Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains

A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.     

Complete sequence of a 184-kilobase catabolic plasmid from Sphingomonas aromaticivorans F199

The complete 184,457-bp sequence of the aromatic catabolic plasmid, pNL1, from Sphingomonas aromaticivorans F199 has been determined. A total of 186 open reading frames (ORFs) are predicted to encode proteins, of which 79 are likely directly associated with catabolism or transport of aromatic compounds. Genes that encode enzymes associated with the degradation of biphenyl, naphthalene, m-xylene, and p-cresol are predicted to be distributed among 15 gene clusters. The unusual coclustering of genes associated with different pathways appears to have evolved in response to similarities in biochemical mechanisms required for the degradation of intermediates in different pathways. A putative efflux pump and several hypothetical membrane-associated proteins were identified and predicted to be involved in the transport of aromatic compounds and/or intermediates in catabolism across the cell wall. Several genes associated with integration and recombination, including two group II intron-associated maturases, were identified in the replication region, suggesting that pNL1 is able to undergo integration and excision events with the chromosome and/or other portions of the plasmid. Conjugative transfer of pNL1 to another Sphingomonas sp. was demonstrated, and genes associated with this function were found in two large clusters. Approximately one-third of the ORFs (59 of them) have no obvious homology to known genes.     

呋喃丹降解菌CDS-1的双标记菌株的构建

用Sau3AI消化呋喃丹降解菌Sphingomonassp.CDS-1的基因组DNA,将所得DNA片段与BamHⅠ酶切的启动子探针载体pRobe-GFP酶连后转化E.coliDH5α感受态细胞,在选择性平板上培养,从大约1×104个菌落中筛选到50个含启动子片段的阳性克隆。挑选其中一个发光强度最强的阳性克隆F7,将它的重组质粒pF7用EcoRⅠ和HindⅢ双酶切后得到包含Sphingomonassp.CDS-1启动子和gfp基因的DNA片段,将该片段克隆到广宿主载体pPZP201上,得到pPZP201-gfp质粒。将pPZP201-gfp通过三亲接合转移至Sphingomonassp.CDS-1中得到GFP标记菌株CDS-gfp,经荧光显微镜观察,gfp基因在CDS-gfp中表达量很高。对标记菌株进行连续传代10次(48h/次),发现pPZP201-gfp依然存在,而且发光明显。通过NotⅠ酶切位点把linA基因连接到pUT/mini-Tn5上构建新的转座子载体pUT/mini-Tn5-linA。以pRK600为辅助质粒将pUT/mini-Tn5-linA引入到CDS-1中,linA基因通过转座作用,插入到CDS-gfp的染色体中,得到双标记菌株CDS-GFP-LinA。该菌株是一株能同时降解γ-六六六和呋喃丹的基因工程菌,本研究的结果为研究Sphingomonassp.CDS-1的生态学行为奠定了基础。     

Use of tagged random hexamer amplification (TRHA) to clone and sequence minute quantities of DNA--application to a 180 kb plasmid isolated from Sphingomonas F199.

We have developed a novel method to clone and sequence minute quantities of DNA. The method was applied to sequence a 180 kb plasmid pNL1. The first step was the production of a size distributed population of DNA molecules that were derived from the 180 kb plasmid pNL1. The first step was accomplished by a random synthesis reaction using Klenow fragment and random hexamers tagged with a T7 primer at the primer 5'-end (T7-dN6, 5'-GTAATACGACTCACTATAGGGCNNNNNN-3'. In the second step, Klenow-synthesized molecules were amplified by PCR using T7 primer (5'-GTAATACGACTCACTATAGGGC-3'). With a hundred nanograms starting plasmid DNA from pNL1, we were able to generate Klenow-synthesized molecules with sizes ranging from 28 bp to >23 kb which were detectable on an agarose gel. The Klenow-synthesized molecules were then used as templates for standard PCR with T7 primer. PCR products of sizes ranging from 0.3 to 1.3 kb were obtained for cloning and sequencing. From the same Klenow-synthesized molecules, we were also able to generate PCR products with sizes up to 23 kb by long range PCR. A total 232.5 kb sequences were obtained from 593 plasmid clones and over twenty putative genes were identified. Sequences from these 593 clones were assembled into 62 contigs and 99 individual sequence fragments with a total unique sequence of 86.3 kb.     

Growth in coculture stimulates metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2

Metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the beta-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that Sphingomonas sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for Sphingomonas sp. SRS2. This suggests that strain SRS1 supplies amino acids to Sphingomonas sp. SRS2, thereby leading to rapid metabolism of (14)C-labeled isoproturon to (14)CO(2) and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by Sphingomonas sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by Sphingomonas sp. strain SRS2 to a similar extent.     

Construction and characterization of a minimized version of the HIV-1 pNL4-3 plasmid and its application for pseudotyping HIV-1 vectors

The pUC-based pNL4-3 plasmid is the most widely used vector for in vitro manipulations of the HIV-1 proviral sequences. We have developed a minimal plasmid (pCHUS) based on pNL4-3, which may be useful to facilitate the design of HIV-based constructions. The strategy that has allowed us to construct pCHUS includes the following steps: (1) pNL-3 digestion by using restriction sites contained within the long terminal repeats (LTRs), (2) recircularization of the fragment containing the pUC18 sequence, (3) amplification of the LTR region restored in the previous step, (4) double digestion of the products obtained in steps 2 and 3, (5) ligation of the fragment containing ColE1+Amp^R with the LTR fragment, (6) linearization of the intermediate plasmid obtained, and (7) insertion of the fragment containing the proviral genome into the linearized vector. The pCHUS plasmid includes essential information for its replication and antibiotic selection in bacteria, but it lacks all the unnecessary sequences. Our results suggest that pCHUS may be more advantageous than pNL4-3 for in vitro manipulation of the HIV-1 proviral genome. In addition, we describe a potential application of this new vector for pseudotyping HIV-1 particles, using a single plasmid transfection, as a more helpful alternative to the traditionally used cotransfection method.     

Induction of aromatic catabolic activity in Sphingomonas aromaticivorans strain F199

Enzyme induction studies with Sphingomonas aromaticivorans F199 demonstrated that both toluene and naphthalene induced expression of both naphthalene and toluene catabolic enzymes. However, neither aromatic compound induced expression of all the enzymes required for complete mineralization of either naphthalene or toluene. Activity measurements in combination with gene sequence analyses indicate that growth on either aromatic substrate in the absence of the other is, therefore, sub-optimal and is predicted to lead to the build-up of metabolites due to imbalance in toluene or naphthalene catabolic enzyme activities. Growth on toluene may be further inhibited by the co-expression of two toluene catabolic pathways, as predicted from gene sequence analyses. One of these pathways may potentially result in the formation of a dead-end intermediate, possibly benzaldehyde. In contrast, either p-cresol or benzoate can support high levels of growth. Analyses of promoter region sequences on the F199 aromatic catabolic plasmid, pNL1, suggest that additional regulatory events are modulated through the interaction of BphR with Sigma54 type promoters and through the binding of a regulator upstream of p-cresol catabolic genes and xylM. We hypothesize that the unusual gene clustering in strain F199 is optimized for simultaneous degradation of multiple aromatic compound classes, possibly in response to the heterogeneous composition of aromatic structures in the fossil organic matter present in the deep Atlantic Coastal Plain sediments from which this bacterium was isolated. Received 26 April 1999/ Accepted in revised form 16 August 1999     

Sphingomonas astaxanthinifaciens sp. nov., a novel astaxanthin-producing bacterium of the family Sphingomonadaceae isolated from Misasa, Tottori, Japan

A red-pigmented, Gram-negative, motile, strictly aerobic, mesophilic, oval- or short rod-shaped bacterium (TDMA-17(T)) was isolated from fresh water collected at Misasa, a radioactive site in Japan. TDMA-17(T) was slightly tolerant against gamma-ray irradiation, and effectively produced carotenoids (2.8 mg g(-1) dry cells) including, astaxanthin and astaxanthin isomers. Phylogenetic analysis based on 16S rRNA gene sequences placed TDMA-17(T) in a distinct lineage in the family Sphingomonadaceae, and the highest degree of sequence similarity determined were to Sphingomonas aerolata NW12(T) (94.5%), Sphingomonas aurantiaca MA101b(T) (94.0%), Sphingomonas melonis DAPP-PG 224(T) (94.0%), Sphingomonas asaccharolytica IFO 15499(T) (93.9%) and Sphingomonas abaci C42(T) (93.9%). The major fatty acids were C(17 : 1)omega6c (33.0%) and C(18 : 1)omega7c (20.8%). The DNA G+C content was 67.7 mol%. The presence of Q-10 as the main ubiquinone, the presence of Sphingomonadaceae-specific sphingoglycolipid in the polar lipid profiles, the presence of 2-hydroxy fatty acids and the absence of 3-hydroxy fatty acids supported the identification of this strain as a member of the genus Sphingomonas sensu stricto. Phylogenetic distinctiveness and unique phenotypic characteristics differentiated strain TDMA-17(T) from the closely related Sphingomonas species. The results of polyphasic taxonomic analyses suggested that TDMA-17(T) represents a novel Sphingomonas species, for which the name Sphingomonas astaxanthinifaciens sp. nov. is proposed. The type strain is TDMA-17(T) (=NBRC 102146=CCUG 53608).     

Isolation and characterization of polycyclic aromatic hydrocarbons-degrading Sphingomonas sp. strain ZL5

A bacterial strain ZL5, capable of growing on phenanthrene as a sole carbon and energy source but not naphthalene, was isolated by selective enrichment from crude-oil-contaminated soil of Liaohe Oil Field in China. The isolate was identified as a Sphingomonas sp. strain on the basis of 16S ribosomal DNA analysis. Strain ZL5 grown on phenanthrene exhibited catechol 2,3-dioxygenase (C23O) activity but no catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxygenase activities. This suggests that the mode of cleavage of phenanthrene by strain ZL5 could be meta via the intermediate catechol, which is different from the protocatechuate way of other two bacteria, Alcaligenes faecelis AFK2 and Nocardioides sp. strain KP7, also capable of growing on phenanthrene but not naphthalene. A resident plasmid (approximately 60 kb in size), designated as pZL, was detected from strain ZL5. Curing the plasmid with mitomycin C and transferring the plasmid to E. coli revealed that pZL was responsible for polycyclic aromatic hydrocarbons degradation. The C23O gene located on plasmid pZL was cloned and overexpressed in E. coli JM109(DE3). The ring-fission activity of the purified C23O from the recombinant E. coli on dihydroxylated aromatics was in order of catechol > 4-methylcatechol > 3-methylcatechol > 4-chlorocatechol > 3,4-dihydroxyphenanthrene > 3-chlorocatechol.     

Carbofuran degradation mediated by three related plasmid systems

Two carbofuran-metabolizing Sphingomonas strains, TA and CD, were isolated from soils with differing histories of exposure to carbofuran. These strains were compared with a previously described strain, Sphingomonas sp. CFO6, with regard to growth rate, formation of metabolites, and plasmid content and structure. Extensive regions of similarity were observed between the three different plasmid systems as evidenced by cross hybridization. In addition, all three systems harbor IS1412, an insertion sequence (IS) element involved in heat-induced loss of carbofuran phenotype in CFO6, and heat-induced carbofuran deficient mutants of all three strains correlated with loss of IS1412. A carbofuran deficient mutant of TA generated by induction of IS elements was complemented by reintroduction of the wild-type plasmid, confirming the presence of genes required for carbofuran metabolism on this plasmid. Carbofuran metabolism in these three strains is clearly linked via plasmids of different numbers and sizes that share extensive common regions, and carbofuran-degrading genes may be associated with active IS elements.     

The function of cytoplasmic flavin reductases in the reduction of azo dyes by bacteria

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an "azo reductase." The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic "azo reductases," which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.     

Changes to the structure of Sphingomonas spp. communities associated with biodegradation of the herbicide isoproturon in soil

The phenyl-urea herbicide isoproturon is a major contaminant of surface and ground-water in agricultural catchments. Earlier work suggested that within-field spatial variation of isoproturon degradation rate resulted from interactions between catabolizing Sphingomonas spp. and pH. In the current study, changes to the structure of Sphingomonas communities during isoproturon catabolism were investigated using Sphingomonas-specific 16S rRNA gene primers. Growth-linked catabolism at high-pH (>7.5) sites was associated with the appearance of multiple new denaturing gradient gel electrophoresis (DGGE) bands. At low-pH sites, there was no change in DGGE banding at sites in which there was cometabolism, but at sites in which there was growth-linked catabolism, degradation was associated with the appearance of a new band not present at high pH sites. Sequencing of DGGE bands indicated that a strain related to Sphingomonas mali proliferated at low pH sites, while strain Sphingomonas sp. SRS2, a catabolic strain identified in earlier work, together with several further Sphingomonas spp., proliferated at high-pH sites. The data indicate that degradation was associated with complex changes to the structure of Sphingomonas spp. communities, the precise nature of which was spatially variable.     

Genetic and molecular analyses of Escherichia coli N-acetylneuraminate lyase gene.

Two plasmids containing the N-acetylneuraminate lyase (NALase) gene (nanA) of Escherichia coli, pNL1 and pNL4, were constructed. Immunoprecipitation analysis indicated that the 35,000-dalton protein encoded in pNL4 was NALase. The synthesis of NALase in E. coli carrying these plasmids was constitutive.     

遗传稳定型六六六、多菌灵降解基因工程菌构建

通过PCR的方法从六六六降解菌Sphingomonas sp.BHC-A扩增出完整的脱氯化氢酶基因linA.将其克隆到含有mini-Tn5的自杀性质粒pUT4K上,构建成质粒pUT/mini-Tn5-linA.通过三亲杂交,在辅助质粒RK600的帮助下,将pUT/mini-Tn5-linA转移到一株高效降解多菌灵菌株Rhodococcus sp.DJL-6中.利用mini-Tn5的转座作用将linA基因整合到DJL-6的染色体DNA上,得到工程菌株DJL-6A.该工程菌具有同时降解多菌灵和六六六的功能,且对于初始浓度为0.05 μg/mL和5 μg/mL的六六六的降解活性与亲本菌株BHC-A相当.在不加任何选择压力的条件下工程菌株进行连续传代,结果证明linA基因可以持续稳定的存在于宿主的染色体DNA上.     

Sequence and analysis of the 46.6-kb plasmid pA1 from Sphingomonas sp. A1 that corresponds to the typical IncP-1beta plasmid backbone without any accessory gene

Sphingomonas sp. A1 (strain A1) is capable of directly incorporating macromolecules (e.g., alginate) through the specialized import system--"super-channel." Here, we report the complete DNA sequence and genetic organization of plasmid pA1 from strain A1. Nucleotide sequence analysis revealed that pA1 comprises 46,557 bp encoding 49 open reading frames (ORFs) with 65% G+C content and abundant GCCG/CGGC motifs. Many predicted pA1 ORFs showed high similarity to pA81 ORFs; pA81 is supposedly a self-transmissible promiscuous incompatibility (Inc) group P-1beta plasmid. Unlike any reported IncP-1 plasmids, pA1 contains no inserted mobile genetic elements. The genetic organization and predicted pA1 ORFs showed greater similarity to the IncP-1beta plasmid backbone than to the IncP-1alpha plasmid backbone. pA1 contains restriction site-associated repeat sequences typical of the IncP-1beta but absent in the IncP-1alpha and delta subgroups. Thus, the overall pA1 structure corresponds to that of the typical IncP-1beta plasmids. Phylogenetic analysis of the replication-associated proteins suggested that pA1 may have diverged later along with the two IncP-1beta plasmids--pA81 and pB4. The 2.4-kb duplicates of stable inheritance genes klcAB and korC in pA1 possibly resulted from insertion and/or recombination events via the repeat sequences flanking these duplicates.     

Isolation and characterization of dibenzofuran-degrading bacteria

Two bacterial strains capable of utilizing dibenzofuran (DF) as a sole carbon source were isolated from soil samples of reclaimed land. The strains designated HL1 and HL7 were identified as Klebsiella sp. and Sphingomonas sp., respectively, on the basis of biochemical characteristics and the sequences of the 16S ribosomal DNA. Sphingomonas sp. strain HL7 degraded non-, mono- and also dichlorinated DF and dibenzo-p-dioxin (DD). Klebsiella sp. strain HL1 was able to degrade non- and monochlorinated DFs and DDs, but not dichlorinated ones. The metabolites formed from DF by strains HL1 and HL7 were similar to those by dioxin-degrading bacteria Sphingomonas sp. strain RW1 except for salicylic acid and catechol. Strain HL7 had a gene homologous to that encoding the dioxin dioxygenase alpha-subunit (dxnA1) gene of Sphingomonas sp. strain RW1. However, Southern hybridization analysis showed that the size of an EcoRV-digested genomic fragment involving the dioxin dioxygenase gene of strain HL7 was smaller than that of strain RW1, and that strain HL1 did not have the homologous gene. Strains HL1 and HL7 provided useful information regarding the dioxygenase genes.     

HIV假病毒用于p24抗原检测试剂研制的研究

为了解决在研制新型HIV检测试剂盒时遇到的p24蛋白阳性对照物保存不便、阳性对照物稳定性不高以及蛋白结构和聚集形式与天然HIV病毒的p24存在差异等问题,通过将改造的HIV-1骨架质粒pNL43 EGFP R-E-和包膜蛋白质粒pGX-C共转染293T细胞后分离细胞培养液,获得具有单轮感染活性的HIV-1假病毒。通过对此假病毒颗粒的天然衣壳蛋白p24和市售HIV检测试剂盒中的p24蛋白阳性对照物的阳性反应有效性和保存稳定性的检测,证明了HIV-1假病毒p24蛋白具有更易保存、更稳定、更接近天然病毒p24蛋白的优点,适宜作为HIV检测试剂盒阳性对照物。     

A second [2Fe-2S] ferredoxin from Sphingomonas sp. Strain RW1 can function as an electron donor for the dioxin dioxygenase

The first step in the degradation of dibenzofuran and dibenzo-p-dioxin by Sphingomonas sp. strain RW1 is carried out by dioxin dioxygenase (DxnA1A2), a ring-dihydroxylating enzyme. An open reading frame (fdx3) that could potentially specify a new ferredoxin has been identified downstream of dxnA1A2, a two-cistron gene (J. Armengaud, B. Happe, and K. N. Timmis, J. Bacteriol. 180:3954-3966, 1998). In the present study, we report a biochemical analysis of Fdx3 produced in Escherichia coli. This third ferredoxin thus far identified in Sphingomonas sp. strain RW1 contained a putidaredoxin-type [2Fe-2S] cluster which was characterized by UV-visible absorption spectrophotometry and electron paramagnetic resonance spectroscopy. The midpoint redox potential of this ferredoxin (E'(0) = -247 +/- 10 mV versus normal hydrogen electrode at pH 8.0) is similar to that exhibited by Fdx1 (-245 mV), a homologous ferredoxin previously characterized in Sphingomonas sp. strain RW1. In in vitro assays, Fdx3 can be reduced by RedA2 (a reductase similar to class I cytochrome P-450 reductases), previously isolated from Sphingomonas sp. strain RW1. RedA2 exhibits a K(m) value of 3.2 +/- 0.3 microM for Fdx3. In vivo coexpression of fdx3 and redA2 with dxnA1A2 confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase.     

Mechanism of bacitracin resistance in gram-negative bacteria that synthesize exopolysaccharides.

Four representative species from three genera of gram-negative bacteria that secrete exopolysaccharides acquired resistance to the antibiotic bacitracin by stopping synthesis of the exopolysaccharide. Xanthomonas campestris, Sphingomonas strains S-88 and NW11, and Escherichia coli K-12 secrete xanthan gum, sphingans S-88 and NW11, and colanic acid, respectively. The gumD gene in X. campestris is required to attach glucose-P to C55-isoprenyl phosphate, the first step in the assembly of xanthan. A recombinant plasmid carrying the gumD gene of X. campestris restored polysaccharide synthesis to bacitracin-resistant exopolysaccharide-negative mutants of X. campestris and Sphingomonas strains. Similarly, a newly cloned gene (spsB) from strain S-88 restored xanthan synthesis to the same X. campestris mutants. However, the intergeneric complementation did not extend to mutants of E. coli that were both resistant to bacitracin and nonproducers of colanic acid. The genetic results also suggest mechanisms for assembling the sphingans which have commercial potential as gelling and viscosifying agents.