Nucleotide sequence of Rhizobium meliloti insertion sequence ISRm1: homology to IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens.

Nucleotide sequencing of Rhizobium meliloti insertion sequence ISRm1 showed that it is 1319 nucleotides long and includes 32/31 nucleotide terminal inverted repeats. Analysis of five different insertion sites using sequencing primers complementary to sequences within the left and right ends demonstrated that ISRm1 generates five bp direct repeats at the sites of insertion. Although ISRm1 has shown a target preference for certain short regions (hot spots), there was no apparent similarity in the DNA sequences near the insertion sites. On one strand ISRm1 contains two contiguous open reading frames (ORFs) spanning most of its length. ISRm1 was found to have over 50% sequence homology to insertion sequences IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens. Their sizes, the sequences of their inverted repeats, and the characteristics of their insertion sites are also comparable, indicating that ISRm1, IS2 and IS426 belong to a class of related insertion sequences. Comparison of the proteins potentially encoded by these insertion sequences showed that the two ORFs found in ISRm1 are also present in IS2 and IS426, suggesting that they may be functional genes.     

Identification of insertion sequence element IS427 in pTiT37 plasmid DNA of an Agrobacterium tumefaciens T37 isolate

The isolation and characterization of an insertion sequence (IS) element, IS427, from Agrobacterium tumefaciens T37 is described. IS427 is present in three nonidentical copies on the pTiT37 plasmid. The copy that was isolated through transposition on the entrapment vector pUCD800 contains at its ends a 16-bp imperfect inverted repeat and generates a 2-bp duplication of the target DNA. IS427 does not show homology with previously characterized IS elements of A. tumefaciens, based on hybridization experiments and/or sequence comparison.     

Nucleotide sequence analysis of IS427 and its target sites in Agrobacterium tumefaciens T37

We have determined the nucleotide sequence of IS427, an insertion sequence from Agrobacterium tumefaciens T37, IS427 is 1271 bp long, contains 16-bp imperfect terminal inverted repeats, and generates a 2-bp target sequence duplication. It is present at three sites in the pTiT37 plasmid and is absent from the chromosome of A. tumefaciens T37. Each of the IS427 elements sequenced was near a site with sequence homology to integration host factor (IHF)-binding sites which suggested that IHF may be involved in IS427 transposition.     

Sequence and distribution of IS1312: evidence for horizontal DNA transfer from Rhizobium meliloti to Agrobacterium tumefaciens.

Two novel insertion sequences, IS1312 and IS1313, were found in pTiBo542, the Ti plasmid of Agrobacterium tumefaciens strains Bo542 and A281. Nucleotide sequencing and Southern hybridization revealed that IS1312 and IS1313 are homologous to Rhizobium meliloti ISRm1 and ISRm2, respectively. IS1312, ISRm1, and another Agrobacterium insertion sequence, IS426, belong to the same IS3 family of insertion sequences; however, IS1312 is more closely related to the Rhizobium ISRm1 than it is to the Agrobacterium IS426. The distribution patterns of these insertion elements and their sequence similarities suggest that IS1312 and IS1313 were horizontally transferred from R. meliloti to A. tumefaciens.     

Purification and characterization of a novel lactonohydrolase from Agrobacterium tumefaciens

A novel lactonohydrolase, catalyzing the stereospecific hydrolysis of L-pantoyl lactone to L-pantoic acid, was purified 2,400-fold to apparent homogeneity with a 1.96% overall recovery from Agrobacterium tumefaciens AKU 316 through a purification procedure including ammonium sulfate fractionation, and column chromatographies on DEAE-Sephacel, phenyl-Sepharose CL-4B, Sephacryl S-200, Mono-Q and alkyl-Superose. The relative molecular mass of the native enzyme estimated on high-pressure gel permeation chromatography was 62,000 Da, and the subunit molecular mass was estimated to 26,500 Da on SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzes several aromatic lactones, such as 3,4-dihydrocoumarin and homogentisic acid lactone, other than L-pantoyl lactone. The Km and Vmax for L-pantoyl lactone were 3.59 mM and 13.7micromol/min/mg, respectively. The enzymatic activity was inhibited by several chelating reagents, Fe2+, Sn2+, Pb2+, and Fe3+.     

The novel insertion sequences IS1417, IS1418, and IS1419 from Burkholderia glumae and their strain distribution

Three insertion sequences, IS1417, IS1418, and IS1419, were isolated from Burkholderia glumae (formerly Pseudomonas glumae), a gram-negative rice pathogenic bacterium, on the basis of their abilities to activate the expression of the neo gene of the entrap vector pSHI1063. The 1335-bp IS1417 element with 17-bp imperfect terminal inverted repeats was found to be flanked by 5-bp direct repeats of the vector sequence. IS1418 is 865 bp in length and carries 15-bp inverted repeats with a target duplication of 3 bp. The 1215-bp IS1419 sequence is bounded by the 36-bp terminal inverted repeats of the element and 7-bp direct repeats of the vector sequence. IS1417 and IS1418 belong to the IS2 subgroup of the IS3 family and the IS427 subgroup of the IS5 family, respectively, whereas IS1419 does not appear to be a member of any known IS family. Southern blot analysis of DNAs from B. glumae field isolates indicated that those IS elements are widely distributed, but the host range of the three IS elements appears to be limited to B. glumae and some other related species such as B. plantarii. The polymorphisms exhibited in B. glumae isolates suggest that those elements are useful for molecular epidemiological studies of B. glumae infections.     

Cloning and nucleotide sequence of the tzs gene from Agrobacterium tumefaciens strain T37.

The trans-zeatin secretion locus (tzs), from the nopaline Ti plasmid of Agrobacterium tumefaciens strain T37, was cloned and the nucleotide sequence determined. This gene is located in the virulence region of pTiT37. The tzs gene is responsible for the secretion of trans-zeatin into bacterial culture medium and in addition has the cytokinin biosynthetic activity, dimethylallylpyrophosphate:AMP dimethylallyltransferase. Sequence analysis showed an open reading frame of 729 nucleotides, capable of encoding a protein of 27,545 daltons. A single new labelled protein of 27,200 daltons was detected in Escherichia coli maxicells expressing the cloned tzs gene. Significant sequence homology was observed between the tzs and the published tmr sequence from pTiT37.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.     

农杆菌介导的灰葡萄孢T-DNA插入突变体库构建及插入位点分析

【目的】利用农杆菌(Agrobacterium tumefaciens)介导法对灰葡萄孢(Botrytis cinerea)进行转化,构建T-DNA插入突变体库,为从分子水平上认识灰葡萄孢的致病机制打下基础。【方法】以含有pCAMBIA 1390双元载体的农杆菌对灰葡萄孢进行转化,利用潮霉素进行筛选。对抗性稳定的转化子进行生物学和形态学观察,采用离体番茄叶片进行致病性测定。利用TAIL-PCR技术对突变体中T-DNA的旁侧序列进行克隆。【结果】得到了一些突变体,表现为生长速率减缓、产孢能力下降、致病力减弱等。克隆并分析了其中一个突变体中T-DNA插入的位置和旁侧序列。【结论】本实验建立了农杆菌介导的灰葡萄孢转化体系,构建了T-DNA插入的灰葡萄孢突变体库。用TAIL-PCR进行突变体中T-DNA旁侧序列的分析是可行的。     

Cloning and sequence of IS1000, a putative insertion sequence from Thermus thermophilus HB8

The complete nucleotide sequences of two copies of a putative insertion sequence IS1000 from Thermus thermophilus HB8 are presented. IS1000 is 1196 base pairs long, contains a long open reading frame which could code for a protein of 317 amino acids, and has imperfect terminal inverted repeats of 6 base pairs (confirmed by the terminal sequencing of 4.5 copies of IS1000), but does not cause a target site duplication. There are at least 6 copies of IS1000 in the genome of T. thermophilus HB8. A search of the GEN-EMBL data base revealed that the putative 317 amino acid protein had significant homology with open reading frames in the transposable elements IS110 of Streptomyces coelicolor and IS492 of Pseudomonas atlantica.     

Agrobactin, a siderophore from Agrobacterium tumefaciens.

A siderophore (microbial iron transport compound) was isolated from low iron cultures of Agrobacterium tumefaciens B6. The substance was characterized as a threonyl peptide of spermidine acylated with 3 residues of 2,3-dihydroxybenzoic acid, the carbonyl group of 1 residue of the latter participating in an oxazoline ring with the beta-hydroxyl of the threonine moiety. The compound, N-[3-(2,3-dihydroxybenzamido)propyl]-N-[4-(2,3-dihydroxybenzamido)butyl]-2-(2,3-dihydroxyphenyl)-trans-5-methyl-oxazoline-4-carboxamide, was given the trivial name agrobactin. Exposure to acid opened the oxazoline ring to afford agrobactin A. Ferric agrobactin A and agrobactin A itself, but not agrobactin or its ferric complex, had some capacity to feed iron to enterobactin-deficient strains of Escherichia coli and Salmonella typhimurium. Agrobactin was produced by A. tumefaciens in response to iron deficiency and was able to reverse the iron starvation in this organism precipitated by the presence of a ferric complexing agent not utilized by the cells.     

Identification and sequencing of a novel insertion sequence, IS1471, in Burkholderia cepacia strain 2a

Abstract Eighty-one isolates of Rhizoctonia solani AG4 were obtained from soil samples with diverse geographic origins in Korea. Forty-five out of 81 isolates (56%) contained at least one dsRNA molecule with their sizes ranging from 2.3 to > 23.1 kb. Nine different sizes of dsRNA molecules were found and extensive variation in banding patterns was observed among isolates. By comparing the sizes and combinations of dsRNAs, 21 distinct banding patterns were observed. Cytoplasmic fractions from 3 isolates showed the same dsRNA band patterns as those from the total cell extracts. The dsRNAs were stable through 10 successive hyphal tip cultures and serial transfers onto the potato dextrose agar supplemented with cycloheximide or emetine. The presence or absence of dsRNAs was not apparently correlated with disease severity, phenol oxidase activity, and geographic origin.     

Sequence analysis and distribution of an IS3-like insertion element isolated from Salmonella enteritidis

The nucleotide sequence of a 3 kb region immediately upstream of the sef operon of Salmonella enteritidis was determined. A 1230 base pair insertion sequence which shared sequence identity (>75%) with members of the IS3 family was revealed. This element, designated IS1230, had almost identical (90% identity) terminal inverted repeats to Escherichia coli IS3 but unlike other IS3-like sequences lacked the two characteristic open reading frames which encode the putative transposase. S. enteritidis possessed only one copy of this insertion sequence although Southern hybridisation analysis of restriction digests of genomic DNA revealed another fragment located in a region different from the sef operon which hybridised weakly which suggested the presence of an IS1230 homologue. The distribution of IS1230 and IS1230-like elements was shown to be widespread amongst salmonellas and the patterns of restriction fragments which hybridised differed significantly between Salmonella serotypes and it is suggested that IS1230 has potential for development as a differential diagnostic tool.     

Isolation and analysis of IS6120, a new insertion sequence from Mycobacterium smegmatis

Insertion sequence IS6120 from Mycobacterium smegmatis was identified by its ability to transpose into different sites in the lambda repressor gene, cl857, carried on an Escherichia coli/mycobacteria shuttle plasmid. IS6120 is a novel 1.5 kb insertion sequence, which has 24-bp imperfect terminal inverted repeats and generates 9-bp duplications of the target DNA following insertion. IS6120 is present in at least three copies in M. smegmatis but was not found in other species, including Mycobacterium tuberculosis. Nucleotide sequence analysis revealed that IS6120 contains two open reading frames, one of which encodes a putative transposase with similarities to those found in IS256 from Staphylococcus aureus, IST2 from Thiobacillus ferrooxidans, and ISRm3 from Rhizobium meliloti. The fact that IS6120 does not recognize a consensus target sequence for insertion and has no homologous sequences in the other strains studied makes IS6120 useful for transposon mutagenesis in mycobacteria.     

IS200: a Salmonella-specific insertion sequence

A new IS element (IS200) has been identified in Salmonella. The sequence was identified as an IS element by the following criteria: its insertion caused the mutation hisD984; six copies of the sequence are present in strain LT2 of S. typhimurium; and transposition of the sequence has been observed on several occasions. IS200 is found in almost all Salmonella species examined but is absent from most other enteric bacteria. The specificity of this element for Salmonella (and the absence of IS1-IS4 from Salmonella) suggest that transfer of insertion sequences between bacterial groups may be less extensive than is commonly believed. Alternatively, the distribution may suggest that these elements play a selectively important role in bacteria.     

Genome sequence of Agrobacterium tumefaciens strain F2, a bioflocculant-producing bacterium

Agrobacterium tumefaciens F2 is an efficient bioflocculant-producing bacterium. But the genes related to the metabolic pathway of bioflocculant biosynthesis in strain F2 are unknown. We present the draft genome of A. tumefaciens F2. It could provide further insight into the biosynthetic mechanism of polysaccharide-like bioflocculant in strain F2.     

Biochemical characterization of a novel hydantoin racemase from Agrobacterium tumefaciens C58

A novel hydantoin racemase gene of Agrobacterium tumefaciens C58 (AthyuA2) has been cloned and expressed in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure and showed an apparent molecular mass of 27000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of approximately 100000 Da, suggesting that the native enzyme is a tetramer. The optimum pH and temperature for hydantoin racemase activity were 7.5 and 55 degrees C, respectively, with L-5-ethylhydantoin as substrate. Enzyme activity was strongly inhibited by Cu(2+) and Hg(2+). No effect on enzyme activity was detected with any other divalent cations, EDTA or DTT, suggesting that it is not a metalloenzyme. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings.