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MvaT proteins are members of the H-NS family of proteins in pseudomonads. The IncP-7 conjugative plasmid pCAR1 carries an mvaT -homologous gene, pmr . In Pseudomonas putida KT2440 bearing pCAR1, pmr and the chromosomally carried homologous genes, turA and turB , are transcribed at high levels, and Pmr interacts with TurA and TurB in vitro . In the present study, we clarified how the three MvaT proteins regulate the transcriptome of P. putida KT2440(pCAR1). Analyses performed by a modified chromatin immunoprecipitation assay with microarray technology (ChIP-chip) suggested that the binding regions of Pmr, TurA, and TurB in the P. putida KT2440(pCAR1) genome are almost identical; nevertheless, transcriptomic analyses using mutants with deletions of the genes encoding the MvaT proteins during the log and early stationary growth phases clearly suggested that their regulons were different. Indeed, significant regulon dissimilarity was found between Pmr and the other two proteins. Transcription of a larger number of genes was affected by Pmr deletion during early stationary phase than during log phase, suggesting that Pmr ameliorates the effects of pCAR1 on host fitness more effectively during the early stationary phase. Alternatively, the similarity of the TurA and TurB regulons implied that they might play complementary roles as global transcriptional regulators in response to plasmid carriage.

The aim of this chapter is to give an overview of aerobic biodegradation of petroleum aromatic compounds. Among the wide variety of petroleum aromatic compounds that occur, BTEX, PAHs, and HACs are the principal components of petroleum and their toxicities are relatively higher than other components, such as alkanes. This chapter will focus on degradation pathways and enzymes used by microorganisms capable of metabolizing BTEX, four representative PAHs, naphthalene, phenanthrene, fluorene, and pyrene and three HACs, carbazole, dibenzothiophene and dibenzofuran.

Shewanella oneidensis MR-1 is capable of reducing extracellular electron acceptors, such as metals and electrodes, through the Mtr respiratory pathway, which consists of the outer membrane cytochromes OmcA and MtrC and associated proteins MtrA and MtrB. These proteins are encoded in the mtr gene cluster (omcA-mtrCAB) in the MR-1 chromosome. Here, we investigated the transcriptional mechanisms for the mtr genes and demonstrated that omcA and mtrC are transcribed from two upstream promoters, P omcA and P mtrC , respectively. In vivo transcription and in vitro electrophoretic mobility shift assays revealed that a cAMP receptor protein (CRP) positively regulates the expression of the mtr genes by binding to the upstream regions of P omcA and P mtrC . However, the expression of omcA and mtrC was differentially regulated in response to culture conditions; specifically, the expression from P mtrC was higher under aerobic conditions than that under anaerobic conditions with fumarate as an electron acceptor, whereas expression from P omcA exhibited the opposite trend. Deletion of the region upstream of the CRP-binding site of P omcA resulted in a significant increase in promoter activity under aerobic conditions, demonstrating that the deleted region is involved in the negative regulation of P omcA . Taken together, the present results indicate that transcription of the mtr genes is regulated by multiple promoters and regulatory systems, including the CRP/cAMP-dependent regulatory system and yet-unidentified negative regulators.

Nucleoid-associated proteins (NAPs) play a role in changing the shape of microbial DNA, making it more compact and affecting the regulation of transcriptional networks in host cells. Genes that encode NAPs include H-NS family proteins (H-NS, Ler, MvaT, BpH3, Bv3F, HvrA, and Lsr2), FIS, HU, IHF, Lrp, and NdpA, and are found in both microbial chromosomes and plasmid DNA. In the present study, NAP genes were distributed among 442 plasmids out of 4602 plasmid sequences, and many H-NS family proteins, and HU, IHF, Lrp, and NdpA were found in plasmids of Alpha-, Beta-, and Gammaproteobacteria, while HvrA, Lsr2, HU, and Lrp were found in other classes including Actinobacteria and Bacilli. Larger plasmids frequently carried multiple NAP genes. In addition, NAP genes were more frequently found in conjugative plasmids than non-transmissible plasmids. Several host cells carried the same types of H-NS family proteins on both their plasmids and chromosome(s), while this was not observed for other NAPs. Recent studies have shown that NAP genes on plasmids and chromosomes play important roles in the physical and regulatory integration of plasmids into the host cell.

SummaryCarbazole 1,9a-dioxygenase (CARDO), a member of the Rieske nonheme iron oxygenase system (ROS), consists of a terminal oxygenase (CARDO-O) and electron transfer components (ferredoxin [CARDO-F] and ferredoxin reductase [CARDO-R]). We determined the crystal structures of the nonreduced, reduced, and substrate-bound binary complexes of CARDO-O with its electron donor, CARDO-F, at 1.9, 1.8, and 2.0 Å resolutions, respectively. These structures provide the first structure-based interpretation of intercomponent electron transfer between two Rieske [2Fe-2S] clusters of ferredoxin and oxygenase in ROS. Three molecules of CARDO-F bind to the subunit boundary of one CARDO-O trimeric molecule, and specific binding created by electrostatic and hydrophobic interactions with conformational changes suitably aligns the two Rieske clusters for electron transfer. Additionally, conformational changes upon binding carbazole resulted in the closure of a lid over the substrate-binding pocket, thereby seemingly trapping carbazole at the substrate-binding site.

Isolated from Pseudomonas resinovorans CA10, pCAR1 is a 199-kb plasmid that carries genes involved in the degradation of carbazole and dioxin. The nucleotide sequence of pCAR1 has been determined previously. In this study, we characterized pCAR1 in terms of its replication, maintenance, and conjugation. By constructing miniplasmids of pCAR1 and testing their establishment in Pseudomonas putida DS1, we show that pCAR1 replication is due to the repA gene and its upstream DNA region. The repA gene and putative oriV region could be separated in P. putida DS1, and the oriV region was determined to be located within the 345-bp region between the repA and parW genes. Incompatibility testing using the minireplicon of pCAR1 and IncP plasmids indicated that pCAR1 belongs to the IncP-7 group. Monitoring of the maintenance properties of serial miniplasmids in nonselective medium, and mutation and complementation analyses of the parWABC genes, showed that the stability of pCAR1 is attributable to the products of the parWAB genes. In mating assays, the transfer of pCAR1 from CA10 was detected in a CA10 derivative that was cured of pCAR1 (CA10dm4) and in P. putida KT2440 at frequencies of 3 × 10 −1 and 3 × 10 −3 per donor strain, respectively. This is the first report of the characterization of this completely sequenced IncP-7 plasmid.

Nocardioides aromaticivorans IC177 is a gram-positive carbazole degrader. The genes encoding carbazole degradation ( car genes) were cloned into a cosmid clone and sequenced partially to reveal 19 open reading frames. The car genes were clustered into the carAaCBaBbAcAd and carDFE gene clusters, encoding the enzymes responsible for the degradation of carbazole to anthranilate and 2-hydroxypenta-2,4-dienoate and of 2-hydroxypenta-2,4-dienoate to pyruvic acid and acetyl coenzyme A, respectively. The conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and mononuclear iron, the Rieske-type [2Fe-2S] cluster, and flavin adenine dinucleotide were found in the deduced amino acid sequences of carAa , carAc , and carAd , respectively, which showed similarities with CarAa from Sphingomonas sp. strain KA1 (49% identity), CarAc from Pseudomonas resinovorans CA10 (31% identity), and AhdA4 from Sphingomonas sp. strain P2 (37% identity), respectively. E s cherichia coli cells expressing CarAaAcAd exhibited major carbazole 1,9a-dioxygenase (CARDO) activity. These data showed that the IC177 CARDO is classified into class IIB, while gram-negative CARDOs are classified into class III or IIA, indicating that the respective CARDOs have diverse types of electron transfer components and high similarities of the terminal oxygenase. Reverse transcription-PCR (RT-PCR) experiments showed that the carAaCBaBbAcAd and carDFE gene clusters are operonic. The results of quantitative RT-PCR experiments indicated that transcription of both operons is induced by carbazole or its metabolite, whereas anthranilate is not an inducer. Biotransformation analysis showed that the IC177 CARDO exhibits significant activities for naphthalene, carbazole, and dibenzo- p -dioxin but less activity for dibenzofuran and biphenyl.

Acinetobacter sp. strain 20B was isolated based on the ability to utilize dimethyl sulfide as the sole sulfur source. Since strain 20B oxidized indole as well as dimethyl sulfide, indigo production by recombinant Escherichia coli clones carrying Acinetobacter DNA was used as a selection for cloning genes encoding dimethyl sulfide oxidation genes. The gene encoding an indole-oxidizing enzyme was also found to oxidize dimethyl sulfide. The dimethyl sulfide-oxidizing enzyme genes consisted of six open reading flames designated dsoABCDEF. The deduced amino acid sequences of dsoABCDEF were homologous with those of the multicomponent phenol hydroxylases. DsoABCDEF oxidized dimethyl sulfide to dimethyl sulfoxide, and dimethyl sulfoxide to dimethyl sulfone.

Terrabacter sp. strain DBF63 is capable of degrading fluorene (FN) to tricarboxylic acid cycle intermediates via phthalate and protocatechuate. Genes were identified for the protocatechuate branch of the β-ketoadipate pathway (pcaR, pcaHGBDCFIJ) by sequence analysis of a 70 kb DNA region of the FN-catabolic linear plasmid pDBF1. RT-PCR analysis of RNA from DBF63 cells grown with FN, dibenzofuran, and protocatechuate indicated that the pcaHGBDCFIJ operon was expressed during both FN and protocatechuate degradation in strain DBF63. The gene encoding β-ketoadipate enol-lactone hydrolase (pcaD) was not fused to the next gene, which encodes γ-carboxymuconolactone decarboxylase (pcaC), in strain DBF63, even though the presence of the pcaL gene (the fusion of pcaD and pcaC) within a pca gene cluster has been thought to be a Gram-positive trait. Quantitative RT-PCR analysis revealed that pcaD mRNA levels increased sharply in response to protocatechuate, and a biotransformation experiment with cis,cis-muconate using Escherichia coli carrying both catBC and pcaD indicated that PcaD exhibited β-ketoadipate enol-lactone hydrolase activity. The location of the pca gene cluster on the linear plasmid, and the insertion sequences around the pca gene cluster suggest that the ecologically important β-ketoadipate pathway genes, usually located chromosomally, may be spread widely among bacterial species via horizontal transfer or transposition events.

Carbazole 1,9a-dioxygenase, which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. The electron-transport complex between CARDO-O and CARDO-F crystallizes at 293 K using hanging-drop vapour diffusion with the precipitant PEG MME 2000 (type I crystals) or PEG 3350 (type II). Blossom-shaped crystals form from a pile of triangular plate-shaped crystals. The type I crystal diffracts to a maximum resolution of 1.90 A and belongs to space group P2(1), with unit-cell parameters a = 97.1, b = 89.8, c = 104.9 A, alpha = gamma = 90, beta = 103.8 degrees. Diffraction data for the type I crystal gave an overall Rmerge of 8.0% and a completeness of 100%. Its VM value is 2.63 A3 Da(-1), indicating a solvent content of 53.2%.

Dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 is comprised of three components, i.e., terminal oxygenase (DbfA1, DbfA2), putative [3Fe-4S] ferredoxin (ORF16b product), and unidentified ferredoxin reductase. We produced DbfA1 and DbfA2 using recombinant Escherichia coli BL21(DE3) cells as a native form and purified the complex to apparent homogeneity. We also produced and purified a putative [3Fe-4S] ferredoxin encoded by ORF16b, which is located 2.5 kb downstream of the dbfA1A2 genes, with E. coli as a histidine (His)-tagged form. The reconstructed DFDO system with three purified components, i.e., DbfA1A2, His-tagged ORF16b product, and His-tagged PhtA4 (which is a tentative reductase derived from the phthalate dioxygenase system of strain DBF63) could convert fluorene to 9-fluorenol (specific activity: 14.4 nmol min(-1) mg(-1)) and convert dibenzofuran to 2,2',3-trihydroxybiphenyl. This indicates that the ORF16b product can transport electrons to the DbfA1A2 complex; and therefore it was designated DbfA3. Based on spectroscopic UV-visible absorption characteristics and electron paramagnetic resonance spectra, DbfA3 was elucidated to contain a [3Fe-4S] cluster. Ferredoxin interchangeability analysis using several types of ferredoxins suggested that the redox partner of the DbfA1A2 complex may be rather specific to DbfA3.

The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 catalyzes the dioxygenation of carbazole; the 9aC carbon bonds to a nitrogen atom and its adjacent 1C carbon as the initial reaction in the mineralization pathway. The CARDO system is composed of ferredoxin reductase (CarAd), ferredoxin (CarAc), and terminal oxygenase (CarAa). CarAc acts as a mediator in the electron transfer from CarAd to CarAa. To understand the structural basis of the protein-protein interactions during electron transport in the CARDO system, the crystal structure of CarAc was determined at 1.9 A resolution by molecular replacement using the structure of BphF, the biphenyl 2,3-dioxygenase ferredoxin from Burkholderia cepacia strain LB400 as a search model. CarAc is composed of three beta-sheets, and the structure can be divided into two domains, a cluster-binding domain and a basal domain. The Rieske [2Fe-2S] cluster is located at the tip of the cluster-binding domain, where it is exposed to solvent. While the overall folding of CarAc and BphF is strongly conserved, the properties of their surfaces are very different from each other. The structure of the cluster-binding domain of CarAc is more compact and protruding than that of BphF, and the distribution of electric charge on its molecular surface is very different. Such differences are thought to explain why these ferredoxins can act as electron mediators in respective electron transport chains composed of different-featured components.

Summary Pseudomonas putida DS1 is able to utilize dimethyl sulphide through dimethyl sulphoxide, dimethyl sulphone (DMSO2), methanesulphonate (MSA) and sulphite as a sulphur source. We previously demonstrated that sfnR encoding a σ54-dependent transcriptional regulator is essential for DMSO2 utilization by P. putida DS1. To identify the target genes of SfnR, we carried out transposon mutagenesis on an sfnR disruptant (DMSO2-utilization-defective phenotype) using mini-Tn5, which contains two outward-facing constitutively active promoters; as a result, we obtained a mutant that restored the ability to utilize DMSO2. The DMSO2-positive mutant carried a mini-Tn5 insertion in the intergenic region between two opposite-facing operons, sfnAB and sfnFG. Both sfnA and sfnB products were similar to acyl-CoA dehydrogenase family proteins, whereas sfnF and sfnG encoded a putative NADH-dependent FMN reductase (SfnF) and an FMNH2-dependent monooxygenase (SfnG). Disruption and complementation of the sfn genes indicated that the sfnG product is essential for DMSO2 utilization by P. putida DS1. Furthermore, an enzyme assay demonstrated that SfnG is an FMNH2-dependent DMSO2 monooxygenase that converts DMSO2 to MSA. It was revealed that the expression of the sfnFG operon is directly activated by the binding of SfnR at its upstream region. Site-directed mutagenesis of the SfnR binding sequences allowed us to define a potential recognition sequence for SfnR. These results provided insight into regulation of sulphate starvation-induced genes in bacteria.

Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with jasmonic acid (JA) for 1/2h yielded a cDNA of a gene tentatively named RERJ1 that is upregulated in response to exogenous JA. Northern blot analysis indicated that the RERJ1 mRNA levels peaked at 1/2-1h after the addition of jasmonic acid and then decreased gradually. RERJ1 encodes a transcriptional regulator with a basic helix-loop-helix motif. The phenotypes of transgenic rice plants overexpressing sense or antisense RERJ1 mRNA demonstrated that RERJ1 is involved in the growth inhibition of rice shoots caused by JA. Other biological functions of RERJ1 are discussed from an evolutionary standpoint.

The carbazole-catabolic plasmid pCAR1 isolated from Pseudomonas resinovorans strain CA10 was sequenced in its entirety; and it was found that pCAR1 carries the class II transposon Tn4676 containing carbazole-degradative genes. In this study, a new plasmid designated pCAR2 was isolated from P. putida strain HS01 that was a transconjugant from mating between the carbazole-degrader Pseudomonas sp. strain K23 and P. putida strain DS1. Southern hybridization and nucleotide sequence analysis of pCAR1 and pCAR2 revealed that the whole backbone structure was very similar in each. Plasmid pCAR2 was self-transmissible, because it was transferred from strain HS01 to P. fluorescens strain IAM12022 at the frequency of 2 x 10(-7) per recipient cell. After the serial transfer of strain HS01 on rich medium, we detected the transposition of Tn4676 from pCAR2 to the HS01 chromosome. The chromosome-located copy of Tn4676 was flanked by a 6-bp target duplication, 5'-AACATC-3'. These results experimentally demonstrated the transferability of pCAR2 and the functionality of Tn4676 on pCAR2. It was clearly shown that plasmid pCAR2 and transposon Tn4676 are active mobile genetic elements that can mediate the horizontal transfer of genes for the catabolism of carbazole.

Among the toxic and persistent compounds that have been released in the environment by human action, the aromatic compounds are of major concern. In spite of their recalcitrance to degradation by most organisms, bacterial strains have adapted to them in consequence of the acquisition of the necessary catabolic abilities to utilize aromatic compounds, and thus rapidly adapting bacteria are regarded as the experimental systems of choice in understanding how catabolic genes end up with regulated expression (8). Carbazole is a recalcitrant N-heterocyclic aromatic compound derived from coal tar and shale oil and possesses mutagenic and toxic activity (1). Pseudomonas resinovorans strain CA10, a gram-negative bacterium isolated from activated sludge, degrades carbazole as the sole source of carbon, nitrogen, and energy (27). In strain CA10, carbazole is initially converted into anthranilate and 2-hydroxypenta-2,4-dienoate through a three-step upper pathway catalyzed by carbazole 1,9a-dioxygenase (CarA), the meta-cleavage enzyme (CarB), and the meta-cleavage compound hydrolase (CarC) (Fig. ​(Fig.1A)1A) (32, 33). CarA is a multicomponent dioxygenase system composed of terminal oxygenase component CarAa, ferredoxin component CarAc, and ferredoxin reductase component CarAd. meta-Cleavage enzyme CarB is a heterotetrameric enzyme (α2β2) composed of CarBa and CarBb subunits. The resulting metabolite, anthranilate, is a naturally occurring compound formed through tryptophan degradation (19) and is also known as an important intermediate in the metabolism of many N-heterocyclic compounds, such as indole (15, 21, 23), o-nitrobenzoate (5, 10), and quinaldine (28). Via dioxygenation catalyzed by class IB (2) two-component anthranilate 1,2-dioxygenase (AntDO) composed of the terminal oxygenase component (AntAB) and the reductase component (AntC), followed by spontaneous deamination and decarboxylation, anthranilate is converted to catechol, which is further degraded to tricarboxylic acid (TCA) cycle intermediates (25, 26). On the other hand, 2-hydroxypenta-2,4-dienoate is metabolized by the meta-cleavage pathway enzymes CarD, CarE, and CarF to form TCA cycle intermediates (26). FIG. 1. (A) Carbazole degradation pathway via anthranilate in P. resinovorans strain CA10. (B) Genetic organization of car and ant gene clusters located on carbazole-degradative plasmid pCAR1. Black and gray pentagons represent the genes and their transcriptional ... As shown in Fig. ​Fig.1B,1B, the genes encoding components (or subunits) of each upper catabolic enzymes constitute the carAaBaBbCAcAd gene cluster, although the carAa gene is tandemly duplicated (32, 33). Just downstream of the upper car gene cluster, there is the lower carDFE gene cluster (26). On the other hand, the antABC gene cluster of strain CA10 is located in the region 21 kb upstream of the car gene cluster, and interestingly, the 5′ portion of the antA gene has been transposed onto the region immediately upstream of the carAa gene along with ISPre1 to form a fusion open reading frame (ORF), ORF9, implying the developmental scheme of the novel genetic structure of the car gene cluster and its flanking region (Fig. ​(Fig.1B)1B) (26). The ant and car genes of strain CA10 exist on carbazole-degradative plasmid pCAR1 (26). Recently, the nucleotide sequence of pCAR1 was completely determined, and both ant and car gene clusters were found within the 73-kb transposon Tn4676 (24). On pCAR1, three ORFs encoding putative transcriptional regulators were found, and two of them (ORF22 and ORF23), both of whose products belong to AraC/XylS family, were also located within Tn4676 (24). It has been known that the expression of antABC genes encoding two-component AntDO is induced in the presence of anthranilate itself in Acinetobacter sp. strain ADP1 (4), but the transcriptional regulation of the antABC genes has not been elucidated yet. In this report, we describe that the ant genes located on pCAR1 are transcribed as a single mRNA that originated from the Pant promoter and that the product of ORF23, designated AntR, is required for the expression from Pant induced by anthranilate. We also describe that another copy of Pant within the transposed region upstream of the carAa gene is functional and that the transcription of carAa is also induced by anthranilate, indicating that transposition of the Pant promoter has made the transcription of car genes induced by anthranilate and regulated by AntR.

Thirteen dibenzofuran (DF)-utilizing bacteria carrying the DF terminal dioxygenase genes homologous to those of Terrabacter sp. strain DBF63 (dbfA1A2) were newly isolated from activated sludge samples. The amplified ribosomal DNA restriction analysis and the hybridization analyses showed that these strains were grouped into five genetically different types of bacteria. The sequence analyses of the 16S rRNA genes and the dbfA1A2 homologues from these five selected isolates revealed that the isolates belonged to the genus Rhodococcus, Terrabacter or Janibacter and that they shared 99-100% conserved dbfA1A2 homologues. We investigated the genetic organizations flanking the dbfA1A2 homologues and showed that the minimal conserved DNA region present in all five selected isolates consisted of an approximately 9.0-kb region and that their outer regions became abruptly non-homologous. Among them, Rhodococcus sp. strain DFA3 possessed not only the 9.0-kb region but also the 6.2-kb region containing dbfA1A2 homologues. Sequencing of their border regions suggested that some genetic rearrangement might have occurred with insertion sequence-like elements. Also, within their conserved regions, some insertions or deletions were observed.

Sphingomonas sp. strain A4 is capable of utilizing acenaphthene and acenaphthylene as sole carbon and energy sources, but it is unable to grow on other polycyclic aromatic hydrocarbons (PAHs). The genes encoding terminal oxygenase components of ring-hydroxylating dioxygenase (arhA1 and arhA2) were isolated from this strain by means of the ability to oxidize indole to indigo of the Escherichia coli clone containing electron transport proteins from phenanthrene-degrading Sphingobium sp. strain P2. The translated products of arhA1 and arhA2 exhibited moderate sequence identity (less than 56%) to large and small subunits of dioxygenase of other ring-hydroxylating dioxygenases. Biotransformation with recombinant E. coli clone revealed the broad substrate specificity of this oxygenase toward several PAHs including acenaphthene, acenaphthylene, naphthalene, phenanthrene, anthracene and fluoranthene. Southern hybridization analysis revealed the presence of a putative arhA1 homologue on a locus different from that of the arhA1 gene. Insertion inactivation of the arhA1 gene in strain A4 suggested that the gene but not the putative homologue one was involved in the degradation of acenaphthene and acenaphthylene in this strain.

4-Hydroxybenzoate polyprenyl diphosphate transferase (4HPT) is the key enzyme that transfers the prenyl side chain to the benzoquione frame in ubiquinone (UQ) biosynthesis. The Arabidopsis AtPPT1 cDNA encoding 4HPT was cloned by reverse transcription-polymerase chain reaction (RT-PCR) based on the information of the Arabidopsis genomic sequence, and the function of the gene was determined. Heterologous expression of the AtPPT1 gene enabled restoration of the respiratory ability and UQ synthesis in a yeast mutant that was defective in 4HPT activity. The mitochondrial fraction that was prepared from the yeast mutant, which expressed the AtPPT1 gene, exhibited 4HPT enzymatic activity with geranyl diphosphate (GPP) as the prenyl substrate. This indicated that the AtPPT1 gene encodes active 4HPT with a broad substrate specificity in terms of the prenyl donor. The AtPPT1 mRNA was predominantly expressed in the flower cluster, and the green fluorescent protein (GFP) fused with the signal peptide of AtPPT1 was translocated into the mitochondria. T-DNA insertion mutation that disrupts the AtPPT1 gene in Arabidopsis resulted in the arrest of embryo development at an early stage of zygotic embryogenesis. These results demonstrate that the AtPPT1 gene involved in the biosynthesis of mitochondrial UQ plays an essential role in embryo development in Arabidopsis .

Southern hybridization analysis of the genomes from the newly-isolated 10 carbazole (CAR)-utilizing bacteria revealed that 8 of the isolates carried gene clusters homologous to the CAR-catabolic car operon of Pseudomonas resinovorans strain CA10. Sequencing analysis showed that two car operons and the neighboring regions of Pseudomonas sp. strain K23 are nearly identical to that of strain CA10. In contrast to strains CA10 and K23, carEF genes did not exist downstream of the car gene cluster of Janthinobacterium sp. strain J3. In the car gene clusters, strains CA10, K23 and J3 have Rieske-type ferredoxin as a component of carbazole dioxygenase, although Sphingomonas sp. strain KA1 possesses a putidaredoxin-type ferredoxin. We confirmed that this putidaredoxin-type ferredoxin CarAc can function as an electron mediator to CarAa of strain KA1. In the upstream regions of the carJ3 and carKA1 gene clusters, ORFs whose deduced amino acid sequences showed homology to GntR-family transcriptional regulators were identified.

Microbial dimethyl sulfide (DMS) conversion is thought to be involved in the global sulfur cycle. We isolated Pseudomonas putida strain DS1 from soil as a bacterium utilizing DMS as a sole sulfur source, and tried to elucidate the DMS conversion mechanism of strain DS1 at biochemical and genetic level. Strain DS1 oxidized DMS to dimethyl sulfone (DMSO(2)) via dimethyl sulfoxide, whereas the oxidation was repressed in the presence of sulfate, suggesting that a sulfate starvation response is involved in DMS utilization by strain DS1. Two of the five DMS-utilization-defective mutants isolated by transposon 5 (Tn 5) mutagenesis had a Tn 5 insertion in the ssuEADCBF operon, which has been reported to encode a two-component monooxygenase system (SsuED), an ABC-type transporter (SsuABC), and a small protein (SsuF), and also to play a key role in utilization of sulfonates and sulfate esters in another bacterium, P. putida strain S-313. Disruption of ssuD and SsuD enzymatic activity demonstrated that methanesulfonate is a metabolic intermediate of DMS and desulfonated by SsuD. Disruption of ssuC or ssuF also led to a DMS-utilization-defective phenotype. Another two mutants had a defect in a gene homologous to pa2354 from P. aeruginosa PAO1, which encodes a putative transcriptional regulator, while the remaining mutant had a defect in cysM encoding O-acetylserine (thiol)-lyase B.

Phthalate is a metabolic intermediate of the pathway of fluorene (FN) degradation via angular dioxygenation. A gene cluster responsible for the conversion of phthalate to protocatechuate was cloned from the dibenzofuran (DF)- and FN-degrading bacterium Terrabacter sp. strain DBF63 and sequenced. The genes encoding seven catabolic enzymes, oxygenase large subunit of phthalate 3,4-dioxygenase (phtA1), oxygenase small subunit of phthalate 3,4-dioxygenase (phtA2), cis-3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase (phtB), [3Fe-4S] or [4Fe-4S] type of ferredoxin (phtA3), ferredoxin reductase (phtA4), 3,4-dihydroxyphthalate decarboxylase (phtC) and putative regulatory protein (phtR), were found in the upstream region of the angular dioxygenase gene (dbfA1A2), encoded in this order. Escherichia coli carrying phtA1A2BA3A4 genes converted phthalate to 3,4-dihydroxyphthalate, and the 3,4-dihydroxyphthalate decarboxylase activity by E. coli cells carrying phtC was finally detected with the introduction of a Shine-Dalgarno sequence in the upstream region of its initiation codon. Homology analysis on the upstream region of the pht gene cluster revealed that there was an insertion sequence (IS) (ISTesp2; ORF14 and its flanking region), part of which was almost 100% identical to the orf1 and its flanking region adjacent to the extradiol dioxygenase gene ( bphC1) involved in the DF degradation of Terrabacter sp. strain DPO360 [Schmid et al. (1997) J Bacteriol 179:53-62]. This suggests that ISTesp2 plays a role in the metabolism of aromatic compounds in Terrabacter sp. strains DBF63 and DPO360.

Five sets of large and small subunits of terminal oxygenase ( ahdA1 [ a – e ] and ahdA2 [ a – e ]) and a single gene set encoding ferredoxin ( ahdA3 ) and ferredoxin reductase ( ahdA4 ) were found to be scattered through 15.8- and 14-kb DNA fragments of phenanthrene-degrading Sphingobium sp. strain P2. RT-PCR analysis indicated the inducible and specific expression of ahdA3 , ahdA4 , and three sets of genes for terminal oxygenase ( ahdA1 [ c – e ] and ahdA2 [ c – e ]) in this strain grown on phenanthrene. The biotransformation experiments with resting cells of Escherichia coli JM109 harboring recombinant ahd genes revealed that AhdA2cA1c, AhdA1dA2d, and AhdA1eA2e can all function as a salicylate 1-hydroxylase which converts salicylate, a metabolic intermediate of phenanthrene, to catechol in cooperation with the electron transport proteins AhdA3A4. The first two oxygenases exhibited a broad range of substrate specificities such that they also catalyzed the hydroxylation of methyl- and chloro-substituted salicylates to produce their corresponding substituted catechols.

The carbazole-degrading (car) operon on the chromosome of Pseudomonas stutzeri strain OM1 showed >99% identity to that in the 72.8 kb catabolic transposon, Tn4676, on plasmid pCAR1. Southern hybridization using probes prepared from the pCAR1 sequence and sequencing analyses showed that the OM1 chromosome contained the 55 kb DNA region, almost all of which was a part of Tn4676, flanked by two copies of novel insertion sequence, ISPst3, and included the car gene.

A recombinant Rhizobium strain, PBK3-IS, that constitutively expressed the oxygenase component of carbazole 1,9a-dioxygenase from Sphingomonas sp. strain KA1, was constructed. In the water-cultured siratro rhizospheres inoculated with strain PBK3-IS, 48% of the dibenzofuran was removed within 3 days (initial substrate, 25 microg). Similar results were obtained in soil-cultured siratro rhizospheres using sterile vermiculite. When non-sterile field soils were used instead of sterile vermiculite, the inoculated recombinant strain could grow on the siratro root in all soils tested, except for wet paddy field.

We studied the degradation of carbazole (CAR) and 2,3-dichlorodibenzo-p-dioxin (2,3-DCDD) in soils inoculated with carbazole- and dioxin-degrader Pseudomonas resinovorans strain CA10. By using Tn5-based transposon delivery systems, this bacterium was chromosomally marked with a tandem green fluorescent protein (gfp) gene. Real-time competitive PCR and direct counting using the (gfp) marker were employed to monitor the total number of carbazole 1,9a-dioxygenase gene (carAa) and survival of CA10 cells in the soil and soil slurry microcosms. Bioaugmentation studies indicated that the survival of the marked CA10 cells in soil microcosms was strongly influenced by pH and organic matter. While the number of the marked CA10 cells decreased rapidly in pH 6 with low organic matter, a high cell density was maintained in pH 7.3 with 2.5% organic matters up to 21 days after inoculation. In pH 7.3 soil, the period needed for complete degradation of CAR (100 microg kg(-1)) was markedly shortened from 21 to 7 days by the inoculation with the CA10 cells. Single inoculation of CA10 cells into the soil slurry system of 2,3-DCDD-contaminated soil enhanced the degradation of 2,3-DCDD from 25.0% to 37.0%. In this system, the population density of CA10 cells and the total number of carAa gene were maintained up to 14 days after inoculation. By repeated inoculation (every 2 days) with CA10 cells each at a density of 10(9) CFU g(-1) of soil, almost all of the 2,3-DCDD (1 microg kg(-1)) was degraded within 14 days. Results of these experiments suggest that P. resinovorans strain CA10 may be an important resource for bioremediation of CAR and chlorinated dibenzo-p-dioxin in contaminated soils.

To obtain basic information towards applying a dibenzofuran (DF)-degrader Terrabacter sp. strain DBF63 to bioremediate dioxin-contaminated soil, we investigated the degradative potential of strain DBF63 for either chlorinated or polychlorinated dibenzo- p -dioxins and dibenzofurans (Cl x DD/Cl x DF) in soil. In the soil slurry system with a soil to water ratio of 1:5 (w/v), the DF-grown DBF63 cells degraded 90% of 1 ppm 2,8-Cl 2 DF, whereas only 40% of 1 ppm 2,3-Cl 2 DD during the 7-day incubation. The degradation rates of 2-ClDF, 2-ClDD, 2,8-Cl 2 DF and 2,3-Cl 2 DF by strain DBF63 in the soil slurry system (5-day incubation) were approximately 89%, 65%, 78% and 32%, respectively. These results suggest that strain DBF63 was able to degrade mono- to dichlorinated dibenzofurans more effectively than mono- to dichlorinated dibenzo- p -dioxins. Using the same soil slurry system, we performed a preliminary bioremediation experiment using the actual dioxin-contaminated soil at an incineration site. We found that approximately 10% of tetra- to hexa-chlorinated congeners was decreased by a single inoculation with DBF63 cells within a 7-day incubation.

The preferred substrates in angular dioxygenation, monooxygenation, and lateral dioxygenation by dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 and carbazole 1,9a-dioxygenase (CARDO) from Pseudomonas resinovorans strain CA10 are shown to be distinctly different. The preferred oxygenation reactions suggest that DFDO evolved from a polycyclic aromatic hydrocarbon dioxygenase and that its most preferred substrates were fluorene and 9-fluorenone. The angular dioxygenases involved in the degradation pathway of dibenzofuran (dioxin) and fluorene are closely related in function, while CARDO is a novel enzyme not only phylogenetically but also functionally.

Nineteen polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from environmental samples in Kuwait, Indonesia, Thailand, and Japan by enrichment with either naphthalene or phenanthrene as a sole carbon source. Sequence analyses of the 16-S rRNA gene indicated that at least seven genera (Ralstonia, Sphingomonas, Burkholderia, Pseudomonas, Comamonas, Flavobacterium, and Bacillus) were present in this collection. Determination of the ability of the isolates to use PAH and its presumed catabolic intermediates suggests that the isolates showed multiple phenotypes in terms of utilization and degradation pathways. The large subunit of the terminal oxygenase gene (phnAc) from Burkholderia sp. strain RP007 hybridized to 32% (6/19) of the isolates, whilst gene probing using the large subunit of terminal oxygenase gene (pahAc) from Pseudomonas putida strain OUS82 revealed no pahAc-like genes amongst the isolates. Using three degenerated primer sets (pPAH-F/NR700, AJ025/26, and RieskeF/R), targeting a conserved region with the genes encoding the large subunit of terminal oxygenase successfully amplified material from 6 additional PAH-degrading isolates. Sequence analyses showed that the large subunit of terminal oxygenase in 4 isolates was highly homologous to the large subunit of naphthalene dioxygenase gene from Ralstonia sp. strain U2. However, we could not obtain any information on the oxygenase system involved in the naphthalene and/or phenathrene degradation by 7 other strains. These results suggest that PAH-degrading bacteria are diverse, and that there are still many unidentified PAH-degrading bacteria.

2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl-HODA) hydrolase (CumD), an enzyme of the cumene biodegradation pathway encoded by the cumD gene of Pseudomonas fluorescens IP01, was purified to homogeneity from an overexpressing Escherichia coli strain. SDS-polyacrylamide gel electrophoresis and gel filtration demonstrated that it is a dimeric enzyme with a subunit molecular mass of 32 kDa. The pH optima for activity and stability were 8.0 and 7.0–9.0, respectively. The enzyme exhibited a biphasic Arrhenius plot of catalysis with two characteristic energies of activation with a break point at 20°C. The enzyme has a K m of 7.3 μM and a k cat of 21 s −1 for 6-isopropyl-HODA (150 mM phosphate, pH 7.5, 25°C), and its substrate specificity covers larger C6 substituents compared with another monoalkylbenzene hydrolase, TodR Unlike TodF, CumD could slightly hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA). A mutant enzyme as to a putative active site residue, S103A, had 10 5 -fold lower activity than that of the wild-type enzyme.

Acinetobacter guillouiae strain 20B can utilize dimethyl sulfide (DMS) as the sole sulfur source and degrade chloroethylenes. We report here the complete 4,648,418-bp genome sequence for this strain, which contains 4,367 predicted coding sequences (CDSs), including a well-characterized DMS degradative operon.

A crystal was obtained of the complex between reduced terminal oxygenase and oxidized ferredoxin components of carbazole 1,9a-dioxygenase. The crystal belonged to space group P21 and diffracted to 2.25 Å resolution., The initial reaction in bacterial carbazole degradation is catalyzed by carbazole 1,9a-dioxygenase, which consists of terminal oxygenase (Oxy), ferredoxin (Fd) and ferredoxin reductase components. The electron-transfer complex between reduced Oxy and oxidized Fd was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant under anaerobic conditions. The crystal diffracted to a maximum resolution of 2.25 Å and belonged to space group P21, with unit-cell parameters a = 97.3, b = 81.6, c = 116.2 Å, α = γ = 90, β = 100.1°. The V M value is 2.85 Å3 Da−1, indicating a solvent content of 56.8%.

Phytoalexins are antimicrobial specialised metabolites that are produced by plants in response to pathogen attack. Momilactones and phytocassanes are major diterpenoid phytoalexins in rice that are synthesised from geranylgeranyl diphosphate that is derived from the methylerythritol phosphate (MEP) pathway. We have previously reported that rice cells overexpressing the basic leucine zipper (bZIP) transcription factor OsTGAP1 exhibit a hyperaccumulation of momilactones and phytocassanes, with hyperinductive expression of momilactone and phytocassane biosynthetic genes and MEP pathway genes, upon response to a chitin oligosaccharide elicitor. For a better understanding of OsTGAP1-mediated regulation of diterpenoid phytoalexin production, we identified OsTGAP1-interacting proteins using yeast two-hybrid screening. Among the OsTGAP1-interacting protein candidates, a TGA factor OsbZIP79 was investigated to verify its physical interaction with OsTGAP1 and involvement in the regulation of phytoalexin production. An in vitro pull-down assay demonstrated that OsTGAP1 and OsbZIP79 exhibited a heterodimeric as well as a homodimeric interaction. A bimolecular fluorescence complementation analysis also showed the interaction between OsTGAP1 and OsbZIP79 in vivo. Intriguingly, whereas OsbZIP79 transactivation activity was observed in a transient reporter assay, the overexpression of OsbZIP79 resulted in suppression of the elicitor-inducible expression of diterpenoid phytoalexin biosynthetic genes, and thus caused a decrease in the accumulation of phytoalexin in rice cells. These results suggest that OsbZIP79 functions as a negative regulator of phytoalexin production triggered by a chitin oligosaccharide elicitor in rice cells, although it remains open under which conditions OsbZIP79 can work with OsTGAP1.

Part 1 Genetic and genomic systems 1. Rhodococcus multiple-enzyme and parallel-degradation system for aromatic compounds 2. Appearance and evolution of gamma-hexachlorocyclohexane-degrading bacteria 3. Diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative genes and degrading bacteria 4. Genetic system of organohalide-respiring bacteria 5. Mobile catabolic genetic elements in pseudomonads 6. Adaptation to xenobiotics and toxic compounds by Cupriavidus and Ralstonia with special reference to Cupriavidus metallidurans CH34 and mobile genetic elements 7. Conjugative elements: Host chromosome function modifiers Part 2 Enzyme systems 8. On-line monitoring of biodegradation processes using enzymatic biosensors 9. Structure and function of aromatic-ring hydroxylating dioxygenase system 10. The protocatechuate 4,5-cleavage pathway: Overview and new findings 11. Toluene tolerance systems in Pseudomonas 12. Diversity and evolution of aromatic degradation pathway enzymes in an activated sludge Part 3 Bacterial behavior in natural environmental systems 13. Syntrophic interactions in biodegradative consortia 14. Strategies to reveal genomic function in natural soil systems 15. Monitoring microbial community dynamics to evaluate bioremediation 16. Selective stimulation of aromatic compound degradation by the indigenous marine bacterium Cycloclasticus for bioremediation of oil spills in the marine environment 17. Biofilm as a multicellular bacterial system Index

UTokyo Research掲載「イネの耐病性向上に成功」 URI: http://www.u-tokyo.ac.jp/ja/utokyo-research/research-news/enhanced-disease-resistance-in-rice-plants/, UTokyo Research "Enhanced disease resistance in rice plants" URI: http://www.u-tokyo.ac.jp/en/utokyo-research/research-news/enhanced-disease-resistance-in-rice-plants/

Enterobacterial H-NS-like proteins and Pseudomonas MvaT-like proteins share low homology at the amino acid sequence level, but both can function as xenogeneic silencers and are included in the H-NS family of proteins. H-NS family members have dimerization/oligomerization and DNA-binding domains connected by a flexible linker and form large nucleoprotein complexes using both domains. Pmr, an MvaT-like protein encoded on the IncP-7 carbazole-degradative plasmid pCAR1, is a key regulator of an interaction between pCAR1 and its host Pseudomonas putida KT2440. KT2440 has two transcribed genes that encode the MvaT-like proteins TurA and TurB. Our previous transcriptome analyses suggested that the functions of Pmr, TurA and TurB are non-equivalent, although the detailed underlying mechanisms remain unclear. In this study, we focused on the protein–protein interactions of Pmr, and assessed the homo-oligomerization capacity of various substituted and truncated Pmr derivatives by protein–protein cross-linking analysis. Six of the seven residues identified as important for homo-oligomerization in Pmr were located near the N-terminus, and the putative flexible linker or the region near that was not involved in homo-oligomerization, suggesting that Pmr homo-oligomerization is different from that of enterobacterial H-NS and that the functional mechanism differs between H-NS-like and MvaT-like proteins. In addition, we assessed homo- and hetero-oligomerization of Pmr by surface plasmon resonance analysis and found that the coupling ratio of TurB-Pmr oligomers is smaller than that of Pmr-Pmr or TurA-Pmr oligomers. These results raised the possibility that composition of the hetero-oligomers of Pmr, TurA, and TurB could explain why the different gene sets were affected by either pmr, turA, or turB disruption in our previous studies.

The fluorogenic probe assay, competitive polymerase chain reaction (PCR) and co-extraction with internal standard cells were combined to develop a rapid, sensitive, and accurate quantification method for the copy number of a target carbazole 1,9a-dioxygenase gene (carAa) and the cell number of Pseudomonas sp. strain CA10. The internal standard DNA was modified by replacement of a 20-bp long region with one for binding a specific probe in fluorogenic PCR (TaqMan). The resultant DNA fragment was similar to the corresponding region of the intact carAa gene in terms of G+C content. When used as a competitor in the PCR reaction, the internal standard DNA was distinguishable from the target carAa gene by two specific fluorogenic probes with different fluorescence labels, and was automatically detected in a single tube using the ABI7700 sequence detection system. To minimize variations in the efficiency of cell lysis and DNA extraction between the samples, the co-extraction method was combined. A mini-transposon was used to introduce competitor DNA into the genome of other pseudomonads, and the resultant construct was used as the standard cell. After the addition of a fixed amount of the internal standard cells to soil samples, total DNA was extracted (co-extraction). Using this method, the copy number of the carAa gene and the cell number of strain CA10 in soil samples could be quantified rapidly.

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the carAaAaBaBbCAc (ORF7) Ad genes of carbazole-degrading Pseudomonas sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of 10 genes ( carAaAaBaBbCAcAdDFE ) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between carD and carFE , makes it quite likely that the carFE genes were recruited from another locus. In the 21-kb region upstream from carAa , aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated antABC . Four homologous insertion sequences, IS 5car1 to IS 5car4 , were identified in the neighboring regions of car and ant genes. IS 5car2 and IS 5car3 constituted the putative composite transposon containing antABC . One-ended transposition of IS 5car2 together with the 5′ portion of antA into the region immediately upstream of carAa had resulted in the formation of IS 5car1 and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed. In addition, it was also revealed that the car and ant gene clusters are located on the megaplasmid pCAR1.

A gram-positive bacterium Terrabacter sp. strain DBF63 is able to degrade dibenzofuran (DF) via initial dioxygenation by a novel angular dioxygenase. The dbfA1 and dbfA2 genes, which encode the large and small subunits of the dibenzofuran 4,4a-dioxygenase (DFDO), respectively, were isolated by a polymerase chain reaction-based method. DbfA1 and DbfA2 showed moderate homology to the large and small subunits of other ring-hydroxylating dioxygenases (less than 40%), respectively, and some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands were conserved in DbfA1. DFDO activity was confirmed in Escherichia coli cells containing the cloned dbfA1 and dbfA2 genes with the complementation of nonspecific ferredoxin and ferredoxin reductase component of E. coli. Under this condition, these cells exhibited angular dioxygenation of DF and dibenzo- p -dioxin, and monooxygenation of fluorene, but not angular dioxygenation of carbazole, xanthene, and phenoxathiin. Phylogenetic analysis revealed that DbfA1 formed a branch with recently reported large subunits of polycyclic aromatic hydrocarbon (PAH) dioxygenase from gram-positive bacteria but did not cluster with that of other angular dioxygenases, i.e., DxnA1 from Sphingomonas sp. strain RW1 [Armengaud, J., Happe, B., and Timmis, K. N. J. Bacteriol. 180, 3954–3966, 1998] and CarAa from Pseudomonas sp. strain CA10 [Sato, S., Nam, J.-W., Kasuga, K., Nojiri, H., Yamane, H., and Omori, T. J. Bacteriol. 179, 4850–4858, 1997].

Marine actinomycetes is a group of bacteria that is highly potential in producing novel bioactive compound. It has unique characteristics and is different from other terrestrial ones. Extreme environmental condition is suspected to lead marine actinomycetes produce different types of bioactive compound found previously. The aim of this study was to explore the presence and diversity of NRPS genes in 14 anticancer-producing actinomycetes isolated from marine sediment in Indonesia. PCR amplification and restriction fragment analysis of NRPS genes with Hae III from 14 marine actinomycetes were done to assess the diversity of NRPS genes. Genome mining of one species of marine actinomycetes (strain GMY01) also was employed towards this goal. The result showed that NRPS gene sequence diversity in 14 marine actinomycetes could be divided into 4 groups based on NRPS gene restriction patterns. Analysis of 16S rRNA gene sequences of representatives from each group showed that all isolates belong to genus of Streptomyces . Genome mining result showed that strain GMY01 harboring 10 different NRPS gene clusters that encode secondary metabolites, as pure NRPS or hybrid between NRPS and other compounds. These results indicated that marine actinomycetes having a high potential to be developed as source of anticancer drugs development.

The transformation efficiency of Methylobacillus sp. strain 12S, using electroporation, was unaffected by the growth phase of the cells but competent cells grown at 21 °C had a 1.9 × 103 times higher transformation efficiency than those grown at 30 °C. Heat shock treatment further increased the transformation efficiency up to 7 times.

We isolated the ipdc gene coding for indole-3-pyruvic acid decarboxylase (IPDC), a key enzyme in the indole-3-pyruvic acid pathway for indole-3-acetic acid biosynthesis, in the plant growth-promoting rhizobacterium Azospirillum lipoferum FS. Gel mobility-shift assay showed the presence of two DNA-binding proteins that might be involved in regulation of the ipdc gene expression.

The impacts of plasmid carriage on the host cell were comprehensively analysed using the conjugative plasmid pCAR1 in three different Pseudomonas hosts, P. putida KT2440, P. aeruginosa PAO1 and P. fluorescens Pf0-1. Plasmid carriage reduced host fitness, swimming motility, and resistance to osmotic or pH stress. Plasmid carriage brought about alterations in primary metabolic capacities in the TCA cycle of the hosts. Differentially transcribed genes in the three hosts associated with plasmid carriage were identified by growth phase-dependent transcriptome analyses. Plasmid carriage commonly showed a greater effect on the host transcriptome at the transition and early stationary phases. The transcriptome alterations were similar between KT2440 and PAO1. Transcriptions of numbers of genes encoding ribosomal proteins, F-type ATPase, and RNAP core in both strains were not suppressed enough in the early stationary phase by plasmid carriage. These responses may have been responsible for the reduction in host fitness, motility and stress resistances. Host-specific responses to plasmid carriage were transcriptional changes of genes on putative prophage or foreign DNA regions. The extents of the impacts on host phenotypes and transcriptomes were similarly greatest in KT2440 and lowest in Pf0-1. These findings suggest that host cell function was actively regulated by plasmid carriage.

Sphingomonas sp. strain P2, which is capable of utilizing phenanthrene as a sole carbon and energy source, was isolated from petroleum-contaminated soil in Thailand. Gas chromatography-mass spectrometry and (1)H and (13)C nuclear magnetic resonance analyses revealed two novel metabolites from the phenanthrene degradation pathway. One was identified as 5,6-benzocoumarin, which was derived by dioxygenation at the 1- and 2-positions of phenanthrene, and the other was determined to be 1,5-dihydroxy-2-naphthoic acid. Other metabolites from phenanthrene degradation were identified as 7, 8-benzocoumarin, 1-hydroxy-2-naphthoic acid and coumarin. From these results, it is suggested that strain P2 can degrade phenanthrene via dioxygenation at both 1,2- and 3,4-positions followed by meta-cleavage.

A CH3OH-utilizing bacterium that has the ability to produce extracellular polysaccharide (EPS) was isolated from a soil sample, and was identified as the obligate methylotroph Methylobacillus sp. strain 12S on the basis of its 16S rDNA sequence and growth-substrate specificity. The EPS produced by strain 12S was purified and the sugar composition was analysed by GC-MS and HPLC to reveal that the EPS was a heteropolymer composed of glucosyl, galactosyl, and mannosyl residues in the molar ratio 3:1:1. In order to produce mono- and/or oligosaccharides by single-step fermentation from CH3OH, stain 12S was mutagenized by transposon 5. Among eleven EPS-deficient mutants, three strains were found to accumulate significant amounts of reducing sugars in the media. The amounts of the reducing sugars produced by the mutants ( > ca. 700 mg glucose equivalent/l) were > 11-22 times higher than those produced by the wild-type strain (

We isolated 11 low-indole-3-acetic acid (IAA)-producing mutants of Bradyrhizobium elkanii by Tn5 mutagenesis. The amount of IAA produced by each mutant was 2.2-13.6% of that of the wild-type. It was found by resting cell reactions that the biosynthetic step to convert indole-3-pyruvic acid to indole-3-acetaldehyde was blocked in all the mutants.

Trichloroethylene (TCE) degradation by the recombinant E. coli JM109 harboring a TCE-degradative plasmid (pIO720 or pIO72K) in continuous culture was studied. The ampicillin-resistant plasmid, pIO720, contained the cumene dioxygenase genes and the dimethyl sulfide monooxygenase genes. pIO72K was constructed according to replacement of an ampicillin resistance gene on pIO720 by a kanamycin resistance gene. In the case of E. coli JM109 (pIO720) in continuous culture, TCE degradation activity decreased rapidly after continuous culture started, and the remaining number of host cells harboring pIO720 also decreased rapidly. In the case of E. coli JM109 (pIO72K) in continuous culture, TCE degradation activity was stable during continuous culture for at least 300 h and the number of the host cells harboring pIO72K did not decrease. TCE degradation activity of E. coli JM109 (pIO72K) was the highest at a dilution rate of 0.2 h−1.

In the uncontaminated farm soil, more than 80% of the supplemented acenaphthene, fluoranthene, and pyrene (100 mg/100 g soil) decreased in 90 days, while ratio of removal was about 20%, 30%, and 0%, respectively, in the Kuwaiti oil-contaminated soil. Simultaneous addition of naphthalene, phenanthrene, and anthrathene (100 mg of each compound/100 g soil) led the acenaphthene to a decrease of about 20% to 45% but not of fluoranthene and pyrene. Addition of the farm soil to the Kuwaiti soil did not enhance the decrease of these three PAHs.