Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation inCorynebacterium glutamicum

Although the protocatechuate branch of the β-ketoadipate pathway in Gram⁻ bacteria has been well studied, this branch is less understood in Gram⁺ bacteria. In this study,Corynebacterium glutamicum was cultivated with protocatechuate,p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locusncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes,ncg12314 andncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). RecombinantEscherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted inC. glutamicum, the ability to degrade and assimilate protocatechuate,p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity ofC. glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded byncg12314 andncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the Gen Bank. The functional identification of genes and their unique organization inC. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.     

Heat Shock Inhibits the Induced Expression of the SOS and SoxRS Regulons in Escherichia coli

Oxidative stress formed in Escherichia coli cells is known to bring about a complex induction of alternative DNA repair processes, including SOS, SoxRS, and heat-shock response (HSR). The modification by heat shock of the expression ofsfiA and soxS genes induced by oxidative agents H₂O₂, menadione and 4-nitroquinoline-1-oxide (4NQO) was studied for the first time. Quantitative parameters of gene expression were examined inE. coli strains with fused genes (promoters) sfiA::lacZ and soxS::lacZ.The expression of these genes induced by cell treatment with H₂O₂, but not menadione or 4NQO, was shown to decrease selectively after exposure to heat shock. Since genetic activity of menadione and 4NQO depends mainly on the formation of superoxide anion ,O^¯ ₂ it is assumed that the effect of selective inhibition by heat-shock of sfiA and soxS gene expression in experiments with H₂O₂ is connected with activity of DnaK heat shock protein, which, unlike other heat-shock proteins, cannot be induced by superoxide anion O^¯ ₂.     

In vitro reconstitution of the catabolic reactions catalyzed by PcaHG,PcaB, and PcaL: the protocatechuate branch of the β-ketoadipate pathway in Rhodococcus jostii RHA1

The β-ketoadipate pathway is a major pathway involved in the catabolism of the aromatic compounds in microbes. The recent progress in genome sequencing has led to a rapid accumulation of genes from the β-ketoadipate pathway in the available genetic database, yet the functions of these genes remain uncharacterized. In this study, the protocatechuate branch of the β-ketoadipate pathway of Rhodococcus jostii was reconstituted in vitro. Analysis of the reaction products of PcaHG, PcaB, and PcaL was achieved by high-performance liquid chromatography. These reaction products, β-ketoadipate enol-lactone, 3-carboxy-cis,cis-muconate, γ-carboxymuconolactone, muconolactone, and β-ketoadipate, were further characterized using LC-MS and nuclear magnetic resonance. In addition, the in vitro reaction of PcaL, a bidomain protein consisting of γ-carboxy-muconolactone decarboxylase and β-ketoadipate enol-lactone hydrolase activities, was demonstrated for the first time. This work provides a basis for analyzing the catalytic properties of enzymes involved in the growing number of β-ketoadipate pathways deposited in the genetic database.     

Cloning of the gene coding for aromatic amino acid aminotransferase from anE. Coli B strain

Summary AnE. coli B strain showing high activity in the transamination of phenylpyruvate to phenylalanine was used as the DNA source for the construction of a cosmid library inE. coli DG30, a strain which is known to be defective in all three major transaminase genes (aspC, ilvE andtyrB). By complementation analysis, cosmid clones could be identified with inserts carrying atyrB gene. The DNA inserts were further subcloned into pAT153 and thetyrB gene fromE. coli B was found to be similar to the gene reported forE. coli K12. Plasmids containing theE. coli BtyrB gene were transformed into the originalE. coli B strain and the recombinant strains assayed for transaminase activity and plasmid stability.Dedicated to Prof. Dr. Heinz Harnisch on the occasion of his 60th birthday.     

Cloning and Genetic Characterization of dca Genes Required for β-Oxidation of Straight-Chain Dicarboxylic Acids in Acinetobacter sp. Strain ADP1

A previous study of deletions in the protocatechuate (pca) region of the Acinetobacter sp. strain ADP1 chromosome revealed that genes required for utilization of the six-carbon dicarboxylic acid, adipic acid, are linked to the pca structural genes. To investigate the genes involved in adipate catabolism, a 33.8-kb SacI fragment, which corrects a deletion spanning this region, was cloned. In addition to containing known pca, qui, and pob genes (for protocatechuate, quinate, and 4-hydroxybenzoate dissimilation), clone pZR8000 contained 10 kb of DNA which was the subject of this investigation. A mutant strain of Escherichia coli DH5α, strain EDP1, was isolated that was able to utilize protocatechuate and 4-hydroxybenzoate as growth substrates when EDP1 cells contained pZR8000. Sequence analysis of the new region of DNA on pZR8000 revealed open reading frames predicted to be involved in β-oxidation. Knockouts of three genes implicated in β-oxidation steps were introduced into the chromosome of Acinetobacter sp. strain ADP1. Each of the mutants was unable to grow with adipate. Because the mutants were affected in their ability to utilize additional saturated, straight-chain dicarboxylic acids, the newly discovered 10 kb of DNA was termed the dca (dicarboxylic acid) region. Mutant strains included one with a deletion in dcaA (encoding an acyl coenzyme A [acyl-CoA] dehydrogenase homolog), one with a deletion in dcaE (encoding an enoyl-CoA hydratase homolog), and one with a deletion in dcaH (encoding a hydroxyacyl-CoA dehydrogenase homolog). Data on the dca region should help us probe the functional significance and interrelationships of clustered genetic elements in this section of the Acinetobacter chromosome.     

Molecular cloning of a gene which regulates the adaptive response to alkylating agents in Escherichia coli

Summary The ada ⁺ gene of E. coli is a regulatory gene of the adaptive response to simple alkylating agents. ada mutants are sensitive to both the mutagenicity and toxicity of alkylating agents, and are unable to induce O⁶-methylguanine DNA methyltransferase and 3-methyladenine DNA glycosylase II. The ada ⁺ gene was cloned from wild type E. coli B by ligating bacterial DNA partially digested with Sau3A into the cosmid vector pJB8. The hybrid cosmid, pCS33, conveyed N-methyl-N-nitro-N-nitrosoguanidine resistance to ada mutants of E. coli B and E. coli K12, and resulted in the constitutive synthesis of the two DNA repair enzymes at high levels. An alk mutation, which results in a deficiency of only the DNA glycosylase, was not complemented by this cosmid. It was concluded that the product of the ada ⁺ gene is a positive regulator of the adaptive response. The cosmid insert DNA was subcloned into the plasmid vector pAT153, and the ada ⁺ plasmids pCS42 and pCS58 selected. The ada ⁺ gene located in PCS58 by transposon mutagenesis and subcloning. Two polypeptides of Mr 37,000 and 27,000, were identified in maxicells as products of the ada ⁺ gene(s). It is as yet unclear whether they represent different forms of the same gene product, or are encoded by separate ada ⁺ genes within the same operon.     

Characterization of Sphingomonas paucimobilis SYK-6 genes involved in degradation of lignin-related compounds

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds. These compounds are degraded via vanillate and syringate by a unique enzymatic system, composed of etherases, O demethylases, ring cleavage oxygenases and side chain cleaving enzymes. These unique and specific lignin modification enzymes are thought to be powerful tools for utilization of the most abundant aromatic biomass, lignin. Here, we focus on the genes and enzymes involved in β-aryl ether cleavage and biphenyl degradation. Two unique etherases are involved in the reductive cleavage of β-aryl ether. These two etherases have amino acid sequence similarity with the glutathione S-transferases, and use glutathione as a hydrogen donor. It was found that 5,5′-dehydrodivanillate, which is a typical lignin-related biphenyl structure, was transformed into 5-carboxyvanillate by the reaction sequence of O-demethylation, meta-ring cleavage, and hydrolysis, and the genes involved in the latter two reactions have been characterized. Vanillate and syringate are the most common intermediate metabolites in lignin catabolism. These compounds are initially O-demethylated and the resulting diol compounds, protocatechuate (PCA) and 3-O-methylgallate, respectively, are subjected to ring cleavage catalyzed by PCA 4,5-dioxygenase. The ring cleavage products generated are further degraded through the PCA 4,5-cleavage pathway. We have isolated and characterized genes for enzymes involved in this pathway. Disruption of a gene for 2-pyrone-4,6-dicarboxylate hydrolase (ligI) in this pathway suggested that an alternative route for 3-O-methylgallate degradation, in which ligI is not involved, would play a role in syringate catabolism. In this article, we describe the genetic and biochemical features of the S. paucimobilis SYK-6 genes involved in degradation of lignin-related compounds. A possible application of the SYK-6 lignin degradation system to produce a valuable chemical material is also described. Received 01 May 1999/ Accepted in revised form 29 July 1999     

Key Aromatic-Ring-Cleaving Enzyme, Protocatechuate 3,4-Dioxygenase, in the Ecologically Important Marine Roseobacter Lineage

Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the α-proteobacteria was investigated. Initial screens suggested that isolates in the Roseobacter lineage can degrade aromatic compounds via the β-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six Roseobacter isolates were screened for the presence of protocatechuate 3,4-dioxygenase, a key enzyme in the β-ketoadipate pathway. All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, benzoate, p-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate). Four of the Roseobacter group isolates had inducible protocatechuate 3,4-dioxygenase activity in cell extracts when grown on p-hydroxybenzoate. The pcaGH genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates, Sagittula stellata E-37 and isolate Y3F, and in both cases the genes could be expressed in Escherichia coli to yield dioxygenase activity. Additional genes involved in the protocatechuate branch of the β-ketoadipate pathway (pcaC, pcaQ, and pobA) were found to cluster with pcaGH in these two isolates. Pairwise sequence analysis of the pca genes revealed greater similarity between the two Roseobacter group isolates than between genes from either Roseobacter strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of pcaH from two additional Roseobacter group isolates, and Southern hybridization indicated the presence of pcaH in the remaining two isolates. This evidence of protocatechuate 3,4-dioxygenase and the β-ketoadipate pathway was found in all six Roseobacter isolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.     

Novel metabolic routes during the oxidation of hydroxylated aromatic acids by the yeast Arxula adeninivorans

Aim: To complete our study on tannin degradation via gallic acid by the biotechnologically interesting yeast Arxula adeninivorans as well as to characterize new degradation pathways of hydroxylated aromatic acids. Methods and Results: With glucose‐grown cells of A. adeninivorans, transformation experiments with hydroxylated derivatives of benzoic acid were carried out. The 12 metabolites were analysed and identified by high performance liquid chromatography and GC/MS. The yeast is able to transform the derivatives by oxidative and nonoxidative decarboxylation as well as by methoxylation. The products of nonoxidative decarboxylation of protocatechuate and gallic acid are substrates for further ring fission. Conclusion: Whereas other organisms use only one route of transformation, A. adeninivorans is able to carry out three different pathways (oxidative, nonoxidative decarboxylation and methoxylation) on one hydroxylated aromatic acid. The determination of the K_M‐values for protocatechuate and gallic acid in crude extracts of cells of A. adeninivorans cultivated with protocatechuate and gallic acid, respectively, suggests that the decarboxylation of protocatechuate and gallic acid may be catalysed by the same enzyme. Significance and Impact of the Study: This transformation pathway of protocatechuate and gallic acid via nonoxidative decarboxylation up to ring fission is novel and has not been described so far. This is also the first report of nonoxidative decarboxylation of gallic acid by a eukaryotic micro‐organism.     

Gene expression in<Emphasis Type="Italic"> Escherichia coli</Emphasis> biofilms

DNA microarrays were used to study the gene expression profile of Escherichia coli JM109 and K12 biofilms. Both glass wool in shake flasks and mild steel 1010 plates in continuous reactors were used to create the biofilms. For the biofilms grown on glass wool, 22 genes were induced significantly (p0.05) compared to suspension cells, including several genes for the stress response (hslS, hslT, hha, and soxS), type I fimbriae (fimG), metabolism (metK), and 11 genes of unknown function (ybaJ, ychM, yefM, ygfA, b1060, b1112, b2377, b3022, b1373, b1601, and b0836). The DNA microarray results were corroborated with RNA dot blotting. For the biofilm grown on mild steel plates, the DNA microarray data showed that, at a specific growth rate of 0.05/h, the mature biofilm after 5 days in the continuous reactors did not exhibit differential gene expression compared to suspension cells although genes were induced at 0.03/h. The present study suggests that biofilm gene expression is strongly associated with environmental conditions and that stress genes are involved in E. coli JM109 biofilm formation.     

鸡源大肠杆菌毒力基因检测及分子流行特征

[目的] 本试验旨在阐明鸡源大肠杆菌致病性及分子流行特性,为探索大肠杆菌流行途径制定合理的防控策略提供新思路。[方法] 2018–2019年在河北省采集病死鸡肝脏样品,通过选择培养基筛选、生化鉴定、血清凝集试验对分离菌株进行系统鉴定,应用PCR方法检测分离株中毒力基因流行情况。参考系统发育群分类方法对大肠杆菌进行分群分析,并参照McMLST网站数据库提供的7对管家基因序列进行多位点序列分型（multilocus sequence typing,MLST）分析。[结果] 结果显示,56株分离株符合大肠杆菌生化特征,分为8个生化表型,B4（30.36%）、B5（25%）和B2（23.21%）为主要生化表型。56株分离株大肠杆菌血清凝集试验均呈阳性,分为11种血清型,O₇₈（26.79%）、O₂（23.21%）、O₁₅₇（17.86%）和O₁（14.29%）为主要流行血清型。56株大肠杆菌共检测出15种肠外大肠杆菌毒力基因,未检出papC、ibeA和ibeB基因。黏附相关基因fimC和抗血清存活因子相关基因ompA携带率为100%。aatA、yijP、irp2、mat、iss,检出率分别为98.21%、98.21%、98.21%、96.43%、92.86%。同时,大肠杆菌与铁转运相关基因iroN、fyuA、iucD、irp2检出率均在80%以上。56株大肠杆菌中有20株属于肠出血型大肠杆菌（enterohemorrhagic E.coli,EHEC）,其次是肠聚集型大肠杆菌（enteroaggregative E.coli,EAEC）（n=4）、肠产毒素型大肠杆菌（Enterotoxigenic E.coli,ETEC）（n=2）。这些菌株D群分离株较多,其次是B2群。通过MLST分型分析,共分为22个ST型,其中ST88（n=7）、ST85（n=6）、ST243（n=6）型为主要流行型。[结论] 结果显示大肠杆菌血清型多样,毒力因子种类繁多,致病性大肠杆菌同时携带多种毒力基因,表明动物源大肠杆菌具有较强的毒力基础。     

Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12.

Summary capR (lon) mutants of Escherichia coli K-12 are mucoid on minimal agar because they produce large quantities of capsular polysaccharide. When such mutants are transformed to tetracycline resistance by plasmid pMC44, a hybrid plasmid that contains a 2 megadalton (Mdal) endonuclease EcoR1 fragment of E. coli K-12 DNA joined to the cloning vehicle-pSC101, capsular polysaccharide synthesis is inhibited and the transformed colonies exhibit a nonmucoid phenotype. Re-cloning of the 2 Mdal EcoR1 fragment onto plasmid pHA105, a min-colE1 plasmid, yielded plasmid pFM100 which also inhibited capsular polysaccharide synthesis in the capR mutants. A comparison of the polypeptides specified by both plasmids pFM100 and pMC44 in minicells demonstrated that seven polypeptide bands were specified by the 2 Mdal DNA, one of which was previously demonstrated to be outer membrane protein a; also known as 3b or M2 (40 kilodaltons, Kdal). Plasmid mutants no longer repressing capsular polysaccharide synthesis were either unable to specify the 40 K dal outer membrane protein a or were deficient in synthesis of 25 K dal and 14.5 K dal polypeptides specified by the 2 Mdal DNA fragment. Studies with a minicell-producing strain that also contained a capR mutation indicated that the capR gene product regulated processing of at least one normal protein, the precursor of outer membrane protein a.     

Ecdysterone selectively stimulates the expression of a 23000-Da heat-shock protein-β-galactosidase hybrid gene in cultured Drosophila cells

To study the regulated expression of cloned heat-shock genes in homologous cells, hybrid Drosophila heat-shock-Escherichia coli β-galactosidase genes were constructed. Segments of the ecdysterone-inducible 23,000-Da heat-shock protein (hsp23) gene and of two other hsp genes (hsp84 and 70), which are not hormone regulated, were functionally linked to the bacterial coding sequence, and the resulting hybrid genes were introduced into cultured, hormone-responsive Drosophila cells by transfection. All hybrid genes directed the synthesis of E. coli-specific β-galactosidase in heat-treated cells. hsp23 hybrid gene expression was stimulated strongly by ecdysterone, while the activities of the other hybrid genes were not affected at all by the hormone. A hybrid gene with only 147 bp of hsp23 promoter sequence could not be activated by either heat or ecdysterone treatment. Thus, far upstream sequences contain signals required for the regulated expression of the hsp23 gene in Drosophila cells.     

Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784

Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites for a P450, a linker sequence, and the P450RhF reductase domain, in this order. The known P450 genes, encoding P450cam from Pseudomonas putida (CYP101A) and P450bzo from an environmental metagenome library (CYP203A), were expressed on vector pRED as soluble fusion enzymes with their natural spectral features in Escherichia coli. These E. coli cells expressing the P450cam and P450bzo genes could convert (+)-camphor and 4-hydroxybenzoate into 5-exo-hydroxycamphor and protocatechuate (3,4-dihydroxybenzoate), respectively (the expected products). Using this system, we also succeeded in directly identifying the function of P450 CYP153A as alkane 1-monooxygenase for the first time, i.e., E. coli cells expressing a P450 CYP153A gene named P450balk, which was isolated form Alcanivorax borkumensis SK2, converted octane into 1-octanol with high efficiency (800 mg/l). The system presented here may be applicable to the functional identification of a wide variety of bacterial cytochromes P450.     

Role of Putrescine in the Regulation of the Expression of the Oxidative Stress Defense Genes of Escherichia coli

The role of putrescine in the adaptive response of Escherichia coligrown aerobically in synthetic M9 medium with glucose to the H₂O₂-induced oxidative stress was studied. Under oxidative stress, the expression of the single-copy reporter gene fusions oxyR"::lacZand katG"::lacZwas found to undergo biphasic changes, which were most pronounced in glucose-starved E. colicells. The concentration-dependent activating effect of putrescine on the expression of the OxyR regulon genes was maximum when theoxyRgene was inhibited by high concentrations of hydrogen peroxide.     

Diversity of genetic lineages among CTX-M-15 and CTX-M-14 producing Escherichia coli strains in a Tunisian hospital

Fourteen broad-spectrum-cephalosporin-resistant Escherichia coli isolates were recovered between June and December 2007 in a Tunisian hospital. Genes encoding extended-spectrum-beta-lactamases (ESBL) and other resistance genes were characterized by PCR and sequencing. The following ESBL genes were identified: bla _CTX-M-15 (12 isolates), bla _CTX-M-14a (one isolate), and bla _CTX-M-14b (one isolate). The bla _OXA-1 gene was detected in 13 bla _CTX-M-producing strains and a bla _TEM-1 gene in 6 of them. The ISEcp1 sequence was found upstream of bla _CTX-M genes in 8 of 14 strains, and orf477 or IS903 downstream of this gene in 13 strains. Nine of the strains carried class 1 integrons and five different gene cassette arrangements were detected, dfrA17–aadA5 being the most common. One of the strains (bla _CTX-M-14a-positive) harbored three class 1 integrons, and one of them was non-previously described containing as gene cassettes new variants of aac(6′)-Ib and cmlA1 genes and it was linked to the bla _CTX-M-14a gene flanked by a truncated ISEcp1 sequence (included in GenBank with accession number JF701188). CTX-M-15-producing strains were ascribed to phylogroup B2 (six isolates) and D (six isolates). Multilocus-sequence-typing revealed ten different sequence-types (STs) among ESBL-positive E. coli strains with prevalence of ST405 (four strains of phylogroup D) and ST131 types (two strains of phylogroup B2 and serogroup O25b). A high clonal diversity was also observed among studied strains by pulsed-field-gel-electrophoresis (11 unrelated profiles). CTX-M-15 is an emergent mechanism of resistance in the studied hospital and the world-disseminated 0:25b-ST131-B2 and ST405-D clones have been identified among CTX-M-15-producing isolates.     

Vanillate Metabolism in Corynebacterium glutamicum

Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the mycolic acids-containing actinomycetes, is able to use the lignin degradation products ferulate, vanillate, and protocatechuate as sole carbon sources. The gene cluster responsible for vanillate catabolism was identified and characterized. The vanAB genes encoding vanillate demethylase are organized in an operon together with the vanK gene, coding for a transport system most likely responsible for protocatechuate uptake. While gene disruption mutagenesis revealed that vanillate demethylase is indispensable for ferulate and vanillate utilization, a vanK mutation does not lead to a complete growth arrest but to a decreased growth rate on protocatechuate, indicating that one or more additional protocatechuate transporter(s) are present in C. glutamicum.     

Cloning and restriction mapping of the yeast URA2 gene coding for the carbamyl phosphate synthetase aspartate-transcarbamylase complex

Summary Two yeast DNA pools inserted in an hybridEscherichia coli-yeast vector pFL1 were used to transformE. coli and yeast aspartate-transcarbamylase-less strains to prototrophy. From the first pool — aBamHI yeast DNA digest — a 6.4 kbBamHI fragment was recovered that gave good complementation of theE. coli auxotrophy but poor complementation of the yeast auxotrophy. From the second pool — a partialSau3A yeast DNA digest — five independent plasmids complementing eitherE. coli, yeast, or both were recovered. Each of the five plasmids possessed sequences in common with the 6.4 kbBamHI fragment. One of these plasmids, which complemented the twoURA2 activities in yeast and which produced a carbamyl-phosphate synthetase, aspartate-transcarbamylase complex sensitive to UTP feedback inhibition contained the fullURA2 gene. A restriction map of theURA2 gene has been constructed and seven different consecutive segments have been recloned in pBR322 to measure their hybridization withURA2 messenger RNA, allowing us to estimate the limits of the gene.