1(Ap lac) insertions in these mutants were genetically mapped between mutS and srl and thus define a new locus we have termed ant (anaerobic electron transport). Recombinant lambda derivatives were isolated which complemented the deficiency of the ant mutants in anaerobic electron transport and also carried a trans-acting region of DNA which reduced expression of the ant-lac fusions under anaerobic conditions; a probe to the ant region was generated from one of these recombinant lambda derivatives. Southern hybridization analysis revealed that the four independent ant::Mu d1(Ap lac) fusions we isolated spanned an approximately 5-kilobase region and that all were transcribed in the same direction, counterclockwise on the E. coli genetic map.     

Role of the chlC gene in formation of the formate-nitrate reductase pathway in Escherichia coli.

Five temperature-sensitive chlC mutants were isolated from Escherichia coli by the technique of localized mutagenesis. All of the mutants produced severely reduced levels of both nitrate reductase and formate dehydrogenase when grown at 43 degrees C. In three of the mutants, the nitrate reductase activity produced at the permissive temperature was shown to be thermolabile compared with the activity produced by the parent wild-type strain, both in membrane preparations and in preparations released from the membrane by deoxycholate. In each case, formate dehydrogenase activity was similar to the wild-type activity in its stability to heat. It is concluded that the chlC gene codes for at least one of the polypeptide chains of nitrate reductase and that the chlC mutations affect indirectly the formation of formate dehydrogenase.     

chlC (nar) operon of Escherichia coli includes structural genes for alpha and beta subunits of nitrate reductase.

The synthesis of the alpha and beta subunits of nitrate reductase by 20 chlC::Tn5 insertion mutants of Escherichia coli was determined by immune precipitation of the subunits from fractions of cell extracts. Only two of the mutants produced either subunit in detectable amounts; these two accumulated the alpha subunit, but no beta subunit. In both cases the alpha subunit was present in the cytosolic fraction, in contrast to wild-type cells, in which both subunits are present mainly in the membrane fraction. EcoRI restriction fragments containing the Tn5 inserts from five of the mutants were cloned into pBR322. The insertions were localized on two contiguous EcoRI fragments spanning a 5.6-kilobase region that overlapped the contiguous ends of the two fragments. An insertion that permitted alpha subunit formation defined one end of the 5.6-kilobase region. The results indicated that the genes encoding the alpha and beta subunits of nitrate reductase were part of a chlC (nar) operon that is transcribed in the direction alpha leads to beta.     

The number of accessible SH-groups in Escherichia coli membrane vesicles is increased by ATP or by formate

The number of accessible SH-groups was determined in membrane vesicles prepared from Escherichia coli growing in fermentation conditions at slightly alkaline pH on glucose with or without added formate. Addition of ATP or formate to the vesicles caused a approximately 1.4-fold increase in the number of accessible SH-groups. The increase was inhibited by treatment with N-ethylmaleimide or the presence of the F(0)F(1)-ATPase inhibitors N,N(')-dicyclohexylcarbodiimide or sodium azide. The increase in accessible SH-groups was also absent in strains with the ATP synthase operon deleted or with the single F(0) domain cysteine Cysb21 changed to Ala. Using hyc and hyf mutants, it was shown that the increase was also largely dependent on hydrogenase 4 or hydrogenase 3, main components of formate hydrogen lyase, when bacteria were grown in the absence or presence of added formate. These results suggest a relationship between the F(0)F(1)-ATP synthase and hydrogenase 4 or hydrogenase 3 under fermentation conditions.     

Biochemical and genetic studies with nitrate reductase C-gene mutants of Escherichia coli

Summary Twenty-eight narC (chlC) mutants of Escherichia coli were isolated and characterised by their resistance to chlorate, inability to use nitrate as terminal electron acceptor and positive gas reaction. The extent of gas production by the majority of mutants was almost normal but quantitative differences ranging from 40 to 100% of wild-type activity were found. Biochemical studies showed that all the mutants lacked nitrate reductase, decreasing gas production was correlated with a simultaneous decrease in formate dehydrogenase activity and the lowest gas production was due to deficiencies in formate dehydrogenase and hydrogenase. The position of narC relative to other loci was determined as: purB ... hemA ... narC ... supIII,C ... galU ... att80 ... tonB ... trp ... cysB by transduction analysis, and the mutant sites of 6 strains representing the complete range of gas reactions were clustered at this position. It is suggested that narC is the structural gene for nitrate reductase and the variations in phenotype may be due to polarity effects on neighbouring genes specifying components of the formate hydrogenlyase system. Transduction of narC by 80 could not be detected but an effect of galU ^– on phage P1kc susceptibility was demonstrated.     

Oxygen-regulated stimulons of Salmonella typhimurium identified by Mu d(Ap lac) operon fusions

Using the technique of Mu d1(Ap lac)-directed lacZ operon fusions, several oxygen-regulated genetic loci were identified in Salmonella typhimurium. Thirteen anaerobically inducible and six aerobically inducible operon fusions were identified. Based on control by the oxrA and oxrB regulatory loci, the anti-lacZ fusions were grouped into three classes: class I loci were regulated by both oxr loci, class II genes were regulated by oxrA only, and class III loci were not affected by either regulatory locus. Several of the anti-lacZ fusions required growth in complex medium before they exhibited the inducible phenotype. While the expression of some of these loci was repressed when organisms were grown in nitrate, others were stimulated by nitrate. Fusions into the hyd and phs loci were identified among the isolated anti-lacZ fusions. Six oxygen-inducible (oxi) operon fusions were also identified. Two of the oxi loci mapped near oxygen-regulatory loci: oxiC near oxrA and oxiE near oxyR. However, neither fusion appeared to occur within the regulatory locus. The data presented serve to further define the aerobic and anaerobic stimulons of S. typhimurium but indicate additional regulatory circuits above those already defined.