Duplicate Copies of Genes Encoding Methanesulfonate Monooxygenase in Marinosulfonomonas methylotropha Strain TR3 and Detection of Methanesulfonate Utilizers in the Environment

Marinosulfonomonas methylotropha strain TR3 is a marine methylotroph that uses methanesulfonic acid (MSA) as a sole carbon and energy source. The genes from M. methylotropha strain TR3 encoding methanesulfonate monooxygenase, the enzyme responsible for the initial oxidation of MSA to formaldehyde and sulfite, were cloned and sequenced. They were located on two gene clusters on the chromosome of this bacterium. A 5.0-kbp HindIII fragment contained msmA, msmB, and msmC, encoding the large and small subunits of the hydroxylase component and the ferredoxin component, respectively, of the methanesulfonate monooxygenase, while a 6.5-kbp HindIII fragment contained duplicate copies of msmA and msmB, as well as msmD, encoding the reductase component of methanesulfonate. Both sets of msmA and msmB genes were virtually identical, and the derived msmA and msmB sequences of M. methylotropha strain TR3, compared with the corresponding hydroxylase from the terrestrial MSA utilizer Methylosulfonomonas methylovora strain M2 were found to be 82 and 69% identical. The msmA gene was investigated as a functional gene probe for detection of MSA-utilizing bacteria. PCR primers spanning a region of msmA which encoded a unique Rieske [2Fe-2S] binding region were designed. These primers were used to amplify the corresponding msmA genes from newly isolated Hyphomicrobium, Methylobacterium, and Pedomicrobium species that utilized MSA, from MSA enrichment cultures, and from DNA samples extracted directly from the environment. The high degree of identity of these msmA gene fragments, compared to msmA sequences from extant MSA utilizers, indicated the effectiveness of these PCR primers in molecular microbial ecology.     

The gene encoding ammonia monooxygenase subunit A exists in three nearly identical copies in Nitrosospira sp. NpAV

Abstract The gene encoding ammonia monooxygenase subunit A (AmoA) was found in three copies in the genome of the chemolithotrophic soil bacterium, Nitrosospira sp. NpAV. The open reading frame and flanking regions of the three copies were isolated from digested and size fractionated genomic DNA using oligodeoxyribonucleotide primers and polymerase chain reaction. The three gene copies of amoA were sequenced and the sequences compared to each other. The open reading frames and the upstream and downstream flanking regions were nearly identical in the three copies. All three copies were expressed in recombinant Escherichia coli strains from the indigenous promoter producing a product of approximately 30 kDa. All amoA copies encode 274 amino acid polypeptides which have similarity to the ammonia monooxygenase acetylene-binding protein from Nitrosomonas europaea .     

Purification and crystallization of the hydroxylase component of the methanesulfonate monooxygenase from Methylosulfonomonas methylovora strain M2

The aim of the work described in this paper was two-fold: (1) the purification of the hydroxylase component of the MSAMO to electrophoretic homogeneity using a four-step chromatographic strategy and (2) the crystallization of the two-component hydroxylase of the MSAMO in order to enhance our understanding of the precise three-dimensional structure of the MSAMO, thus yielding an insight into the nature of the active site of this enzyme. Optimised crystallization conditions were identified allowing growth of crystals of the hydroxylase component of the MSAMO within five days. Crystals exhibited a brown colour suggesting the presence on an intact Rieske-iron sulfur centre and diffracted to 7.0A when a few degrees of data were evaluated on a beam line X11.     

Metabolism of phenylbenzoate by Pseudomonas sp. strain TR3

A bacterial isolate, tentatively identified as Pseudomonas sp. strain TR3, was found to utilize the diaryl ester phenylbenzoate as sole source of carbon and energy. This strain has the ability to productively degrade phenylbenzoate and some substituted derivatives by a catabolic sequence which was characterized biochemically. The biodegradation of phenylbenzoate is thus initiated by an inducible esterase, effectively hydrolyzing the diaryl esters to produce stoichiometric amounts of two monoaromatic metabolites, identified as benzoate and phenol in the case of phenylbenzoate. The diaryl ester p-tolylbenzoate was hydrolyzed to yield benzoate and 4-methylphenol while 4-chlorophenylbenzoate gave rise to the production of benzoate and 4-chlorophenol. These monoaromatic catabolites were further degraded via the oxoadipate pathway.     

Cloning and expression of the genes encoding the propene monooxygenase from Xanthobacter, Py2

Xanthobacter Py2 grows on propene as sole carbon source, converting propene to propene oxide (epoxypropane) using an alkene-specific monooxygenase, as the first step in catabolism. Four mutants, NZ1–4, with a propene^– propene oxide⁺ phenotype were isolated by 1-methyl-3-nitro-1-nitrosoguanidine mutagenesis or by enrichment with the suicide substrate vinylidene chloride, and were shown to have lost the ability to convert alkenes to epoxides. All four mutants were complemented by a number of clones of Xanthobacter Py2 chromosomal DNA in the broad-host-range cosmid pLAFR5, some of which appeared to be non-overlapping. Representatives of the different clones obtained were transferred into Xanthobacter autotrophicus JW33 and one, pNY2, the most frequently isolated clone, was shown to express an inducible, fully functional propene monooxygenase. Subcloning revealed that all four mutants were complemented by a 2.4-kb EcoRI-PstI fragment situated at one end of the cosmid insert. However, activity in X. autotrophicus JW33 could only be expressed from pNY2, containing the complete insert (25 kb), suggesting a large operon or some form of long-range control. pNY2 failed to express in E. coli. In X. autotrophicus JW33 [pNY2] at least three new polypeptides were evident after induction with propene compared with a control carrying only the cosmid pLAFR5.     

Molecular evolution of duplicate copies of genes encoding cytosolic glutamine synthetase in Pisum sativum

Here, we describe two nearly identical expressed genes for cytosolic glutamine synthetase (GS3A and GS3B) in Pisum sativum L. RFLP mapping data indicates that the GS3A and GS3B genes are separate loci located on different chromosomes. DNA sequencing of the GS3A and GS3B genes revealed that the coding regions are 99% identical with only simple nucleotide substitutions resulting in three amino acid differences. Surprisingly, the non-coding regions (5 non-coding leader, the 11 introns, and 3 non-coding tail) all showed a high degree of identity (96%). In these non-coding regions, 25% of the observed differences between the GS3A and GS3B genes were deletions or duplications. The single difference in the 3 non-coding regions of the GS3A and GS3B genes was a 25 bp duplication of an AU-rich element in the GS3B gene. As the GS3B mRNA accumulates to lower levels than the GS3A gene, we tested whether this sequence which resembles an mRNA instability determinant functioned as such in the context of the GS mRNA. Using the GS3B 3 tail as part of a chimeric gene in transgenic plants, we showed that this AU-rich sequence has little effect on transgene mRNA levels. To determine whether the GS3A/GS3B genes represent a recent duplication, we examined GS3-like genes in genomic DNA of ancient relatives of P. sativum. We observed that several members of the Viceae each contain two genomic DNA fragments homologous to the GS3B gene, suggesting that this is an ancient duplication event. Gene conversion has been invoked as a possible mechanism for maintaining the high level of nucleotide similarity found between the GS3A and GS3B genes. Possible evolutionary reasons for the maintenance of these twin GS genes in pea, and the general duplication of genes for cytosolic GS in all plant species are discussed.     

Oligonucleotide primers for detection and amplification of the emoA gene encoding bacterial ethylenediaminetetraacetate monooxygenase

A system of primers was designed on the basis of analysis of nucleotide sequences of the emoA gene encoding ethylenediaminetetraacetate (EDTA) monooxygenase, which are deposited in GenBank. This system of primers makes it possible to obtain emoA gene fragments approximately 750 bp long for bacterial destructors of EDTA. Polymerase chain reaction (PCR) of total DNA isolated from enrichment and pure cultures showed that this system can be effectively used for detecting the emoA gene in representatives of Alpha- and Gammaproteobacteria. Partial sequences of emoA genes of bacteria of the genera Chelativorans and Stenotrophomonas, which are able to degrade this pollutant, have been sequenced and deposited in GenBank.     

2-萘酸加单氧酶基因及NADH:黄素还原酶基因的克隆与分析

伯克霍尔德氏菌(Burkholderiasp.)JT1500对2-萘酸(2-naphthoate)生物降解的关键步骤之一是通过2-萘酸加单氧酶羟化2-萘酸生成1-羟基-2-萘酸(1-hydroxy-2-naphthoate)。在已确定2-萘酸加单氧酶基因及其功能的基础上对含有该基因的一个4.8kb长度的基因簇进行了克隆测序。该序列上含有4个可能的阅读框orfB、orfC、orfD、orfA。序列比对发现,orfA序列与JaponicumUSDA110和RalstoniaeutrophaHF39中的加单氧酶基因同源性较高,orfB序列与BordetllapertussisTohamaI、RalstoniasolanacearumGMI1000和BordetellabronchisepticaRB50等菌中的黄素还原酶基因有一定的同源性。酶活分析发现只含基因orfA的重组大肠杆菌SA细胞提取液有很低的加氧活性,含基因orfB的重组子SB细胞提取液没有加氧活性,但在反应体系中同时加入SA和SB的细胞提取液后,其加氧活性显著增强,包含片段orfB orfA的重组子SB A在黄素(FMN、FAD)存在的情况下也表现出很强的加氧活性;在厌氧条件下,能检测出SB细胞提取液的黄素还原活性。基于以上信息,认为2-萘酸加单氧酶基因簇含有两个重要的组分黄素还原酶基因(nmoB)和加单氧酶基因(nmoA)。2-萘酸加单氧酶Nmo羟化2-萘酸的过程为先由黄素还原酶(NmoB)在NADH存在的条件下将黄素(FMN、FAD)还原为还原型黄素(FMNH2、FADH2),然后加单氧酶(NmoA)利用还原型黄素和O2羟化底物2-萘酸,生成1-羟基-2-萘酸。NmoB是NmoA的偶联蛋白。     

System of oligonucleotide primers for detection and amplification of the emoA gene encoding bacterial ethylenediaminetetraacetate monooxygenase

A system of primers was designed on the basis of analysis of nucleotide sequences of the emoAgene encoding ethylenediaminetetraacetate (EDTA) monooxygenase, which are deposited in GenBank. This system of primers makes it possible to obtain emoA gene fragments approximately 750 bp long for bacterial destructors of EDTA. Polymerase chain reaction (PCR) of total DNA isolated from enrichment and pure cultures showed that this system can be effectively used for detecting the emoAgene in representatives of Alpha- and Gammaproteobacteria. Partial sequences of emoA genes of bacteria of the genera Chelativorans and Stenotrophomonas, which are able to degrade this pollutant, have been sequenced and deposited in GenBank.     

Identification and characterization of genes encoding carbazole 1,9a-dioxygenase in Pseudomonas sp. strain CA10.

Nucleotide sequence analysis of the flanking regions of the carBC genes of Pseudomonas sp. strain CA10 revealed that there were two open reading frames (ORFs) ORF4 and ORF5, in the upstream region of carBC. Similarly, three ORFs, ORF6 to ORF8, were found in the downstream region of carBC. The deduced amino acid sequences of ORF6 and ORF8 showed homologies with ferredoxin and ferredoxin reductase components of bacterial multicomponent dioxygenase systems, respectively. ORF4 and ORF5 had the same sequence and were tandemly linked. Their deduced amino acid sequences showed about 30% homology with large (alpha) subunits of other terminal oxygenase components. Functional analysis using resting cells harboring the deleted plasmids revealed that the products of ORF4 and -5, ORF6, and ORF8 were terminal dioxygenase, ferredoxin, and ferredoxin reductase, respectively, of carbazole 1,9a-dioxygenase (CARDO), which attacks the angular position adjacent to the nitrogen atom of carbazole, and that the product of ORF7 is not indispensable for CARDO activity. Based on the results, ORF4, ORF5, ORF6, and ORF8 were designated carAa, carAa, carAc, and carAd, respectively. The products of carAa, carAd, and ORF7 were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be polypeptides with molecular masses of 43, 36, and 11 kDa, respectively. However, the product of carAc was not detected in Escherichia coli. CARDO has the ability to oxidize a wide variety of polyaromatic compounds, including dibenzo-p-dioxin, dibenzofuran, biphenyl, and polycyclic aromatic hydrocarbons such as naphthalene and phenanthrene. Since 2,2',3-trihydroxydiphenyl ether and 2,2',3-trihydroxybiphenyl were identified as metabolites of dibenzo-p-dioxin and dibenzofuran, respectively, it was considered that CARDO attacked at the angular position adjacent to the oxygen atom of dibenzo-p-dioxin and dibenzofuran as in the case with carbazole.     

Cloning, expression, and site-directed mutagenesis of the propene monooxygenase genes from Mycobacterium sp. strain M156

Propene monooxygenase has been cloned from Mycobacterium sp. strain M156, based on hybridization with the amoABCD genes of Rhodococcus corallinus B276. Sequencing indicated that the mycobacterial enzyme is a member of the binuclear nonheme iron monooxygenase family and, in gene order and sequence, is most similar to that from R. corallinus B-276. Attempts were made to express the pmoABCD operon in Escherichia coli and Mycobacterium smegmatis mc(2)155. In the former, there appeared to be a problem resolving overlapping reading frames between pmoA and -B and between pmoC and -D, while in the latter, problems were encountered with plasmid instability when the pmoABCD genes were placed under the control of the hsp60 heat shock promoter in the pNBV1 vector. Fortuitously, constructs with the opposite orientation were constitutively expressed at a level sufficient to allow preliminary mutational analysis. Two PMO active-site residues (A94 and V188) were targeted by site-directed mutagenesis to alter their stereoselectivity. The results suggest that changing the volume occupied by the side chain at V188 leads to a systematic alteration in the stereoselectivity of styrene oxidation, presumably by producing different orientations for substrate binding during catalysis. Changing the volume occupied by the side chain at A94 produced a nonsystematic change in stereoselectivity, which may be attributable to the role of this residue in expansion of the binding site during substrate binding. Neither set of mutations changed the enzyme's specificity for epoxidation.     

Purification and properties of the NADH reductase component of alkene monooxygenase from Mycobacterium strain E3.

Alkene monooxygenase, a multicomponent enzyme system which catalyzes the epoxidation of short-chain alkenes, is induced in Mycobacterium strain E3 when it is grown on ethene. We purified the NADH reductase component of this enzyme system to homogeneity. Recovery of the enzyme was 19%, with a purification factor of 920-fold. The enzyme is a monomer with a molecular mass of 56 kDa as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is yellow-red with absorption maxima at 384, 410, and 460 nm. Flavin adenine dinucleotide (FAD) was identified as a prosthetic group at a FAD-protein ratio of 1:1. Tween 80 prevented irreversible dissociation of FAD from the enzyme during chromatographic purification steps. Colorimetric analysis revealed 2 mol each of iron and acid-labile sulfide, indicating the presence of a [2Fe-2S] cluster. The presence of this cluster was confirmed by electron paramagnetic resonance spectroscopy (g values at 2.011, 1.921, and 1.876). Anaerobic reduction of the reductase by NADH resulted in formation of a flavin semiquinone.     

Construction and characterization of an Azotobacter vinelandii strain with mutations in the genes encoding flavodoxin and ferredoxin I.

Flavodoxin and ferredoxin I have both been implicated as components of the electron transport chain to nitrogenase in the aerobic bacterium Azotobacter vinelandii. Recently, the genes encoding flavodoxin (nifF) and ferredoxin I (fdxA) were cloned and sequenced and mutants were constructed which are unable to synthesize either flavodoxin (DJ130) or ferredoxin I (LM100). Both single mutants grow at wild-type rates under N2-fixing conditions. Here we report the construction of a double mutant (DJ138) which does not synthesize either flavodoxin or ferredoxin I. When plated on ammonium-containing medium, this mutant had a very small colony size when compared with the wild type, and in liquid culture with ammonium, this double mutant grew three times slower than the wild type or single mutant strains. This demonstrated that there is an important metabolic function unrelated to nitrogen fixation that is normally carried out by either flavodoxin or ferredoxin. If either one of these proteins is missing, the other can substitute for it. The double mutant phenotype can now be used to screen site-directed mutant versions of ferredoxin I for functionality in vivo even though the specific function of ferredoxin I is still unknown. The double mutant grew at the same slow rate under N2-fixing conditions. Thus, A. vinelandii continues to fix N2 even when both flavodoxin and ferredoxin I are missing, which suggests that a third as yet unidentified protein also serves as an electron donor to nitrogenase.     

PCR detection of genes encoding nitrite reductase in denitrifying bacteria

Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplify cd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships of nir genes were also established. The cd1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.     

Cloning and expression in Escherichia coli of Pseudomonas strain LB400 genes encoding polychlorinated biphenyl degradation.

Pseudomonas strain LB400 is able to degrade an unusually wide variety of polychlorinated biphenyls (PCBs). A genomic library of LB400 was constructed by using the broad-host-range cosmid pMMB34 and introduced into Escherichia coli. Approximately 1,600 recombinant clones were tested, and 5 that expressed 2,3-dihydroxybiphenyl dioxygenase activity were found. This enzyme is encoded by the bphC gene of the 2,3-dioxygenase pathway for PCB-biphenyl metabolism. Two recombinant plasmids encoding the ability to transform PCBs to chlorobenzoic acids were identified, and one of these, pGEM410, was chosen for further study. The PCB-degrading genes (bphA, -B, -C, and -D) were localized by subcloning experiments to a 12.4-kilobase region of pGEM410. The ability of recombinant strains to degrade PCBs was compared with that of the wild type. In resting-cell assays, PCB degradation by E. coli strain FM4560 (containing a pGEM410 derivative) approached that of LB400 and was significantly greater than degradation by the original recombinant strain. High levels of PCB metabolism by FM4560 did not depend on the growth of the organism on biphenyl, as it did for PCB metabolism by LB400. When cells were grown with succinate as the carbon source, PCB degradation by FM4560 was markedly superior to that by LB400.     

Tick-borne encephalitis virus: primary structure of DNA copies of the genes for strain 205 structural proteins

The nucleotide sequences of the cDNAs of the genes for the structural proteins C, preM, M and E of the tick-borne encephalitis viral strain 205 have been determined. The complete nucleotide sequence of the 5'-end nonstructural region of the viral genome has been studied for the first time. The difference in the amino acids sequences of the structural proteins from different strains (205, Sofiin and Neidorf) of the virus is discussed.     

Development and evaluation of functional gene arrays for detection of selected genes in the environment.

To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (nirS and nirK) genes, ammonia mono-oxygenase (amoA) genes, and methane mono-oxygenase (pmoA) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65 degrees C). The detection limit for nirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (r(2) = 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution of nir and amoA and pmoA gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.     

Repression of the expression of genes encoding xylanolytic enzymes in Aspergillusoryzae by introduction of multiple copies of the xynF1 promoter

A xylanase gene, xynF1, was cloned and characterized from a shoyu koji mould Aspergillus oryzae KBN616. The xynF1 gene was found to be comprised of 1484 bp with ten introns. The deduced amino acid sequence encodes a protein consisting of 327 amino acids (35,402 Da) which is very similar to the fungal family F xylanases such as Aspergillus nidulans XlnC, Aspergillus kawachii XynA and Penicillium chrysogenum XylP. The intron/exon organization of xynF1 is very similar to that of the fungal family F xylanase genes. Plasmid pXPR64, which contains 64 copies of the xynF1 promoter region (PxynF1) in the same direction, was constructed and introduced into A. oryzae. This led to reduced expression of both xylanase and β-xylosidase genes in the transformants. Received: 18 May 1998 / Received revision: 7 July 1998 / Accepted: 9 July 1998