Biochemical characterization of NfsA, the Escherichia coli major nitroreductase exhibiting a high amino acid sequence homology to Frp, a Vibrio harveyi flavin oxidoreductase.

We identified the nfsA gene, encoding the major oxygen-insensitive nitroreductase in Escherichia coli, and determined its position on the E. coli map to be 19 min. We also purified its gene product, NfsA, to homogeneity. It was suggested that NfsA is a nonglobular protein with a molecular weight of 26,799 and is associated tightly with a flavin mononucleotide. Its amino acid sequence is highly similar to that of Frp, a flavin oxidoreductase from Vibrio harveyi (B. Lei, M. Liu, S. Huang, and S.-C. Tu, J. Bacteriol. 176:3552-3558, 1994), an observation supporting the notion that E. coli nitroreductase and luminescent-bacterium flavin reductase families are intimately related in evolution. Although no appreciable sequence similarity was detected between two E. coli nitroreductases, NfsA and NfsB, NfsA exhibited a low level of the flavin reductase activity and a broad electron acceptor specificity similar to those of NfsB. NfsA reduced nitrofurazone by a ping-pong Bi-Bi mechanism possibly to generate a two-electron transfer product.     

Oxygen-insensitive nitroreductases NfsA and NfsB of Escherichia coli function under anaerobic conditions as lawsone-dependent Azo reductases

Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E. coli and thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.     

Characterization of enteroaggregative Escherichia coli isolates

Forty enteraggregative Escherichia coli (EAggEC) previously characterized by their ability to adhere to HEp-2 cells or/and their hybridization with the 1-kb EAggEC DNA probe were investigated for the presence of adherence factors and heat-stable enterotoxin (EAST1)-encoding genes. Only 45% of the isolates harbored the EAST1-encoding genes as detected by polymerase chain reaction. None of them hybridized with an AAF/II-encoding gene specific DNA probe and 35% (14/40) were positive in a PCR assay using primers specific for aggC, an accessory gene of the AAF/I-encoding operon. Cloning and sequence analysis of the aggA variant from one isolate, EAggEC 457, revealed 68.9% identity between its deduced amino acid sequence and those of the aggA product from the AAF/I-producing reference strain, E. coli 17.2. No major protein subunit was detected at the surface of EAggEC 457 compared to the bacterial surface extract of E. coli 17.2.     

Cloning and expression in Escherichia coli of the dnaK gene of Zymomonas mobilis.

The DnaK protein of Zymomonas mobilis (DnaKz) was identified and found to be 80% identical to the DnaK protein of Escherichia coli on the basis of the sequence of the N-terminal 21 amino acids. The dnaKz gene was cloned and found to be expressed in a thermosensitive dnaK mutant of Escherichia coli. Expression of the foreign gene restored a thermoresistant phenotype but failed to modulate the heat shock response in E. coli.     

Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7

The eae (Escherichia coli attaching and effacing) gene from enteropathogenic Escherichia coli (EPEC) was previously shown to be essential for production of the 'attaching and effacing' histopathology characteristic of EPEC infections (Jerse et al., 1990). We have now cloned the eae gene from enterohaemorrhagic E. coli (EHEC) which, in addition to producing Shiga-like cytotoxins, also produces the attaching and effacing effect. The sequence homology between the EPEC and EHEC sequences was 86% and 83% at the nucleotide and amino acid levels, respectively. The predicted amino acid sequence of the EHEC eae gene shared 31% identity and 51% similarity with invasin of Yersinia pseudotuberculosis. Alignment of the EPEC and EHEC Eae proteins and the Y. pseudotuberculosis and Y. enterocolitica invasins shows striking regions of identity with the greatest divergence at the C-terminal end, the putative receptor-binding portion of invasin.     

Evidence for a new Escherichia coli protein resembling a lysyl-tRNA synthetase.

The amino acid sequence deduced from the nucleotide sequence of an open reading frame adjacent to the frdA gene of Escherichia coli shows 30.5% identity with the C terminus of Escherichia coli lysyl-tRNA synthetases. The three motifs characteristic of aminoacyl-tRNA synthetases of class 2 are recognizable within this sequence.     

大肠杆菌ubiC基因的克隆及序列分析

研究以克隆得到正确序列的大肠杆菌ubiC基因目的,实验通过PCR方法从大肠杆菌基因组中扩增得到了ubiC基因,扩增产物克隆到pUC118载体,转化大肠杆菌JM109,DNA序列分析结果表明克隆得到的大肠杆菌ubiC基因碱基序列正确。     

Cloning and overproduction of biodegradative threonine deaminase from Escherichia coli W strain.

We have cloned the structural gene (tdcB) of biodegradative threonine deaminase from Escherichia coli W strain by utilizing the polymerase chain reaction. The JM109/pUCTDA strain, which was obtained by transforming E. coli JM109 with a vector plasmid (pUCTDA) containing the cloned tdcB gene, produced a large amount of the enzyme corresponding to more than 5% of the total soluble protein. Amino acid sequence analysis of this recombinant enzyme showed that the amino acid sequence is identical to the nucleotide-deduced sequence of biodegradative threonine deaminase from E. coli K-12.     

Overexpression and purification of asparagine synthetase from Escherichia coli.

Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUC18, in E. coli. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15% of the total soluble protein in the E. coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene. Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.     

Homology between the photoreactivation genes of Saccharomyces cerevisiae and Escherichia coli

A cloned fragment of Saccharomyces cerevisiae chromosomal DNA carrying the photoreactivation gene (PHR) has been sequenced. The fragment contains a 1695-bp intronless open reading frame (ORF) coding for a polypeptide of 564 amino acids (aa). The phr gene of Escherichia coli was also sequenced, and the sequence is in agreement with the published data. The yeast PHR gene has a G + C content of 36.2%, whereas 53.7% was found for the E. coli gene. Despite the difference in G + C content there is a 35% homology between the deduced aa sequences. This homology suggests that both genes have originated from a common ancestral gene.     

High level secretion of recombinant staphylokinase into periplasm of Escherichia coli

The staphylokinase (SAK) gene from Staphylococcus aureus NCTC10033 was inserted into an expression vector, pKK-ompA, having a tac promoter and an ompA signal sequence. Escherichia coli JM109 carrying the recombinant plasmid produced and secreted the recombinant SAK (rSAK) at 15ug/ml into periplasm and 5ug/ml to extracellular media, respectively. The rSAK was purified with 59% yield by simple procedures from the periplasm of E. coli. The amino-terminal sequence and human plasminogen activating activity of rSAK were coincided with the authentic SAK.     

Cloning and expression of the Erwinia herbicola tyrosine phenol-lyase gene in Escherichia coli.

The tyrosine phenol-lyase (TPL) gene of Erwinia herbicola was cloned and expressed in Escherichia coli, and the complete nucleotide sequence of the gene determined. The TPL gene comprises 1368 bp, encoding 456 amino acids which have 90% amino acid identity with TPL from Citrobacter freundii. After replacing the 5'-flanking region of the TPL gene with the E. coli lac promoter, TPL protein could be hyperproduced constitutively in E. coli without induction by L-tyrosine.     

Escherichia coli produces a cytoplasmic alpha-amylase, AmyA.

In the gap between two closely linked flagellar gene clusters on the Escherichia coli and Salmonella typhimurium chromosomes (at about 42 to 43 min on the E. coli map), we found an open reading frame whose sequence suggested that it encoded an alpha-amylase; the deduced amino acid sequences in the two species were 87% identical. The strongest similarities to other alpha-amylases were to the excreted liquefying alpha-amylases of bacilli, with > 40% amino acid identity; the N-terminal sequence of the mature bacillar protein (after signal peptide cleavage) aligned with the N-terminal sequence of the E. coli or S. typhimurium protein (without assuming signal peptide cleavage). Minicell experiments identified the product of the E. coli gene as a 56-kDa protein, in agreement with the size predicted from the sequence. The protein was retained by spheroplasts rather than being released with the periplasmic fraction; cells transformed with plasmids containing the gene did not digest extracellular starch unless they were lysed; and the protein, when overproduced, was found in the soluble fraction. We conclude that the protein is cytoplasmic, as predicted by its sequence. The purified protein rapidly digested amylose, starch, amylopectin, and maltodextrins of size G6 or larger; it also digested glycogen, but much more slowly. It was specific for the alpha-anomeric linkage, being unable to digest cellulose. The principal products of starch digestion included maltotriose and maltotetraose as well as maltose, verifying that the protein was an alpha-amylase rather than a beta-amylase. The newly discovered gene has been named amyA. The natural physiological role of the AmyA protein is not yet evident.     

Nucleotide sequence of the ethidium efflux gene from Escherichia coli

The nucleotide sequence of the gene specifying the ethidium efflux system of Escherichia coli has been determined. The translated open reading frame has identified a membrane-bound polypeptide of 110 amino acids (11,960 Da) which shares 42% identity with a staphylococcal protein specifying resistance to ethidium.     

Conversion of NfsB, a minor Escherichia coli nitroreductase, to a flavin reductase similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri, by a single amino acid substitution.

NfsB is an oxygen-insensitive nitroreductase of Escherichia coli with significant amino acid sequence homology to the major flavin reductase (FRase I) of Vibrio fischeri. Here, we show that NfsB is convertible to an FRase I-like flavin reductase three times as active as the authentic FRase I by a single amino acid substitution in the least-conserved region.     

High-level expression of a semisynthetic dam gene in Escherichia coli

We constructed a semisynthetic gene encoding a DNA-adenine-methyltransferase (Dam) that codes for the same amino acid sequence as the wild type (wt) Escherichia coli dam gene. Since for unknown reasons the entire wt sequence, from the start codon to the end of the gene, could not be cloned, a gene was constructed consisting of a chemically synthesized 5' portion and a 3' portion from the E. coli chromosome. Introduction of this semisynthetic gene into a suitable vector allows overproduction of E. coli Dam in mg amounts per liter E. coli culture, with optimum expression of the gene in the vector pJLA503. This plasmid places the target gene under control of the strong, tandemly arranged pR pL promoters from bacteriophage lambda, regulated by a temperature-sensitive lambda repressor. A rapid, two-column purification protocol is described that allows for very fast purification of the protein. The 32-kDa recombinant protein methylates the sequence GATC.     

Actinobacillus pleuropneumoniae hlyX gene homology with the fnr gene of Escherichia coli.

The hlyX gene from Actinobacillus pleuropneumoniae, which confers a hemolytic phenotype on Escherichia coli, was sequenced, and its role in regulation of gene expression was investigated. No similarity was found between the hlyX sequence and sequences of known hemolysin or cytotoxin genes. However, the hlyX sequence was very similar to that of the fnr gene of Escherichia coli which encodes the global regulatory protein, FNR. Comparison of the deduced amino acid sequence of the hlyX gene product (HlyX) with that of FNR revealed a high degree of well-aligned sequence correlation throughout the polypeptide chain. For example, 23 of 24 amino acids in the DNA-binding region of FNR are identical in the corresponding region of HlyX. Four cysteine residues in the amino-terminal region are also conserved. The promoter region of hlyX is very similar to that of fnr. It has a putative -10 sequence which closely resembles the E. coli -10 consensus sequence. This sequence is overlapped by a potential operator which is very similar to the FNR-binding-site consensus sequence. Functional homology between HlyX and FNR was also demonstrated. Plasmids carrying hlyX complemented the nutritional lesion of an fnr deletion strain of E. coli. These data suggest that HlyX may regulate, rather than mediate, hemolytic activity in E. coli, but the possibility that HlyX is both a regulator of gene expression and a hemolysin cannot be excluded.