Characterization of Insertions of IS 476 and Two Newly Identified Insertion Sequences, IS 1478 and IS 1479 , in Xanthomonas campestris pv. campestris

Abstract

Thirty-two plasmid insertion mutants were independently isolated from two strains of Xanthomonas campestris pv. campestris in Taiwan. Of the 32 mutants, 14 (44%), 8 (25%), and 4 (12%) mutants resulted from separate insertions of an IS3 family member, IS476, and two new insertion sequences (IS), IS1478 and IS1479. While IS1478does not have significant sequence homology with any IS elements in the EMBL/GenBank/DDBJ database, IS1479 demonstrated 73% sequence homology with IS1051 in X. campestrispv. dieffenbachiae, 62% homology with IS52 inPseudomonas syringae pv. glycinea, and 60% homology with IS5 in Escherichia coli. Based on the predicted transposase sequences as well as the terminal nucleotide sequences, IS1478 by itself constitutes a new subfamily of the widespread IS5 family, whereas IS1479, along with IS1051, IS52, and IS5, belongs to the IS5 subfamily of the IS5 family. All but one of the IS476 insertions had duplications of 4 bp at the target sites without sequence preference and were randomly distributed. An IS476 insertion carried a duplication of 952 bp at the target site. A model for generating these long direct repeats is proposed. Insertions of IS1478 and IS1479, on the other hand, were not random, and IS1478 and IS1479 each showed conservation of PyPuNTTA and PyTAPu sequences (Py is a pyrimidine, Pu is a purine, and N is any nucleotide) for duplications at the target sites. The results of Southern blot hybridization analysis indicated that multiple copies of IS476, IS1478, and IS1479 are present in the genomes of all seven X. campestris pv. campestris strains tested and several X. campestrispathovars.

Conversion ofShigella flexneriserotype 2a to serotype Y in a shigellosis patient due to a single amino acid substitution in the protein product of the bacterial glucosyltransferasegtrIIgene

“…Genomic and plasmid DNA extraction, cloning and plasmid transformation into E. coli were carried out by the methods previously described [18,19]. DNA fragments were purified from gels using Gene Clean III (Bio101).…”

The IS1elements inShigella boydii: horizontal transfer, vertical inactivation and target duplication

“…S. boydii (ATCC 8700) and E. coli W3110 (ATCC 27325) were purchased from the American Type Culture Collection (USA). E. coli DH5α, DH1 and HB101 have been previously described [7,20]. All bacteria were grown at 37°C in Luria–Bertani (LB) broth or on LB plates containing the appropriate antibiotics at the following concentrations: ampicillin 50 μg ml −1 ; chloramphenicol 25 μg ml −1 ; kanamycin 50 μg ml −1 ; streptomycin 200 μg ml −1 .…”

“…Genomic and plasmid DNA were extracted as previously described [20]. Cloning and transformation were carried out in E. coli DH5α according to Maniatis et al [21].…”

“…Cloning and transformation were carried out in E. coli DH5α according to Maniatis et al [21]. Polymerase chain reaction (PCR) amplification, DNA labelling and Southern hybridization were carried out as previously described [6,20]. DNA sequencing was performed using the ABI Prism big dye termination cycle sequencing kit (Applied Biosystems).…”

Análise estrutural e comparativa do genoma de Leifsonia xyli subsp. xyli

“…parainfluenzae (Kauc & Goodgal, 1989 (Philipp et al, 1996) e a análise do genoma de Xyllela fastidiosa (Frohme, et al, 2000). O tamanho do genoma circular de Clostridium sacchaobutylicum NPC 262 foi estimado em 5,3 Mb com uma média de resolução aproximada em 140 kb a partir de fragmentos gerados por enzimas de restrição, separados por PFGE (Keis et al, 2001 (Mahillon & Chandler, 1998;Chen et al, 1999;Schneider, 2002). As ISs são amplamente distribuídas ao longo do genoma de bactérias e suas atividades causam mutações, promovem diversidade genética e algumas vezes adaptações (Schneider et al, 2002).…”