Functional analysis of unique class II insertion sequence IS1071

Various xenobiotic-degrading genes on many catabolic plasmids are often flanked by two copies of an insertion sequence, IS1071. This 3.2-kb IS element has long (110-bp) terminal inverted repeats (IRs) and a transposase gene that are phylogenetically related to those of the class II transposons. However, the transposition mechanism of IS1071 has remained unclear. Our study revealed that IS1071 was only able to transpose at high frequencies in two environmental beta-proteobacterial strains, Comamonas testosteroni and Delftia acidovorans, and not in any of the bacteria examined which belong to the alpha- and gamma-proteobacteria. IS1071 was found to have the functional features of the class II transposons in that (i) the final product of the IS1071 transposition was a cointegrate of its donor and target DNA molecules connected by two directly repeated copies of IS1071, one at each junction; (ii) a 5-bp duplication of the target sequence was observed at the insertion site; and (iii) a tnpA mutation of IS1071 was efficiently complemented by supplying the wild-type tnpA gene in trans. Deletion analysis of the IS1071 IR sequences indicated that nearly the entire region of the IRs was required for its transposition, suggesting that the interaction between the transposase and IRs of IS1071 might be different from that of the other well-characterized class II transposons.     

工业废水中阿特拉津降解细菌的遗传和生理多样性

【目的】通过遗传学和生理学实验,揭示分离自工业废水的阿特拉津降解细菌具有遗传和生理多样性,为阐明阿特拉津生物降解的分子机理和阿特拉津降解细菌在污染环境生物修复中的应用提供新见解。【方法】用普通PCR方法检测菌株的阿特拉津降解基因,分析其降解基因组成;用基因组重复序列PCR技术(rep-PCR)分析降解菌株的基因组类型;用Western blot方法检测菌株阿特拉津降解途径的第一个酶三嗪水解酶(TrzN);用不同氮源(阿特拉津、莠灭净、扑草净、西玛津、氰草净、阿特拉通和氰尿酸)和碳源(蔗糖、葡萄糖、麦芽糖、乳糖、柠檬酸钠、乙酸钠和琥珀酸钠)培养降解菌株,通过检测培养液的OD600值,证明菌株能够利用的氮源和碳源种类。【结果】对分离自工业废水的27个阿特拉津降解菌株所进行的阿特拉津降解基因PCR检测表明,其降解基因组成分别为trzN-atzBC、trzN-atzABC和atzADEF;通过rep-PCR实验将27个阿特拉津降解菌株分为7个群;Western blot结果表明,27个菌株中有24个含有三嗪水解酶TrzN;氮源利用实验表明,2个菌株能够利用所有7种氮源生长,其余25个菌株只能利用其中的2-6种;碳源利用实验表明,10个菌株能够利用所有7种碳源生长,其余17个菌株只能利用其中的3-6种。【结论】分离自某工业废水的27株阿特拉津降解功能菌存在相当广泛的遗传和生理学上的多样性,trzN-atzABC降解基因组成为首次发现。     

Real-time reverse transcription PCR analysis of expression of atrazine catabolism genes in two bacterial strains isolated from soil

The level of expression of highly conserved, plasmid-borne, and widely dispersed atrazine catabolic genes (atz) was studied by RT-qPCR in two telluric atrazine-degrading microbes. RT-qPCR assays, based on the use of real-time PCR, were developed in order to quantify atzABCDEF mRNAs in Pseudomonas sp. ADP and atzABC mRNAs in Chelatobacter heintzii. atz gene expression was expressed as mRNA copy number per 10(6) 16S rRNA. In Pseudomonas sp. ADP, atz genes were basally expressed. It confirmed atrazine-degrading kinetics indicating that catabolic activity starts immediately after adding the herbicide. atz gene expression increased transitorily in response to atrazine treatment. This increase was only observed while low amount of atrazine remained in the medium. In C. heintzii, only atzA was basally expressed. atzA and atzB expression levels were similarly and significantly increased in response to atrazine treatment. atzC was not expressed even in the presence of high amounts of atrazine. This study showed that atz genes are basally expressed and up-regulated in response to atrazine treatment. atz gene expression patterns are different in Pseudomonas ADP and C. heintzii suggesting that the host may influence the expression of plasmid-borne atrazine-catabolic potential.     

Fitness drift of an atrazine-degrading population under atrazine selection pressure

Pseudomonas sp. ADP harbouring the atrazine catabolic plasmid ADP1 was subcultured in liquid medium containing atrazine as sole source of nitrogen. After approximately 320 generations, a new population evolved which replaced the initial population. This newly evolved population grew faster and degraded atrazine more rapidly than the initial population. Plasmid profiles and Southern blot analyses revealed that the evolved strain, unlike the ancestral strain, presented a tandem duplication of the atzB gene encoding the second enzyme of the atrazine catabolic pathway responsible for the transformation of hydroxyatrazine to N-isopropylammelide. This duplication resulted from a homologous recombination that occurred between two direct repeats of 6.2 kb flanking the atzB gene and constituted by the insertion sequences IS 1071 , IS Pps1 and a pdhL homologous sequence. This study highlights the IS-mediated plasticity of atrazine-degrading potential and demonstrates that insertion sequences not only help to disperse the atrazine-degrading gene but also improve the fitness of the atrazine-degrading population.     

Characterization of a class II defective transposon carrying two haloacetate dehalogenase genes from Delftia acidovorans plasmid pUO1

The two haloacetate dehalogenase genes, dehH1 and dehH2, on the 65-kb plasmid pUO1 from Delftia acidovorans strain B were found to be located on transposable elements. The dehH2 gene was carried on an 8.9-kb class I composite transposon (TnHad1) that was flanked by two directly repeated copies of IS1071, IS1071L and IS1071R. The dehH1 gene was also flanked by IS1071L and a truncated version of IS1071 (IS1071N). TnHad1, dehH1, and IS1071N were located on a 15.6-kb class II transposon (TnHad2) whose terminal inverted repeats and res site showed high homology with those of the Tn21-related transposons. TnHad2 was defective in transposition because of its lacking the transposase and resolvase genes. TnHad2 could transpose when the Tn21-encoded transposase and resolvase were supplied in trans. These results demonstrated that Tn Had2 is a defective Tn21-related transposon carrying another class I catabolic transposon.     

Characterization of an atrazine-degrading Pseudaminobacter sp. isolated from Canadian and French agricultural soils

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Fourteen bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from soils obtained from two farms in Canada and two farms in France. These strains were indistinguishable from each other based on repetitive extragenic palindromic PCR genomic fingerprinting performed with primers ERIC1R, ERIC2, and BOXA1R. Based on 16S rRNA sequence analysis of one representative isolate, strain C147, the isolates belong to the genus Pseudaminobacter in the family Rhizobiaceae. Strain C147 did not form nodules on the legumes alfalfa (Medicago sativa L.), birdsfoot trefoil (Lotus corniculatus L.), red clover (Trifolium pratense L.), chickpea (Cicer arietinum L.), and soybean (Glycine max L.). A number of chloro-substituted s-triazine herbicides were degraded, but methylthio-substituted s-triazine herbicides were not degraded. Based on metabolite identification data, the fact that oxygen was not required, and hybridization of genomic DNA to the atzABC genes, atrazine degradation occurred via a series of hydrolytic reactions initiated by dechlorination and followed by dealkylation. Most strains could mineralize [ring-U-(14)C]atrazine, and those that could not mineralize atrazine lacked atzB or atzBC. The atzABC genes, which were plasmid borne in every atrazine-degrading isolate examined, were unstable and were not always clustered together on the same plasmid. Loss of atzB was accompanied by loss of a copy of IS1071. Our results indicate that an atrazine-degrading Pseudaminobacter sp. with remarkably little diversity is widely distributed in agricultural soils and that genes of the atrazine degradation pathway carried by independent isolates of this organism are not clustered, can be independently lost, and may be associated with a catabolic transposon. We propose that the widespread distribution of the atrazine-degrading Pseudaminobacter sp. in agricultural soils exposed to atrazine is due to the characteristic ability of this organism to utilize alkylamines, and therefore atrazine, as sole sources of carbon when the atzABC genes are acquired.     

recA-independent recombination between repeated IS50 elements is not caused by an IS50-encoded function.

Certain pBR322-related plasmids containing direct repeats of the insertion element IS50 appear to be unstable in recA Escherichia coli because smaller recombinant derivatives accumulate rapidly in plasmid DNA populations. We show here that (i) this instability is plasmid specific, but not IS50 specific; (ii) it is due to a detrimental effect exerted by these plasmids on bacterial growth; and (iii) the growth impairment is alleviated in cells harboring the smaller recombinant plasmids. Although a recent report had concluded that accumulation of recombinants reflected an IS50-specific recombination function, when correction is made for the relative growth rates of cells containing the parental and recombinant plasmids the evidence for such a recombination function disappears.     

Cloning of the genes for degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine from Rhodococcus sp. strain TE1.

The degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine (2-chloro-4-ethyl-amino-6-isopropylamino-1,3,5-triazine) is associated with an indigenous plasmid in Rhodococcus sp. strain TE1. Plasmid DNA libraries of Rhodococcus sp. strain TE1 were constructed in a Rhodococcus-Escherichia coli shuttle vector, pBS305, and transferred into Rhodococcus sp. strain TE3, a derivative of Rhodococcus sp. strain TE1 lacking herbicide degradation activity, to select transformants capable of growing on EPTC as the sole source of carbon (EPTC+). Analysis of plasmids from the EPTC+ transformants indicated that the eptA gene, which codes for the enzyme required for EPTC degradation, residues on a 6.2-kb KpnI fragment. The cloned fragment also harbored the gene required for atrazine N dealkylation (atrA). The plasmid carrying the cloned fragment could be electroporated into a number of other Rhodococcus strains in which both eptA and atrA were fully expressed. No expression of the cloned genes was evident in E. coli strains. Subcloning of the 6.2-kb fragment to distinguish between EPTC- and atrazine-degrading genes was not successful.     

The role of a groundwater bacterial community in the degradation of the herbicide terbuthylazine

A bacterial community in an aquifer contaminated by s- triazines was studied. Groundwater microcosms were treated with terbuthylazine at a concentration of 100 μg L⁻¹ and degradation of the herbicide was assessed. The bacterial community structure (abundance and phylogenetic composition) and function (carbon production and cell viability) were analysed. The bacterial community was able to degrade the terbuthylazine; in particular, Betaproteobacteria were involved in the herbicide biotransformation. Identification of some bacterial isolates by PCR amplification of the 16S rRNA gene revealed the presence of two Betaproteobacteria species able to degrade the herbicide: Advenella incenata and Janthinobacterium lividum . PCR detection of the genes encoding s -triazine-degrading enzymes indicated the presence of the atz A and atz B genes in A. incenata and the atz B and atz C genes in J. lividum . The nucleotide sequences of the PCR fragments of the atz genes from these strains were 100% identical to the homologous genes of the Pseudomonas sp. strain ADP. In conclusion, the results show the potential for the use of a natural attenuation strategy in the treatment of aquifers polluted with the terbuthylazine. The two bacteria isolated could facilitate the implementation of effective bioremediation protocols, especially in the case of the significant amounts of herbicide that can be found in groundwater as a result of accidental spills.     

Isolation and characterization of an atrazine-degrading bacterium from industrial wastewater in China

AIMS: To isolate and characterize atrazine-degrading bacteria in order to identify suitable candidates for potential use in bioremediation of atrazine contamination. METHODS AND RESULTS: A high efficiency atrazine-degrading bacterium, strain AD1, which was capable of utilizing atrazine as a sole nitrogen source for growth, was isolated from industrial wastewater. 16S rDNA sequencing identified AD1 as an Arthrobacter sp. The atrazine chlorohydrolase gene (atzA) isolated from strain AD1 differed from that found in the Pseudomonas sp. ADP by only one nucleotide. However, it was found located on the bacterial chromosome rather than on plasmids as previously reported for other bacteria. CONCLUSIONS: Atrazine chlorohydrolase gene, atzA, either encoded by chromosome or plasmid, is highly conserved. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison analysis of atrazine degradation gene structure and arrangement in this and other bacteria provides insight into our understanding of the ecology and evolution of atrazine-degrading bacteria.     

Visualization and enumeration of bacteria carrying a specific gene sequence by in situ rolling circle amplification

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 10(6)-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.     

Conjugative plasmids and the degradation of arylsulfonates in Comamonas testosteroni.

Comamonas testosteroni T-2 degrades p-toluenesulfonate (TSA) via p-sulfobenzoate (PSB) and protocatechuate and degrades toluenecarboxylate via terephthalate (TER) and protocatechuate. The appropriate genes are expressed in at least five regulatory units, some of which are also found in C. testosteroni PSB-4 (F. Junker, R. Kiewitz, and A. M. Cook, J. Bacteriol. 179:919-927, 1997). C. testosteroni T-2 was found to contain two plasmids, pTSA (85 kbp) and pT2T (50 kbp); a TSA- mutant (strain TER-1) contained only plasmid pT2T. C. testosteroni PSB-4, which does not degrade TSA, contained one plasmid, pPSB (85 kbp). The type strain contained no plasmids. Conjugation experiments showed that plasmid pTSA (possibly in conjunction with pT2T) was conjugative, and the single copy of the TSA operon (tsaMBCD) with its putative regulator gene (tsaR) in strain T-2 was found on plasmid pTSA, which also carried the PSB genes (psbAC) and presumably transport for both substrates. Plasmid pTSA was assigned to the IncP1 beta group and was found to carry two copies of insertion element IS1071. Plasmid pPSB (of strain PSB-4), which could be maintained in strains with plasmid pTSA or pT2T, was also conjugative and was found to carry the PSB genes as well as to contain two copies of IS1071. In attempted conjugations with the type strain, no plasmid was recovered, but the PSB+ transconjugant carried two copies of IS1071 in the chromosome. We presume the PSB genes to be located in a composite transposon. The genes encoding the putative TER operon and degradation of protocatechuate, with the meta cleavage pathway, were attributed a chromosomal location in strains T-2 and PSB-4.     

Genetic diversity of dioxygenase genes in polycyclic aromatic hydrocarbon-degrading bacteria isolated from mangrove sediments

To investigate the diversity of dioxygenase genes involved in polycyclic aromatic hydrocarbon (PAH)-degradation, a total of 32 bacterial strains were isolated from surface mangrove sediments, from the genera Mycobacterium, Sphingomonas, Terrabacter, Sphingopyxis, Sphingobium and Rhodococcus. Two sets of PCR primers were constructed to detect the nidA-like and nahAc-like sequences of the alpha subunit of the PAH ring-hydroxylating dioxygenase. PCR amplified the DNA fragments from all Gram-positive bacteria by using nidA-like primers and from all Gram-negative bacteria, except two, by using nahAc-like primers. The nidA-like primers showed three subtypes of nidA-like gene: (i) fadA1, clustering with nidA3 from M. vanbaalenii PYR-1, (ii) nidA, clustering with nidA from PYR-1, and (iii) fadA2 clustering with dioxygenase from Arthrobacter sp. FB24. The amplicons detected by nahAc-like primers had high sequence homologies to phnA1a from Sphingomonas sp. CHY-1 and were amplifiable from 8 of the 16 Gram-negative isolates. The primer also generated amplicons that had a 32-36% similarity to phnA1a and 53-93% identity to p-cumate dioxygenase. These results suggest that the nidA-like and nahAc-like genes are prevalent in the PAH-degrading bacteria and that they are useful for determining the presence of PAH-dioxygenase genes in environmental samples.     

Conservation of key elements of natural competence in Lactococcus lactis ssp

Natural competence is active in very diverse species of the bacterial kingdom and probably participates in horizontal gene transfer. Recently, the genome sequence of various species, including Lactococcus lactis, revealed the presence of homologues of competence genes in bacteria, which were not previously identified as naturally transformable. We investigated the conservation among lactococcal strains of key components of the natural competence process in streptococci: (i) comX which encodes a sigma factor, allowing the expression of the late competence genes involved in DNA uptake, (ii) its recognition site, the cin-box and (iii) dprA which encodes a protein shown to determine the fate of incoming DNA. The comX and dprA genes and the cin-box appeared conserved among strains, although some L. lactis ssp. lactis strains presented an inactivated dprA gene. We established that ComX controls the expression of the late competence genes in L. lactis. In conclusion, our work strongly suggests that ComX has the same role in streptococci and L. lactis, i.e. the regulation of late competence genes. It also allowed the identification of a set of L. lactis strains and the construction of a comX overexpression system, which should facilitate the investigation of the natural competence activity in lactococci.     

Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 10⁶-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.     

阿特拉津降解菌株DNS32的降解特性及分类鉴定与降解途径研究

【目的】研究阿特拉津降解菌株DNS32的菌种分类、降解特性及降解途径,丰富阿特拉津降解菌菌种资源。【方法】在长期施用阿特拉津的东北地区寒地黑土中筛选出一株以阿特拉津为唯一氮源生长的降解菌株DNS32,测定其基本降解特性,通过16S rRNA序列分析进行分类鉴定,并利用阿特拉津降解基因PCR扩增技术及降解产物生成量的测定,进一步揭示其降解途径。【结果】实验结果发现DNS32菌株具有较好的降解能力,且在相对较低温度下也具有一定的降解能力。16S rRNA序列分析结果表明DNS32与鲁氏不动杆菌(Acinetobacter lwoffii)16S rRNA序列同源性高达99%。成功地扩增降解基因trzN、atzB及atzC,实验结果表明DNS32遵循Arthrobacter aurescens TC1的降解模式,可将阿特拉津降解为氰尿酸,降解产物的生成量测定也证明了这一点。【结论】实验结果丰富了阿特拉津降解菌菌种资源,为不动杆菌属的阿特拉津降解菌研究提供了参考。     

Identification of the Escherichia coli deoR and cytR gene products.

The protein products encoded by the Escherichia coli deoR and cytR structural genes have been identified based on results obtained from E. coli maxicells harboring (i) recombinant plasmids carrying wild-type deoR and cytR genes, (ii) deletion derivatives of the deoR+ and cytR+ plasmids, (iii) plasmids containing site-specific mutations in the deoR and cytR structural genes, and (iv) plasmids which have transposon Tn1000 inserted into the deoR and cytR structural genes. Analysis of the protein profiles obtained from all the maxicell experiments demonstrated that the deoR gene encodes a protein with a subunit molecular weight of 30,500 and that the product of the cytR gene is a protein with a subunit molecular weight of 37,000.     

Cloning and expression of the chloroplast-encoded rbcL and rbcS genes from the marine diatom Cylindrotheca sp. strain N1

Both the rbcL and rbcS genes, encoding the large and small subunits, respectively, of ribulose 1,5-bisphosphate carboxylase/oxygenase, have been found to be encoded by chloroplast DNA in the marine diatom Cylindrotheca sp. N1. The rbcS gene in this diatom was found to be adjacent to the rbcL gene by a combination of: (i) Southern-blotting analyses, using heterologous probes; (ii) examination of recombinant proteins synthesized in Escherichia coli, directed by cloned rbcL/rbcS genes; and (iii) synthesis of enzymatically active heterologous Rubisco protein in vivo by recombinant DNA procedures using large subunits of Anacystis nidulans and small subunits of Cylindrotheca sp. N1. It appears that two copies of rbcL and rbcS genes are encoded by the chloroplast DNA of this diatom.