Nucleotide sequence of the gene for monoamine oxidase (maoA) from Escherichia coli

We found that the structural gene for monoamine oxidase was located at 30.9 min on the Escherichia coli chromosome. Deletion analysis showed that two amine oxidase genes are located in this region. The nucleotide sequence of one of the two genes was determined. The peptide sequence of the first 40 amino acids from the N terminus of monoamine oxidase purified from E. coli agrees with that deduced from the nucleotide sequence of the gene. The leader peptide extends over 30 amino acids. The nucleotide sequence of the gene and amino acid sequence of the predicted mature enzyme (M.W. 81,295) were highly homologous to those of the maoA_K gene and monoamine oxidase from Klebsiella aerogenes, respectively. From these results and analysis of the enzyme activity, we concluded that the gene encodes for monoamine oxidase (maoA_E). The tyrosyl residue, which may be converted to topa quinone in the E. coli enzyme, was located by comparison with amino acid sequences at the cofactor sites in other copper/topa quinone-containing amine oxidases.     

Cloning of the pullulanase gene and overproduction of pullulanase in Escherichia coli and Klebsiella aerogenes.

The pullulanase gene (pul) of Klebsiella aerogenes was cloned into a pBR322 vector in Escherichia coli. Deletion analysis of the recombinant plasmid showed that the pul coding sequence, probably with the regulator gene, was located entirely within a 4.2-kilobase segment derived from the chromosomal DNA of K. aerogenes. E. coli cells carrying the recombinant plasmids produced about three- to sevenfold more pullulanase than did the wild-type strain of K. aerogenes W70. When the cloned cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Transfer of the plasmid containing the pul gene into K. aerogenes W70 resulted in about a 20- to 40-fold increase in total production of pullulanase, and the intracellular enzyme level was about 100- to 150-fold higher than that of the parent strain W70. The high level of pullulanase activity in K. aerogenes cells carrying the recombinant plasmid was maintained for at least 2 weeks.     

Cloning of the pullulanase gene and overproduction of pullulanase in Escherichia coli and Klebsiella aerogenes

The pullulanase gene (pul) of Klebsiella aerogenes was cloned into a pBR322 vector in Escherichia coli. Deletion analysis of the recombinant plasmid showed that the pul coding sequence, probably with the regulator gene, was located entirely within a 4.2-kilobase segment derived from the chromosomal DNA of K. aerogenes. E. coli cells carrying the recombinant plasmids produced about three- to sevenfold more pullulanase than did the wild-type strain of K. aerogenes W70. When the cloned cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Transfer of the plasmid containing the pul gene into K. aerogenes W70 resulted in about a 20- to 40-fold increase in total production of pullulanase, and the intracellular enzyme level was about 100- to 150-fold higher than that of the parent strain W70. The high level of pullulanase activity in K. aerogenes cells carrying the recombinant plasmid was maintained for at least 2 weeks.     

The Klebsiella aerogenes glutamate dehydrogenase (gdhA) gene: cloning, high-level expression and hybrid enzyme formation in Escherichia coli

Summary The NADP-dependent glutamate dehydrogenase gene of Klebsiella aerogenes was cloned in E. coli in the expression plasmid pRK9. The cloned gene shows a high level of expression in E. coli in the hybrid plasmid pKG3 and such expression is independent of the vector promoter, as shown by experiments in which the promoter was deleted. Active hybrid GDH hexamers were shown in cell-free extracts of an E. coli strain carrying cloned gdhA genes of both E. coli and K. aerogenes. The nucleotide sequence of the N-terminal coding region of the K. aerogenes gdhA gene was determined and found to be strongly homologous with that of E. coli.Abbreviations GDH glutamate dehydrogenase - PMS phenazine methosulphate - MTT 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium-bromide - PMSF phenylmethylsulphonylfluoride - SSC standard saline citrate - DTT dithiothreitol - bp base pairs - kbp kilo base pairs - dNTP deoxynucleoside triphosphate     

Purification,Characterization, and Crystallization of Monoamine Oxidase from Escherichia coli K-12

The gene for monoamine oxidase (MAO) was cloned from an Escherichia coli genomic library and MAO was overproduced in the periplasmic space. The enzyme was purified to homogeneity by preparation of a periplasmic fraction, followed by ammonium sulfate fractionation and DEAE-cellulose column chromatography. Crystals were obtained by the hanging drop method using sodium citrate as a precipitant. The enzyme was found to be a dimer of identical subunits with a molecular weight of 80,000, and showed the highest activity at pH 7.5 and 45°C. The enzyme was inhibited by a MAO specific inhibitor, hydroxylamine, hydrazine, phenelzine, isoniazid, and tranycypromine. The enzyme oxidized tyramine, phenethylamine, and tryptamine at higher rates, but not oxidized diamine and polyamines such as putrecine and spermine. The antibody against E. coli MAO cross-reacted with purified MAO A from Klebsiella aerogenes.     

In Vivo Cloning of Arylsulfatase-Tyramine Oxidase Genes in rec Strains of Klebsiella aerogenes

A general, genetic technique for in vivo cloning of bacterial genes is presented. We previously introduced the Mu phage into various genera of bacteria including Klebsiella aerogenes with RP4 : : Mu. Using these strains carrying RP4 : : Mu cts and thermo-inducible Mu prophage in the chromosome, we cloned in vivo the arylsulfatase (ats) and tyramine oxidase (tyn) genes by partial thermo-induction. The donor strains carrying the recombinant plasmids were conjugated with K. aerogenes rec strains, which were isolated as UV-sensitive mutants. The resultant recombinant plasmids, pAT1 and pAT2, were purified and used for the transformation of mutant strains deficient in the ats and tyn genes. The ats-tyn genes seemed to be transposed into the RP4::Mu cts plasmid together with other chromosomal DNA fragments. This in vivo cloning method is applicable to a wide variety of gram-negative bacteria.     

Enzymatic Formation of Sphingenine from Ketosphinganine in Rat Liver Particulates

Mutants of Klebsiella aerogenes that synthesized tyramine oxidase and arylsulfatase constitutively were isolated. The properties of four of seven constitutive mutants isolated were unstable, segregating spontaneously to the parental type at high frequency. Some of these segregants also lost arylsulfatase (AtsA^?) or tyramine oxidase (TynA^?) spontaneously. These unstable constitutive mutations were shown by genetic analysis to involve mutations of tynP and tynR, and transductants receiving the tynP gene were also unstable. These results showed that the instability was due to unstable tynP gene, which may be the promoter region for tyramine oxidase.     

Immunological study of the regulation of cellular arylsulfatase synthesis in Klebsiella aerogenes.

Regulation of cellular arylsulfatase synthesis in Klebsiella aerogenes was analyzed by immunological techniques. Antibody directed against the purified arylsulfatase from K. aerogenes W70 was obtained from rabbits and characterized by immunoelectrophoresis, double-diffusion, quantitative precipitation, and enzyme neutralization tests. Arylsulfatase was located in the periplasmic space when the wild-type strain was cultured with methionine or with inorganic sulfate plus tyramine, but not with inorganic sulfate without tyramine, as the sole sulfur source. Tyramine oxidase was retained in the membrane fraction prepared from cells grown in the presence of tyramine. Arylsulfatase protein was not synthesized in the presence of tyramine and inorganic sulfate by mutant K611, which is deficient in tyramine oxidase (tynA). We conclude that the expression of the arylsulfatase gene (atsA) is regulated by the expression of tynA and that inorganic sulfate serves as a corepressor. In addition, strains mutated in the atsA gene were analyzed by using antibody.     

Gene transfer between Escherichia coli and Enterobacter aerogenes

Summary Transfer of chromosomal genes between Escherichia coli K12 and Enterobacter aerogenes was carried out by P1-mediated transduction as well as by transformation of genes cloned in vitro on plasmid vectors. The efficient expression of E. coli genes in E. aerogenes probably reflects the existance of a poor restriction system in the latter, and suggests that this strain might be useful as a recipient of genetic information from E. coli.     

psi, a plasmid-linked Rhizobium phaseoli gene that inhibits exopolysaccharide production and which is required for symbiotic nitrogen fixation

Summary A strain of R. phaseoli cured of its symbiotic plasmid, pRP2JI, retained the ability to make exopolysaccharide (EPS). However, a region of pRP2JI, when cloned at an increased copy number in wide host-range vectors and transferred to this and other strains of Rhizobium, inhibited EPS synthesis. The gene responsible was termed psi (polysaccharide inhibition) and was located in a region of the symbiotic plasmid close to nodulation and nitrogen fixation genes. psi is important in the symbiosis since a wild-type strain containing psi cloned on a multicopy plasmid failed to form Phaseolus nodules, and mutant strains containing psi::Tn5 mutations failed to fix nitrogen in Phaseolus nodules. It is proposed that the function of psi may be to repress in the bacteriod the expression of genes such as those for EPS synthesis which are normally expressed in free-living culture.     

Quantitative Analysis of the Sex Pheromone of Several Phycitid Moths by Electron-capture Gas Chromatography

β-Phenetyl alcohol and procaine hydrochloride are known to alter membrane structure. Their effects on the syntheses of tyramine oxidase and arylsulfatase were studied in Klebsiella aerogenes. β-Phenetyl alcohol inhibited the syntheses of membrane-bound tyramine oxidase and arylsulfatase, located in the periplasm, under non-repressing and derepressing conditions, but did not affect the syntheses of β-galactosidase and histidase, which are located internally. In contrast, procaine hydrochloride stimulated the synthesis of tyramine oxidase and derepressed the synthesis of arylsulfatase, but inhibited non-repressed synthesis of arylsulfatase. Thus, derepressed synthesis of cellular arylsulfatase was affected by the level of tyramine oxidase synthesis. Structural alterations in the cell membrane seem to impair the formation of active-arylsulfatase protein in the periplasmic space.     

Alteration of hydrogen metabolism of ldh-deleted Enterobacter aerogenes by overexpression of NAD(+)-dependent formate dehydrogenase

The NAD⁺-dependent formate dehydrogenase FDH1 gene (fdh1), cloned from Candida boidinii, was expressed in the ldh-deleted mutant of Enterobacter aerogenes IAM1183 strain. The plasmid of pCom10 driven by the PalkB promoter was used to construct the fdh1 expression system and thus introduce a new dihydronicotinamide adenine dinucleotide (NADH) regeneration pathway from formate in the ldh-deleted mutant. The knockout of NADH-consuming lactate pathway affected the whole cellular metabolism, and the hydrogen yield increased by 11.4% compared with the wild strain. Expression of fdh1 in the ldh-deleted mutant caused lower final cell concentration and final pH after 16 h cultivation, and finally resulted in 86.8% of increase in hydrogen yield per mole consumed glucose. The analysis of cellular metabolites and estimated redox state balance in the fdhl-expressed strain showed that more excess of reducing power was formed by the rewired NADH regeneration pathway, changing the metabolic distribution and promoting the hydrogen production.     

Gene manipulation of catabolic activities for production of intermediates of various biphenyl compounds

Summary A bioconversion process was demonstrated by manipulation of catabolic genes. Catabolic intermediates of various biphenyl compounds could be efficiently produced by Pseudomonas aeruginosa carrying recombinant plasmids containing a set of cloned bph genes. A dihydrodiol compound was produced by the strain carrying plasmid pMFB4 containing bphA (encoding biphenyl dioxygenase) gene. A dihydroxy compound was produced from 4-chlorobiphenyl by the strain carrying plasmid pMFB6 containing bphA and bphB (encoding dihydrodiol dehydrogenase) genes. Tetrahydroxybiphenyl was accumulated as the final product via dihydroxybiphenyl from biphenyl by the same pMFB6 carrying strain. Meta-cleavage yellow compounds were produced from biphenyl and its derivatives substituted with methyl, chloro, bromo, or nitro group on one of the biphenyl rings by the strain carrying plasmid pMFB2 containing bphA, bphB and bphC (encoding dihydroxybiphenyl dioxygenase) genes.     

Immunological Studies on Antisera of Mice Immunized with Dried or DEPC-treated Ehrlich Ascites Tumor Cells

Intracellular arylsulfatases from Klebsiella aerogenes W70 cells grown in methionine medium (M enzyme) and inorganic sulfate medium containing tyramine (T enzyme) were purified respectively by fractionation with (NH₄)₂SO₄, followed by successive chromatographies on DEAE cellulose, hydroxylapatite, Sephadex G-100 and DEAE Sephadex A-25. On polyacrylamide gel electrophoresis, the two enzymes gave single bands with the same mobilities. Molecular weights of both, determined by SDS gel electrophoresis and by Sephadex G-100 chromatography, were 47,000 and 45,000, respectively. Their activities were maximal at pH 7.5. The affinities of the enzymes (M and T enzymes) for their substrate (Km) and the maximum velocity of hydrolysis (V_max) were enhanced by addition of electron withdrawing substituents. The enzymes were inhibited by inorganic phosphate, cyanide, hydroxylamine and tyramine. The inhibition by tyramine was competitive (Ki = 1.0 × 10^?4 m). These results show that the two enzymes were identical. This was confirmed by the fact that mutant strains, which were unable to synthesize arylsulfatase when grown with methionine, could also not synthesize the enzyme when grown with tyramine.     

Possible involvement of the ugpA gene product in the stable maintenance of mini-F plasmid in Escherichia coli

Summary The seg-3 mutant Escherichia coli does not support the maintenance of mini-F plasmid at 42° C. We cloned the chromosomal DNA segment of the wild-type strain W3110 that complements the Seg^– phenotype of this mutant. Cleavage mapping of this segment showed that it was derived from the 76-min region of the E. coli chromosome map. Complementation tests using plasmids carrying subcloned DNA segments suggested that the seg-3 mutant carried two mutations that additively affected the maintenance of mini-F plasmid; one was in the ugpA gene and the other was presumably in the rpoH gene. We generated a disrupted ugpA null mutant and found that the mini-F plasmid was unstable in this ugpA null mutant even at 30° C. This suggests that the ugpA gene product is required for the stable maintenance of mini-F plasmid.     

Purification, cloning, and three-dimensional structure prediction of Micrococcus luteus FAD-containing tyramine oxidase

The FAD-containing tyramine oxidase enzyme and gene from the Gram (+) bacterium Micrococcus luteus were isolated, and computer prediction was used to propose a preliminary 3D model of the protein. A 2.8-kb Sau3AI fragment containing the structural gene of tyramine oxidase was cloned from a M. luteus genomic DNA library. The 1332 bp gene encodes a protein of 443 amino acids, with a calculated molecular mass of 49.1 kDa. The enzyme was found to be a homodimer with a molecular weight of 49,000. It oxidizes tyramine, adrenaline, 3-hydroxytyramine, dopamine, and noradrenaline, and was reversibly inhibited by FAD-containing monoamine oxidase A and B specific inhibitors. Sequence comparison show that tyramine oxidase is smaller than other FAD-amine oxidases but that it contains well-conserved amino acid residues reported in all other FAD-amine oxidases. A hypothetical three-dimensional structure of tyramine oxidase has also been proposed based on secondary structure predictions, threading, and comparative modeling.     

Cloning and nucleotide sequence of a negative regulator gene for Klebsiella aerogenes arylsulfatase synthesis and identification of the gene as folA.

A negative regulator gene for synthesis of arylsulfatase in Klebsiella aerogenes was cloned. Deletion analysis showed that the regulator gene was located within a 1.6-kb cloned segment. Transfer of the plasmid, which contains the cloned fragment, into constitutive atsR mutant strains of K. aerogenes resulted in complementation of atsR; the synthesis of arylsulfatase was repressed in the presence of inorganic sulfate or cysteine, and this repression was relieved, in each case, by the addition of tyramine. The nucleotide sequence of the 1.6-kb fragment was determined. From the amino acid sequence deduced from the DNA sequence, we found two open reading frames. One of them lacked the N-terminal region but was highly homologous to the gene which codes for diadenosine tetraphosphatase (apaH) in Escherichia coli. The other open reading frame was located counterclockwise to the apaH-like gene. This gene was highly homologous to the gene which codes for dihydrofolate reductase (folA) in E. coli. We detected 30 times more activity of dihydrofolate reductase in the K. aerogenes strains carrying the plasmid, which contains the arylsulfatase regulator gene, than in the strains without plasmid. Further deletion analysis showed that the K. aerogenes folA gene is consistent with the essential region required for the repression of arylsulfatase synthesis. Transfer of a plasmid containing the E. coli folA gene into atsR mutant cells of K. aerogenes resulted in repression of the arylsulfatase synthesis. Thus, we conclude that the folA gene codes a negative regulator for the ats operon.     

Catabolite repression and derepression of arylsulfatase synthesis in Klebsiella aerogenes

When a mutant (Mao(-)) of Klebsiella aerogenes lacking an enzyme for tyramine degradation (monoamine oxidase) was grown with d-xylose as a carbon source, arylsulfatase was repressed by inorganic sulfate and repression was relieved by tyramine. When the cells were grown on glucose, tyramine failed to derepress the arylsulfatase synthesis. When grown with methionine as the sole sulfur source, the enzyme was synthesized irrespective of the carbon source used. Addition of cyclic adenosine monophosphate overcame the catabolite repression of synthesis of the derepressed enzyme caused by tyramine. Uptake of tyramine was not affected by the carbon source. We isolated a mutant strain in which derepression of arylsulfatase synthesis by tyramine occurred even in the presence of glucose and inorganic sulfate. This strain also produced beta-galactosidase in the presence of an inducer and glucose. These results, and those on other mutant strains in which tyramine cannot derepress enzyme synthesis, strongly suggest that a protein factor regulated by catabolite repression is involved in the derepression of arylsulfatase synthesis by tyramine.     

High Expression of the Second Lysine Decarboxylase Gene,ldc, in Escherichia coli WC196 Due to the Recognition of the Stop Codon (TAG), at a Position Which Corresponds to the 33th Amino Acid Residue of σ38, as a Serine Residue by the Amber Suppressor,supD

Escherichia coli WC196, which was obtained from the strain W3110 by nitrosoguanidine mutagenesis as an overproducer of lysine, produced approximately twenty times more cadaverine than did W3110, and had a twenty fold higher level of rpoS gene product, σ³⁸, than in W3110. Both WC196 and W3110 had a stop codon (TAG) in rpoS at position which corresponds to the 33th residue of σ³⁸ protein. In addition, WC196 but not W3110 had a mutation in the gene encoding Ser-tRNA (SerU), called, supD. Analysis of the amino acid sequence of a σ³⁸ preparation from WC196 showed that the 33th residue of σ³⁸ is a serine residue. The ΔrpoS ΔcadA mutant of E. coli W3110 harboring the plasmid containing rpoS, in which the TAG codon was converted to a TCG codon for serine-33 residue of σ³⁸, expressed a significant amount of Ldc and accumulated a large amount of σ³⁸. However, the ΔrpoS ΔcadA mutant of W3110 with the plasmid containing the intact rpoS from W3110 could synthesize neither σ³⁸ nor Ldc significantly.     

Tyramine,as an Active Factor for the Production of Phenolsulphatase by Aerobacter aerogenes

An active factor for the production of phenolsulphatase by some strains of Aerobacter aerogenes was separated as pure crystals from a commercial peptone preparation by ion exchange chromatographic techniques. Properties of this substance were studied, and it was found to be identical with tyramine. Even when pure tyramine in a concentration of 1 × 10^?5M was added to the culture medium, its effect on the level of enzymatic activity could be observed. In addition to tyramine, hordenine was also found effective but this, only in the case of A. aerogenes strain 9621. This specific activity of tyramine appears to be an induction of enzyme production.