The gene-protein database of Escherichia coli is both an index relating a gene to its protein product on two-dimensional gels, and a catalog of information about the function, regulation, and genetics of individual proteins obtained from two-dimensional gel analysis or collated from the literature. Edition 5 has 102 new entries--a 15% increase in the number of annotated two-dimensional gel spots. The large increase in this edition was accomplished in part by the use of a new method for expression analysis of ordered segments of the E. coli genome, which has resulted in linking 50 gel spots to their genes (or open reading frames) and another 45 to specific regions of the chromosome awaiting the availability of DNA sequence information. Communication of information from the scientific community resulted in additional identifications and regulatory information. To increase accessibility of the database it has been placed in the repository at the National Center for Biotechnology Information (NCBI) at the National Library of Medicine under the name ECO2DBASE. It will be updated twice yearly. This edition of the gene-protein database is estimated to contain entries for one-sixth of the protein-encoding genes of E. coli.

No abstract

Production of active hydrogenase by Escherichia coli requires several gene products. One of the essential genes, hydB, is encoded by a DNA fragment of approximately 1.0 kilobase. The hydB gene produced a protein with an apparent molecular weight of 32,000. The hydB gene was transcribed only under anaerobic conditions. Oxygen and nitrate repressed transcription of this gene. hydB gene transcription also required sigma 60, the product of the rpoN gene.

Fig. 1, and many of these plasmids were described previously (23). Strains SE-1174 is a hyd+ fhlA102::Tn1O derivative of strain SE-38 (hydB103). This strain was constructed by transducing strain SE-38 to hyd+ by using bacteriophage P1 grown in an E. coli mutant pool obtained after random mutagenesis with transposon TnJO. Hyd+ transductants of strain SE-38 were tested for fhIA character and tetracycline resistance. The resulting strain, JhlA::TnlO, was named strain SE-1174, and its genotype was fhIA102. Strain SE-9, parent of strain SE-68, was previously described as carrying hydB108 andfdv (23). The results presented here show that the genotype of strain SE-68 is hydB108 and JhlA101.All other methods, including enzyme assays, were as described previously (14,23,24

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.

The novel transcription system of bacteriophage T7 was used to express Escherichia coli genes preferentially with a new low-copy-number plasmid vector, pFN476, to minimize toxic gene effects. Selected E. coli chromosomal fragments from an ordered genomic library (Y. Kohara, K. Ikiyama, and K. Isono, Cell 50:495-508, 1987) were recloned into this vector, and their genes were preferentially expressed in vivo utilizing its T7 promoter. The protein products were analyzed by two-dimensional gel electrophoresis. By using DNA sequence information, the gel migration was predicted for the protein products of open reading frames from these segments, and this information was used to identify gene products visualized as spots on two-dimensional gels. Even in the absence of DNA sequence information, this approach offers the opportunity to identify all gene products of E. coli and map their genes to within 10 kb on the E. coli genome; with sequence information, this approach can produce a definitive expression map of the E. coli genome.

A new gene whose product is essential for production of all three hydrogenase isoenzymes in Escherichia coli has been identified. This gene, termed hydF, mapped at 59 min in the E. coli chromosome and resided next to the hydB gene. The map order of these genes was hydE, hydF, hydB, fhUA, and fdv. The hydF gene was transcribed from its own promoter and coded for a protein with an apparent molecular weight of 43,000 to 44,000. Expression of the hydF operon was enhanced by anaerobic growth conditions. Partial products of the hydF gene were capable of supporting various levels of hydrogenase activity in a hydF mutant in the presence of the fllA gene product, also produced from multicopy plasmids. In the presence of a second mutation in an unidentified, unlinked gene, hydrogenase activity in a hydF mutant was restored by plasmids which carried incomplete hydF and hydB+ genes. These results suggest that the products of hydF andlhlA interact with each other and with yet one other gene product.

Escherichja 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-Ill 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'eptablished 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-kilobasepair DNA (pSE-130) or fragments derived from this DNA in different orientations inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, straip K-10, but only during an early induction period. These results, based on complementation analysjs 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, ffl4 andfdv are also njeded, All four genes map between 58 and 59 min in the E. coli chromosome. SE-5, SE-6, SE-7, and SE-6}, respectively. Table 1 provides the pedigree and hyd genotype of the hydrogenase-defective mutant strains of E. coli used in this study, and these mutant strains were isolated by E$V selection (22).Media and growth conditions. Luria broth, glucose minimal medium, and HF have been described previously (22).Ampicillin and tetracycliqp, when present, were added to the medium after autoclaving to a final concentration of 100 and 15 p.g/ml, respectively. Cultures for enzyme assays were 353 on July 31, 2020 by guest