Characterization of a plasmid carrying the Escherichia coli K-12 entD, fepA, fes, and entF genes

Abstract The plasmid pMS101 carries Escherichia coli K-12 genes ( entD, fes, entF ) essential for enterochelin-mediated iron transport [Laird, A.J. and Young, I.G., Gene 11 (1980) 359–366]. We have further characterized pMS101 and shown that it also contains the gene ( fepA ) for the 81 000 Da outer membrane ferrienterochelin receptor. Subcloning experiments in conjunction with complementation and maxicell studies demonstrated the gene order to be entD fepA fes entF . The entF - and fes -encoded polypeptides were found to be 115 000 and 42 000-Da soluble proteins, respectively. Plasmid-borne enterochelin cluster genes were overexpressed in iron-deficient conditions and their products were undetectable under iron-replete conditions.     

The entD gene of the Escherichia coli K12 enterobactin gene cluster

The Escherichia coli entD gene encodes a product necessary for the synthesis of the iron-chelating and transport molecule enterobactin (Ent); cells harbouring entD mutations fail to grow in iron-deficient environments. For unknown reasons, it has not been possible to identify the entD product. The nucleotide sequence of the entD region has now been determined. An open reading frame extending in the same direction as the adjacent fepA gene and capable of encoding an approximately 24 kDa polypeptide was found; it contained a high percentage of rare codons and two possible translational start sites. Complementation data suggested that EntD proteins truncated at the carboxy terminus retain some activity. Two REP sequences were present upstream of entD and an IS186 sequence was observed downstream. RNA dot-blot hybridizations demonstrated that entD is transcribed from the strand predicted by the sequencing results. An entD-lacZ recombinant plasmid was constructed and shown to express low amounts of a fusion protein of the anticipated size (approximately 125 kDa). The evidence suggests a number of possible explanations for difficulties in detecting the entD product. Sequence data indicate that if entD has its own promoter, it is weak; the REP sequences suggest that entD mRNA may be destabilized; and translation may be slow because of the frequency of rare codons and a possible unusual start codon (UUG). The data are also consistent with previous evidence that the entD product is unstable.     

High-level synthesis in Escherichia coli of the SV40 small-t antigen under control of the bacteriophage lambda pL promoter

Several plasmids were constructed in which the SV40 small-t antigen gene was inserted in close proximity downstream from the thermoinducible leftward promoter (pL) of bacteriophage lambda. Upon temperature induction the best of our constructions expressed a small-t-related 19 000-dalton polypeptide in an amount corresponding to approx. 2.5% of total de novo protein synthesis. This 19 000-dalton protein was identified as small-t by specific immunoprecipitation with anti-T serum and by two-dimensional fingerprint analysis. In addition to the 19 000-dalton product, representative plasmids expressed fairly large amounts (up to 7% of total de novo protein synthesis) of a protein with an apparent Mr of 14 500. This 14 500-dalton polypeptide was shown to be related to authentic small-t. Presumably the secondary structure of the mRNA starting at pL is such that translation initiation at an internal AUG codon of the small-t gene is favored over initiation at the true initiating codon.     

Identification of the AraE transport protein of Escherichia coli.

1. Two arabinose-inducible proteins are detected in membrane preparations from strains of Escherichia coli containing arabinose-H+ (or fucose-H+) transport activity; one protein has an apparent subunit relative molecular mass (Mr) of 36 000-37 000 and the other has Mr 27 000. 2. An araE deletion mutant was isolated and characterized; it has lost arabinose-H+ symport activity and the arabinose-inducible protein of Mr 36 000, but not the protein of Mr 27 000. 3. An araE+ specialized transducing phage was characterized and used to re-introduce the araE+ gene into the deletion strain, a procedure that restores both arabinose-H+ symport activity and the protein of Mr 36,000. 4. N-Ethylmaleimide inhibits arabinose transport and partially inhibits arabinose-H+ symport activity. 5. N-Ethylmaleimide modifies an arabinose-inducible protein of Mr 36 000-38 000, and arabinose protects the protein against the reagent. 6. These observations identify an arabinose-transport protein of Escherichia coli as the product of the araE+ gene. 7. The protein was recognized as a single spot staining with Coomassie Blue after two-dimensional gel electrophoresis.     

The FER gene is evolutionarily conserved and encodes a widely expressed member of the FPS/FES protein-tyrosine kinase family.

We have recently isolated human and rat cDNAs (designated FER and flk, respectively) which encode nonreceptor protein-tyrosine kinases which are very similar to one another and related in sequence and domain structure to the c-fps/fes gene product. We show that FER and flk are human and rat counterparts of an evolutionarily conserved gene, hereafter termed FER regardless of species. The human and rat FER genes encode a widely expressed 94-kilodalton protein-tyrosine kinase which is antigenically related to the fps/fes protein-tyrosine kinase. The structural and antigenic similarities between the FER and fps/fes proteins suggest that they are members of a new family of nonreceptor protein-tyrosine kinases.     

Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: relationship between B. subtilis sfpo and Escherichia coli entD genes.

In response to iron deprivation, Bacillus subtilis secretes a catecholic siderophore, 2,3-dihydroxybenzoyl glycine, which is similar to the precursor of the Escherichia coli siderophore enterobactin. We isolated two sets of B. subtilis DNA sequences that complemented the mutations of several E. coli siderophore-deficient (ent) mutants with defective enterobactin biosynthesis enzymes. One set contained DNA sequences that complemented only an entD mutation. The second set contained DNA sequences that complemented various combinations of entB, entE, entC, and entA mutations. The two sets of DNA sequences did not appear to overlap. AB. subtilis mutant containing an insertion in the region of the entD homolog grew much more poorly in low-iron medium and with markedly different kinetics. These data indicate that (i) at least five of the siderophore biosynthesis genes of B. subtilis can function in E. coli, (ii) the genetic organization of these siderophore genes in B. subtilis is similar to that in E. coli, and (iii) the B. subtilis entD homolog is required for efficient growth in low-iron medium. The nucleotide sequence of the B. subtilis DNA contained in plasmid pENTA22, a clone expressing the B. subtilis entD homolog, revealed the presence of at least two genes. One gene was identified as sfpo, a previously reported gene involved in the production of surfactin in B. subtilis and which is highly homologous to the E. coli entD gene. We present evidence that the E. coli entD and B. subtilis sfpo genes are interchangeable and that their products are members of a new family of proteins which function in the secretion of peptide molecules.     

Secretion, but not overall synthesis, of catecholate siderophores contributes to virulence of extraintestinal pathogenic Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) use siderophores to sequester iron during infection. Enterobactin and salmochelins are catecholate siderophores produced by some ExPEC strains and other pathogenic enterobacteria. Siderophore export and synthesis mutants of avian ExPEC strain χ7122 were tested in a chicken infection model. In single-strain infections, siderophore-negative (ΔentDΔiuc), ΔentS and ΔentSΔiroC export mutants were attenuated in tissues and blood, whereas the ΔiroC export mutant was only attenuated in blood. Interestingly, the ΔentD mutant, producing only aerobactin, retained full virulence, and loss of entD in the ΔentSΔiroC mutant restored virulence. LC-MS/MS quantification of siderophores in export mutants demonstrated that loss of entS impaired enterobactin and mono-glucosylated enterobactin secretion, whereas loss of iroC impaired di- and tri-glucosylated enterobactin secretion. Loss of entS and/or iroC resulted in intracellular accumulation and increased secretion of siderophore monomers. Catecholate siderophore export mutants also demonstrated decreased fitness in a co-challenge infection model. By contrast, catecholate siderophore synthesis mutants (ΔentD and ΔiroB) competed as well as the wild-type strain. Results establish that EntS and IroC mediate specific export of catecholate siderophores and the role of these exporters for ExPEC virulence is contingent on enterobactin synthesis, which is not required when other siderophores like aerobactin are functional.     

The human c-fps/fes gene product expressed ectopically in rat fibroblasts is nontransforming and has restrained protein-tyrosine kinase activity.

A 13-kilobase EcoRI genomic restriction fragment containing the human c-fps/fes proto-oncogene locus was expressed transiently in Cos-1 monkey cells and stably in Rat-2 fibroblasts. In both cases, human c-fps/fes directed synthesis of a 92-kilodalton protein-tyrosine kinase (p92c-fes) indistinguishable from a tyrosine kinase previously identified with anti-fps antiserum which is specifically expressed in human myeloid cells. Transfected Rat-2 cells containing approximately 50-fold more human p92c-fes than is found in human leukemic cells remained morphologically normal and failed to grow in soft agar. Synthesis of p92c-fes in this phenotypically normal line exceeded that of the P130gag-fps oncoprotein in a v-fps-transformed Rat-2 line. Despite this elevated expression, human p92c-fes induced no substantial increase in cellular phosphotyrosine and was not itself phosphorylated on tyrosine. In contrast, p92c-fes immunoprecipitated from these Rat-2 cells or expressed as an enzymatically active fragment in Escherichia coli from a c-fps/fes cDNA catalyzed tyrosine phosphorylation with an activity similar to that of v-fps/fes polypeptides. Thus, p92c-fes is not transforming when ectopically overexpressed in Rat-2 fibroblasts. This lack of transforming activity correlates with a restriction imposed on the kinase activity of the normal c-fps/fes product in vivo which is apparently lifted for v-fps/fes oncoproteins, suggesting that regulatory interactions within the host cell modify fps/fes protein function and normally restrain its oncogenic potential.     

Isolation, characterization and regulation of expression of the nuclear genes for the core II and Rieske iron-sulphur proteins of the yeast ubiquinol-cytochrome c reductase

Cloning and mapping of the yeast nuclear genes for the core II (Mr 40 000) and Rieske iron-sulphur proteins of the mitochondrial ubiquinol-cytochrome c reductase, and comparison with the genomic regions in nuclear DNA from which they are derived, show that the genes are likely to be present in single copies and that they are not closely linked. They have been reintroduced into yeast cells on multi-copy plasmids and, similar to results obtained for the Mr 11 000 subunit [Van Loon et al., EMBO J. 2 (1983) 1765-1770], increase in the dosage of either gene prompts discoordinate synthesis of the encoded protein. Quantitative analysis of transformants carrying extra copies of the gene for the iron-sulphur protein shows that messenger RNA level, rate of synthesis and steady-state concentration of the protein correlate well with each other. This indicates that its level, in contrast to that of the Mr 11 000 subunit, is only determined by the concentration of its messenger RNA. Over-production of these proteins does not interfere with mitochondrial function as judged from growth rates of transformed cells on non-fermentable media. The excess Mr 40 000 protein is imported into the mitochondrion, showing that import of this subunit is not obligatorily coupled to complex assembly.     

Purification and characterization of the indole-3-glycerolphosphate synthase/anthranilate synthase complex of Saccharomyces cerevisiae

The indole-3-glycerolphosphate synthase/anthranilate synthase complex from Saccharomyces cerevisiae was purified to apparent homogeneity. The native complex with Mr approximately equal to 130 000 consists of two different subunits, the TRP2 gene product with Mr = 64 000 and the TRP3 gene product with Mr = 58 000. The larger polypeptide was identified as anthranilate synthase and is active in vitro with ammonia as cosubstrate without need of complex formation. The smaller polypeptide carries both glutamine amidotransferase activity and indole-3-glycerolphosphate synthase activity. Various steady-state kinetic parameters as well as the amino acid composition of the two polypeptides were determined.     

Characterization and androgenic regulation of major mRNAs coding for epididymal proteins in a lizard (Lacerta vivipara)

During the reproductive period (spring) under the control of testosterone the epididymis of the viviparous lizard secretes a group of major proteins with an approximate Mr of 19,000 named L protein(s). These proteins are recognized by a specific immunoserum and bind to the heads of spermatozoa. During spring, translation in reticulocyte lysate of RNA from secreting epididymis (stage 6) produced 5 immunoprecipitable bands with Mr values from 21,500 to 25,000. Such synthesis is undetectable during sexual rest in summer (stage 1). The 5 bands disappear when translation is performed in the presence of dog pancreas microsomes although a new band of Mr 19 000 becomes prominent. This suggests that synthesis of L protein involves two steps, i.e. synthesis of precursors (L preproteins) followed by a maturation process. At least 11 translation products (including L-preproteins) are involved in annual variations that follow the differentiation of the epididymal epithelial cells and their androgen dependency was studied by castration and in-vitro stimulation by testosterone. In these conditions, testosterone is able to control accumulation of RNA corresponding to L preproteins and to a translation product of Mr 29 000.     

Nucleotide sequence of the gene ereA encoding the erythromycin esterase in Escherichia coli

We have cloned and determined the nucleotide sequence of the gene ereA of plasmid pIP1100 which confers high-level resistance to erythromycin (Em) in Escherichia coli. The gene was defined by initiation and termination codons and by in vitro insertion-inactivation into an open reading frame (ORF) of 1032 bp corresponding to a product with an Mr of 37 765. However, the enzyme, an Em esterase, displayed an apparent Mr of 43 000 upon electrophoresis of a minicell extract on the SDS-polyacrylamide gels. The G + C content (50.5%) of the gene ereA and the preferential codon usage in its ORF suggest that this resistance determinant should be indigenous to E. coli.     

Mu-induced polarity in the Escherichia coli K-12 ent gene cluster: evidence for a gene (entG) involved in the biosynthesis of enterochelin.

A strain of Escherichia coli K-12 has been isolated that carries a Mu bacteriophage-induced mutation in the ent gene cluster. Nutritional tests together with examination of the compounds accumulated by the mutant strain indicated that the mutant was blocked both in the synthesis of 2,3-dihydroxy-benzoate and its subsequent conversion into enterochelin. Enzymic complementation assays of the mutant with several mutants each affected in one of the ent genes showed that the Mu-induced mutant was entA-, entB-, entC+, entD+, entE+, and entF+. Since the mutant produced the entD, entE, and entF gene products but was unable to produce enterochelin from 2,3-dihydroxybenzoate, it must therefore be affected in an additional protein concerned with this conversion. It is therefore postulated that the Mu-induced mutation affects a previously unrecognized gene, entG. Genetic experiments indicate that the mutation in strain AN462 which affects the three ent genes is the result of a single insertion of Mu in the ent gene cluster. This polarity mutant therefore provides evidence that three of the ent genes are part of an operon.