Sequence of the fhuE outer-membrane receptor gene of Escherichia coli K12 and properties of mutants

The fhuE gene of Escherichia coli codes for an outer-membrane receptor protein required for the uptake of iron(III) via coprogen, ferrioxamine B and rhodotorulic acid. The amino acid sequence, deduced from the nucleotide sequence, consisted of 729 residues. The mature form, composed of 693 residues, has a calculated molecular weight of 77,453, which agrees with the molecular weight of 76,000 determined by polyacrylamide gel electrophoresis. The FhuE protein contains four regions of homology with other TonB-dependent receptors. A valine to proline exchange in the 'TonB box' abolished transport activity. Phenotypic revertants with substitutions of arginine, glutamine, or leucine at the valine position exhibited increasing iron-coprogen transport rates. Point mutations resulting in the replacement of glycine (127) in the second homology region with either alanine, aspartate, valine, asparagine or histidine exhibited decreased transport rates (listed in descending order). A truncated FhuE protein lacking 24 amino acids at the C-terminal end was exported to the periplasm but failed to be inserted into the outer membrane.     

Characterization of the mcrBC region of Escherichia coli K-12 wild-type and mutant strains.

We have carried out an analysis of the Escherichia coli K-12 mcrBC locus in order to (1) elucidate its genetic organization, (2) to identify the proteins encoded by this region, and (3) to characterize their involvement in the restriction of DNA containing methylated cytosine residues. In vitro expression of recombinant plasmids carrying all or portions of the mcrBC region revealed that the mcrB and mcrC genes are organized as an operon. The mcrBC operon specifies five proteins, as evident from parallel in vitro and in in vivo expression studies. Three proteins of 53, 35 and 34 kDa originate from mcrB expression, while two proteins of 37 and 16 kDa arise from mcrC expression. Products of both the mcrB and mcrC genes are required to restrict the methylated substrate DNA used in this study. We also determined the nature of mutant mcrBC loci in comparison to the E. coli K-12 wild-type mcrBC locus. A major goal of these studies was to clarify the nature of the mcrB-1 mutation, which is carried by some strains employed in previous analyses of the E. coli K-12 McrBC system. Based on our analyses the mutant strains investigated could be divided into different complementation groups. The mcrB-1 mutation is a nonsense or frameshift mutation located within mcrB. It causes premature termination of mcrB gene product synthesis and reduces the level of mcrC gene expression. This finding helps to understand an existing conflict in the literature. We also describe temperature-sensitive McrA activity in some of the strains analysed and its relationship to the previously defined differences in the tolerance levels of E. coli K-12 mcrBC mutants to cytosine methylation.     

Uroporphyrin- and coproporphyrin I-accumulating mutant of Escherichia coli K12.

A new type of haem-deficient mutant was isolated in Escherichia coli K12 by neomycin selection. The mutant was deficient in uroporphyrinogen III cosynthase activity as indicated by the accumulation of uroporphyrin I and coproporphyrin. The mapping of the corresponding hemD gene by P1-mediated transduction showed that the new gene was located between ilv and cya, at min 83 on the chromosomal map of Escherichia coli K12.     

Sequence of the ebgA gene of Escherichia coli: comparison with the lacZ gene

We have sequenced the ebgA (evolved beta-galactosidase) gene of Escherichia coli K12. The sequence shows 50% nucleotide identity with the E. coli lacZ gene, demonstrating that the two genes are related by descent from a common ancestral gene. Comparison of the two sequences suggests that the ebgA gene has recently been under selection. A significant excess of identical, rather than synonymous, codons used to encode identical amino acids at the same positions in the aligned sequences implies that some form of selection is operating directly at the DNA level. This selection is independent of, and in addition to, selection based on codon usage or on function of the gene products.      

Multiple regulator gene control of the galactose operon in Escherichia coli K-12

Previous studies showed that nonsense mutations in either of two genes (capR or capS) or an undefined mutation in a third gene (capT) led to pleiotropic effects: (i) increased capsular polysaccharide synthesis (mucoid phenotype); (ii) increased synthesis of enzymes specified by at least four spatially separated operons involved in synthesis of capsular polysaccharide including the product of the galE gene, UDP-galactose-4-epimerase (EC 5.1.3.2) in capR mutants. The present study demonstrated that the entire galactose (gal) operon (galE, galT, and galK) is derepressed by mutations in either the capR or the capT genes, but not by mutation in capS. Double mutants (capR9 capT) were no more derepressed than the capR9 mutant, indicating that capR9 and capT regulate the gal operon via a common pathway. Isogenic double mutants containing either galR(+), galR(-), galR(s), or galO(c) in combination with either capR(+) or capR9 were prepared and analyzed for enzymes of the gal operon. The results demonstrated that capR9 caused derepression as compared to capR(+) in all of the combinations. Strains with a galR(s) mutation are not induced, for the gal operon, by any galactose compound including d-fucose, and this was confirmed in the present study using d-fucose. Nevertheless, the derepression of galR(s) capR9 compared to galR(s) capR(+) was four- to sixfold. The same derepression was observed when galR(+)capR9 was compared to galR(+)capR(+). The data eliminate the explanation that internal induction of the gal operon by a galactose derivative was causing increased gal operon enzyme synthesis in capR or capT mutants. Furthermore, the same data suggest that the galR and capR genes are acting independently to derepress the gal operon. A modified model for the structure of the gal operon is proposed to explain these results. The new feature of the model is that two operator sites are suggested, one to combine with the galR repressor and one to combine with the capR repressor.     

磷脂酰丝氨酸合成酶基因pss的克隆与表达

磷脂酰丝氨酸合成酶能催化转酯反应,是定向合成特定磷脂类物质特别是磷脂酰丝氨酸的工具酶,但出发菌株产量低,很大程度上限制了酶法合成磷脂酰丝氨酸的工业化应用。利用表达载体pET-22b,实现了大肠杆菌磷脂酰丝氨酸合成酶基因在大肠杆菌BL21(DE3)中的同源高效表达。利用镍亲和柱对表达产物进行纯化,并用HPLC法对纯化后的重组酶的活力进行检测。结果表明,目的蛋白可在短时间内进行大量表达,蛋白含量是出发菌株的100倍,同时经6h的转酯反应转化率达到33%,重组磷脂酰丝氨酸合成酶活力达到69U/mg蛋白。     

A DNA replication gene maps near terC in Escherichia coli K12

Summary Temperature-sensitive mutants that filamented at the non-permissive temperature were isolated by specific mutagenesis of the terminus region of the Escherichia coli chromosome. Two of them, mapping at about 35 min, failed to divide due to inhibition of DNA replication. Further characterization indicated that these mutants are temperature-sensitive for DNA chain elongation.     

Expression of cDNAs encoding wild-type and mutant neuromodulins in Escherichia coli: comparison with the native protein from bovine brain

Murine cDNA that encodes neuromodulin, a neurospecific calmodulin binding protein, was inserted into the plasmid pKK223-3 for expression in Escherichia coli. After being transformed into E. coli strain SG20252 (lon-), the expression vector directed the synthesis of a protein that was recognized by polyclonal antibodies raised against bovine neuromodulin. The recombinant protein expressed in E. coli was found to be tightly associated with insoluble cell material and was extractable only with guanidine hydrochloride or sodium dodecyl sulfate. Following solubilization with guanidine hydrochloride, the protein was purified to apparent homogeneity by a single CaM-Sepharose affinity column step with a yield of 0.2 mg of protein/L of E. coli culture. The availability of the purified recombinant neuromodulin made it possible to answer several specific questions concerning the structure and function of the protein. Despite the fact that murine neuromodulin is 12 amino acid residues shorter than the bovine protein and the recombinant protein expressed in E. coli may lack any posttranslational modifications, the two proteins displayed similar biochemical properties in almost all respects examined. They both had higher affinity for CaM-Sepharose in the absence of Ca2+ than in its presence; they were both phosphorylated in vitro by protein kinase C in a Ca2+- and phospholipid-dependent manner; neither form of the proteins was autophosphorylated, and the phosphorylated form of the proteins did not bind calmodulin. The recombinant neuromodulin and neuromodulin purified from bovine brain had similar, but not identical, affinities of calmodulin, indicating that the palmitylation of the protein that occurs in animal cells is not crucial for calmodulin interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Transport of galactose, glucose and their molecular analogues by Escherichia coli K12.

1. Strains of Escherichia coli K12 were made that are unable to assimilate glucose by the phosphotransferase system, since they lack the glucose-specific components specified by the genes ptsG and ptsM. 2. Derivative organisms lacking the methyl galactoside or galactose-specific transport system were examined for their ability to transport galactose, d-fucose, methyl beta-D-galactoside, glucose, 2-deoxy-D-glucose and methyl alpha-D-glucoside. 3. Galactose, glucose and to a lesser extent fucose are substrates for both transport systems. 4. 2-Deoxyglucose is transported on the galactose-specific but not the methyl galactoside system. 5. The ability of sugars to elicit anaerobic proton transport is associated with the galactose-specific, but not with the methyl galactoside transport activity. Hence a chemiosmotic mechanism of energization is likely to apply to the former but not to the latter. Alternatively the methyl galactoside system may be switched off under certain conditions, which would indicate a novel regulatory mechanism. 6. Details of the procedure for the derivation of strains may be obtained from the authors, and have been deposited as Supplementary Publication SUP 50074 (8 pages at the) British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1977), 161,1.