Escherichia coli genes whose products are involved in selenium metabolism.

Mutants of Escherichia coli were isolated which were affected in the formation of both formate dehydrogenase N (phenazine methosulfate reducing) (FDHN) and formate dehydrogenase H (benzylviologen reducing) (FDHH). They were analyzed, together with previously characterized pleiotropic fdh mutants (fdhA, fdhB, and fdhC), for their ability to incorporate selenium into the selenopolypeptide subunits of FDHN and FDHH. Eight of the isolated strains, along with the fdhA and fdhC mutants, maintained the ability to selenylate tRNA, but were unable to insert selenocysteine into the two selenopolypeptides. The fdhB mutant tested had lost the ability to incorporate selenium into both protein and tRNA. fdhF, which is the gene coding for the 80-kilodalton selenopolypeptide of FDHH, was expressed from the T7 promoter-polymerase system in the pleiotropic fdh mutants. A truncated polypeptide of 15 kilodaltons was formed; but no full-length (80-kilodalton) gene product was detected, indicating that translation terminates at the UGA codon directing the insertion of selenocysteine. A mutant fdhF gene in which the UGA was changed to UCA expressed the 80-kilodalton gene product exclusively. This strongly supports the notion that the pleiotropic fdh mutants analyzed possess a lesion in the gene(s) encoding the biosynthesis or the incorporation of selenocysteine. The gene complementing the defect in one of the isolated mutants was cloned from a cosmid library. Subclones were tested for complementation of other pleiotropic fdh mutants. The results revealed that the mutations in the eight isolates fell into two complementation groups, one of them containing the fdhA mutation. fdhB, fdhC, and two of the new fdh isolates do not belong to these complementation groups. A new nomenclature (sel) is proposed for pleiotropic fdh mutations affecting selenium metabolism. Four genes have been identified so far: selA and selB (at the fdhA locus), selC (previously fdhC), and selD (previously fdhB).     

Developmental regulation of the gene for formate dehydrogenase in Neurospora crassa.

We have isolated and characterized a gene, fdh, from Neurospora crassa which is developmentally regulated and which produces formate dehydrogenase activity when expressed in Escherichia coli. The gene is closely linked (less than 0.6 kb apart) to the leu-5 gene encoding mitochondrial leucyl-tRNA synthetase; the two genes are transcribed convergently from opposite strands. The expression patterns of these genes differ: fdh mRNA is found only during conidiation and early germination and is not detectable during mycelial growth, while leu-5 mRNA appears during germination and mycelial growth. The structure of the fdh gene was determined from the sequence of cDNA and genomic DNA clones and from mRNA mapping studies. The gene encodes a 375-amino-acid-long protein with sequence similarity to NAD-dependent dehydrogenases of the E. coli 3-phosphoglycerate dehydrogenase (serA gene product) subfamily. In particular, there is striking sequence similarity (52% identity) to formate dehydrogenase from Pseudomonas sp. strain 101. All of the residues thought to interact with NAD in the crystal structure of the Pseudomonas enzyme are conserved in the N. crassa enzyme. We have further shown that expression of the N. crassa gene in E. coli leads to the production of formate dehydrogenase activity, indicating that the N. crassa gene specifies a functional polypeptide.     

The cobalamin (coenzyme B12) biosynthetic genes of Escherichia coli.

The enteric bacterium Escherichia coli synthesizes cobalamin (coenzyme B12) only when provided with the complex intermediate cobinamide. Three cobalamin biosynthetic genes have been cloned from Escherichia coli K-12, and their nucleotide sequences have been determined. The three genes form an operon (cob) under the control of several promoters and are induced by cobinamide, a precursor of cobalamin. The cob operon of E. coli comprises the cobU gene, encoding the bifunctional cobinamide kinase-guanylyltransferase; the cobS gene, encoding cobalamin synthetase; and the cobT gene, encoding dimethylbenzimidazole phosphoribosyltransferase. The physiological roles of these sequences were verified by the isolation of Tn10 insertion mutations in the cobS and cobT genes. All genes were named after their Salmonella typhimurium homologs and are located at the corresponding positions on the E. coli genetic map. Although the nucleotide sequences of the Salmonella cob genes and the E. coli cob genes are homologous, they are too divergent to have been derived from an operon present in their most recent common ancestor. On the basis of comparisons of G+C content, codon usage bias, dinucleotide frequencies, and patterns of synonymous and nonsynonymous substitutions, we conclude that the cob operon was introduced into the Salmonella genome from an exogenous source. The cob operon of E. coli may be related to cobalamin synthetic genes now found among non-Salmonella enteric bacteria.     

The nucleotide sequence of recG, the distal spo operon gene in Escherichia coli K-12.

A gene is identified in the Escherichia coli K-12 spo operon as recG. Previously identified genes in the spo operon were spoS, alias rpoZ, encoding the omega (omega) subunit of RNA polymerase, as well as the spoT gene encoding the major cellular source of guanosine 3',5'-bispyrophosphate hydrolase activity. The gene order within the spo operon is: spoS (rpoZ), spoT, spoU, recG. A convergent gltS gene is present beyond the spo operon. Mutants bearing recG deletion-insertion alleles display mild sensitivities to both ultraviolet irradiation and to mitomycin C, which is expected to be due to a known recG insertion allele. Deletion-insertion mutations in upstream operon genes (spoT and spoU) show polar effects on these assays of recG function. The deduced 693-amino acid (aa) RecG sequence shows a weak, but significant, relatedness to aa sequence motifs previously reported for putative helicases involved in replication, recombination, and DNA repair.     

Efficient degradation of trichloroethylene by a hybrid aromatic ring dioxygenase.

Engineering of hybrid gene clusters between the toluene metabolic tod operon and the biphenyl metabolic bph operon greatly enhanced the rate of biodegradation of trichloroethylene. Escherichia coli cells carrying a hybrid gene cluster composed of todC1 (the gene encoding the large subunit of toluene terminal dioxygenase in Pseudomonas putida F1), bphA2 (the gene encoding the small subunit of biphenyl terminal dioxygenase in Pseudomonas pseudoalcaligenes KF707), bphA3 (the gene encoding ferredoxin in KF707), and bphA4 (the gene encoding ferredoxin reductase in KF707) degraded trichloroethylene much faster than E. coli cells carrying the original toluene dioxygenase genes (todC1C2BA) or the original biphenyl dioxygenase genes (bphA1A2A3A4).     

Nitrate assimilation gene cluster from the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

A region of the genome of the filamentous, nitrogen-fixing, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 that contains a cluster of genes involved in nitrate assimilation has been identified. The genes nir, encoding nitrite reductase, and nrtABC, encoding elements of a nitrate permease, have been cloned. Insertion of a gene cassette into the nir-nrtA region impaired expression of narB, the nitrate reductase structural gene which together with nrtD is found downstream from nrtC in the gene cluster. This indicates that the nir-nrtABCD-narB genes are cotranscribed, thus constituting an operon. Expression of the nir operon in strain PCC 7120 is subjected to ammonium-promoted repression and takes place from an NtcA-activated promoter located 460 bp upstream from the start of the nir gene. In the absence of ammonium, cellular levels of the products of the nir operon are higher in the presence of nitrate than in the absence of combined nitrogen.