Characterization of transposon Tn5469 from the cyanobacterium Fremyella diplosiphon.

A transposon, designated Tn5469, was isolated from mutant strain FdR1 of the filamentous cyanobacterium Fremyella diplosiphon following its insertion into the rcaC gene. Tn5469 is a 4,904-bp noncomposite transposon with 25-bp near-perfect terminal inverted repeats and has three tandemly arranged, slightly overlapping potential open reading frames (ORFs) encoding proteins of 104.6 kDa (909 residues), 42.5 kDa (375 residues), and 31.9 kDa (272 residues). Insertion of Tn5469 into the rcaC gene in strain FdR1 generated a duplicate 5-bp target sequence. On the basis of amino acid sequence identifies, the largest ORF, designated tnpA, is predicted to encode a composite transposase protein. A 230-residue domain near the amino terminus of the TnpA protein has 15.4% amino acid sequence identity with a corresponding domain for the putative transposase encoded by Lactococcus lactis insertion sequence S1 (ISS1). In addition, the sequence for the carboxyl-terminal 600 residues of the TnpA protein is 20.0% identical to that for the TniA transposase encoded by Tn5090 on Klebsiella aerogenes plasmid R751. The TnpA and TniA proteins contain the D,D(35)E motif characteristic of a recently defined superfamily consisting of bacterial transposases and integrase proteins of eukaryotic retroelements and retrotransposons. The two remaining ORFs on Tn5469 encode proteins of unknown function. Southern blot analysis showed that wild-type F. diplosiphon harbors five genomic copies of Tn5469. In comparison, mutant strain FdR1 harbors an extra genomic copy of Tn5469 which was localized to the inactivated rcaC gene. Among five morphologically distinct cyanobacterial strains examined, none was found to contain genomic sequences homologous to Tn5469.     

中华根瘤菌自体诱导物合成酶基因的筛选及其在大肠杆菌中的表达

通过携带有mariner转座子的质粒pJZ290随机插入诱变中华根瘤菌(Sinorhizobium meliloti)建立突变子文库,并从中筛选到自体诱导物(autoinducer,AI)部分缺失突变株YW1。Arbitrary PCR扩增、DNA测序得到YW1基因组DNA中mariner转座子两端侧翼序列,经DNA序列拼接在GenBank上进行同源性分析后获得一个621bp的完整的开放阅读框(ORF),该ORF编码的酶具有206个氨基酸,与草木樨中华根瘤菌(Sinorhizobium medicae)WSM419的LuxI类自体诱导物合成酶(autoinducer synthase)TraI的同源性高达99%。因此,也将该基因命名为traⅠ。将该基因克隆到广宿主范围表达载体pYC12并在大肠杆菌Escherichia coli DH5α中成功表达,C18反相薄层层析(TLC)在阳性重组子培养上清中检测到四种自体诱导物分子,其中的两种正是AI缺失突变株YW1所缺失的AI,这些结果表明该traⅠ基因在苜蓿中华根瘤菌负责合成两种自体诱导物分子,为进一步研究其群体感应系统奠定了理论基础。     

Characterization of a Chlamydomonas Transposon, Gulliver, Resembling Those in Higher Plants

While pursuing a chromosomal walk through the mt+ locus of linkage group VI of Chlamydomonas reinhardtii, I encountered a 12-kb sequence that was found to be present in approximately 12 copies in the nuclear genome. Comparison of various C. reinhardtii laboratory strains provided evidence that the sequence was mobile and therefore a transposon. One of two separate natural isolates interfertile with C. reinhardtii, C. smithii (CC-1373), contained the transposon, but at completely different locations in its nuclear genome than C. reinhardtii; and a second, CC-1952 (S1-C5), lacked the transposon altogether. Genetic analysis indicated that the transposon was found at dispersed sites throughout the genome, but had a conserved structure at each location. Sequence homology between the termini was limited to an imperfect 15-bp inverted repeat. An 8-bp target site duplication was created by insertion; transposon sequences were completely removed upon excision leaving behind both copies of the target site duplication, with minor base changes. The transposon contained an internal region of unique repetitive sequence responsible for restriction fragment length heterogeneity among the various copies of the transposon. In several cases it was possible to identify which of the dozen transposons in a given strain served as the donor when a transposition event occurred. The transposon often moved into a site genetically linked to the donor, and transposition appeared to be nonreplicative. Thus the mechanism of transposition and excision of the transposon, which I have named Gulliver, resembles that of certain higher plant transposons, like the Ac transposon of maize.     

Characterization of RAT,an autolysis regulator in Staphylococcus aureus

In trying to identify genetic loci involved in the regulation of cap5 genes in Staphylococcus aureus, we isolated a transposon mutant that exhibited a growth defect, enhanced autolysis and increased sensitivity to Triton X-100 and penicillin, attributable in part to increased murein hydrolase activity. Analysis of the chromosomal sequence flanking the transposon insertion site revealed that the gene disrupted in the mutant encodes an open reading frame of 147 amino acids. We named this gene rat, which stands for regulator of autolytic activity. Sequence analysis indicated that Rat is homologous to the MarR and, to a lesser extent, the SarA protein families. Mutations in rat resulted in decreased expression of known autolytic regulators lytSR, lrgAB and arlRS. Gel shift studies indicated that Rat binds to the lytRS and arlRS promoters, thus confirming Rat as a DNA-binding protein to these known repressors of autolytic activity. As anticipated, rat appears to be a negative regulator of autolysin genes including lytM and lytN. These data suggest that the rat gene product is an important regulator of autolytic activity in S. aureus.     

Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257

Trimethoprim resistance mediated by the Staphylococcus aureus multi-resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789-790 bp), the outside two of which are flanked by directly repeated 8-bp target sequences. IS257 has imperfect terminal inverted repeats of 27-28 bp and encodes for a putative transposase with two potential alpha-helix-turn-alpha-helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram-negative bacteria and with ISS1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim-resistant DHFRs from Gram-negative bacteria and with chromosomally encoded DHFRs from Gram-positive and Gram-negative bacteria. 5' to dfrA is a thymidylate synthetase gene, designated thyE.     

Isolation, characterization, and nucleotide sequence of IS1202, an insertion sequence of Streptococcus pneumoniae.

A comparative hybridization protocol was used to isolate a small segment of DNA present in the Streptococcus pneumoniae type 19F strain SSZ but absent from strain Rx1, a nonencapsulated derivative of the type 2 strain D39. This segment of DNA is a 1,747-bp insertion sequence, designated IS1202, flanked by 23-bp imperfect inverted repeats and containing a single open reading frame sufficient to encode a 54.4-kDa polypeptide. A 27-bp target sequence is duplicated at either end of the element. IS1202 is not related to any of the currently known insertion elements and is the first reported for S. pneumoniae. Although found predominantly in type 19F strains in up to five copies, it has also been shown to be present in the chromosomes of pneumococci belonging to other serotypes. One of the four IS1202 copies in the encapsulated strain SSZ is located 1,009 bp downstream of the dexB gene, and transformation studies reveal that it is also closely linked to the type 19F capsular polysaccharide synthesis (cps) locus.     

IS1675, a novel lactococcal insertion element, forms a transposon-like structure including the lacticin 481 lantibiotic operon

Two copies of IS1675, a novel lactococcal insertion element from the IS4 family, are present on a 70-kb plasmid, where they frame the lantibiotic lacticin 481 operon. The whole structure could be a composite transposon designated Tn5721. This study shows that the lacticin 481 operon does not include any regulatory gene and provides a new example of a transposon-associated bacteriocin determinant. We identified five other IS1675 copies not associated with the lacticin 481 operon. The conservation of IS1675 flanking sequences suggested a 24-bp target site.     

LRP1, a gene expressed in lateral and adventitious root primordia of arabidopsis.

We describe a gene that is expressed in lateral and adventitious root primordia of Arabidopsis. The gene was identified by expression of a transposon-borne promoterless beta-glucuronidase gene in lateral root primordia. The gene, designated LRP1 for lateral root primordium 1, and its corresponding cDNA were cloned and sequenced. The expression pattern of the gene in lateral root primordia was confirmed by in situ hybridization with LRP1 cDNA probes. The LRP1 gene encodes a novel protein. LRP1 expression is activated during the early stages of root primordium development and is turned off prior to the emergence of lateral roots from the parent root. Insertion of the transposon in the LRP1 gene disrupted its expression. To evaluate the homozygous insertion line for a mutant phenotype, several aspects of wild-type lateral root development were analyzed. A mutant phenotype has not yet been identified in the insertion line; however, there is evidence that the gene belongs to a small gene family. LRP1 provides a molecular marker to study the early stages of lateral and adventitious root primordium development.     

Propionyl coenzyme A carboxylase is required for development of Myxococcus xanthus.

A dcm-1 mutant, obtained by transposon mutagenesis of Myxococcus xanthus, could aggregate and form mounds but was unable to sporulate under nutrient starvation. A sequence analysis of the site of insertion of the transposon showed that the insertion lies within the 3' end of a 1,572-bp open reading frame (ORF) designated the M. xanthus pccB ORF. The wild-type form of the M. xanthus pccB gene, obtained from a lambdaEMBL library of M. xanthus, shows extensive similarity to a beta subunit of propionyl coenzyme A (CoA) carboxylase, an alpha subunit of methylmalonyl-CoA decarboxylase, and a 12S subunit of transcarboxylase. In enzyme assays, extracts of the dcm-1 mutant were deficient in propionyl-CoA carboxylase activity. This enzyme catalyzes the ATP-dependent carboxylation of propionyl-CoA to yield methylmalonyl-CoA. The methylmalonyl-CoA rescued the dcm-1 mutant fruiting body and spore development. During development, the dcm-1 mutant cells also had reduced levels of long-chain fatty acids (C16 to C18) compared to wild-type cells.     

Identification of a Serratia entomophila genetic locus encoding amber disease in New Zealand grass grub (Costelytra zealandica).

Serratia entomophila UC9 (A1MO2), which causes amber disease in the New Zealand grass grub Costelytra zealandica, was subjected to transposon (TnphoA)-induced mutagenesis. A mutant (UC21) was found to be nonpathogenic (Path-) to grass grub larvae in bioassays and was shown, by Southern hybridization, to contain a single TnphoA insertion. This mutant failed to adhere to the gut wall (Adn-) of the larvae and also failed to produce pili (Pil-). A comparative study of the total protein profiles of wild-type S. entomophila UC9 and mutant UC21 revealed that the mutant lacked an approximately 44-kDa protein and overexpressed an approximately 20-kDa protein. Transfer of cosmids containing homologous wild-type sequences into mutant strain UC21 restored wild-type phenotypes (Path+, Pil+, and Adn+). One of the complementing cosmids (pSER107) conferred piliation on Pil- Escherichia coli HB101. The TnphoA insertion in UC21 was mapped within an 8.6-kb BamHI fragment common to the complementing cosmids, and we designated this gene locus amb-1. Six gene products with molecular masses of 44, 36, 34, 33, 20, and 18 kDa were detected in E. coli minicells exclusive to the cloned 8.6-kb fragment (pSER201A). The 44-kDa gene product was not detected in E. coli minicells containing the cloned mutant fragment. Saturation mutagenesis of this fragment produced four unlinked insertional mutations with active fusions to TnphoA. These active fusions disrupted the expression of one or more gene products encoded by amb-1. The 8.6-kb fragment cloned in the opposite orientation (pSER201B) expressed only a 20-kDa protein. We propose that these are the products of structural and/or regulatory genes involved in adhesion and/or piliation which are prerequisites in the S. entomophila-grass grub interaction leading to amber disease.     

Characterization of Streptococcus criceti insertion sequence ISScr1

A novel insertion sequence element of the IS982 family, ISScr1, was previously identified in Streptococcus criceti strain E49 as a disrupted paaB gene encoding an antigen I/II homologous protein. In this study, we identified two divergent inserted regions of ISScr1 in S. criceti E49 by inverse polymerase chain reaction, the gene-walking method, and screening of the partial plasmid library. Nucleotide sequence analysis indicated the possible explanation that transposition generated 8- and 9-bp direct repeat sequences. DNA hybridization analysis revealed that an identical hybridization pattern to ISScr1 was observed in the four S. criceti strains studied and that at least three copies of ISScr1 were preserved in S. criceti strains. In addition, we found different susceptibility to erythromycin and diverse agglutination properties induced by dextran in S. criceti. Furthermore, DNA hybridization analysis showed that no ISScr1-like copy was detected in the other 14 strains of oral streptococci tested.     

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.     

Inactivation of a glycyl-tRNA synthetase leads to an arrest in plant embryo development.

Embryo formation is the first patterning process during vegetative plant growth. Using transposons as insertional mutagens in Arabidopsis, we identified the mutant edd1 that shows embryo-defective development. The insertion mutation is lethal, arresting embryo growth between the globular and heart stages of embryonic development. The mutant phenotype cosegregates with a transposed Dissociation element. Sequences flanking the transposed element were isolated and used to isolate a full-length cDNA clone representing the wild-type EDD1 gene. Complementation of the mutant through Agrobacterium-mediated gene transfer of an EDD1 wild-type copy as well as loss of the transposon concomitant with phenotypic reversion demonstrated that the transposon had caused the mutation. Based on homology to Escherichia coli, the EDD1 gene is predicted to encode a novel glycyl-tRNA synthetase (GlyRS) that has not been identified previously in higher plants. An N-terminal portion of the plant protein is able to direct a marker protein into pea chloroplasts. Thus, the gene identified by the embryo-defective insertion mutation encodes a GlyRS homolog, probably acting within the plastidic compartment.     

Horizontal gene transfer and host specificity of beta-haemolytic streptococci: the role of a putative composite transposon containing scpB and lmb

Beta-haemolytic streptococci are important human and animal pathogens: their genetic traits that are associated with the ability to infect human hosts remain, however, unclear. The surface protein, Lmb, mediates the adherence of Streptococcus agalactiae to human laminin. For further analysis of the corresponding gene, the adjacent genomic regions were sequenced. Lmb is localized on a putative composite transposon of 16 kb and is flanked by two copies of a novel insertion sequence element (ISSag2). It harbours the genes scpB and lmb, which are 98% identical with the respective genes of Streptococcus pyogenes. Analysis of the distribution of these genes and ISSag2 among 131 streptococcal strains revealed that all of the human isolates, but only 20% (12 of 61) of the animal isolates, contained scpB and lmb or their homologues. To investigate if the putative transposon can be mobilized, an erythromycin resistance marker was incorporated into the lmb gene of S. agalactiae. Screening for mutant strains with a regained susceptibility for erythromycin identified strains with a deletion of scpB, lmb, and one copy of ISSag2. We hypothesize that a horizontal gene transfer caused the exchange of scpB and lmb and that the ability of S. pyogenes, S. agalactiae and group C and G streptococcal strains to colonize or infect human hosts is dependent on their presence.     

Characterization of IS1515, a Functional Insertion Sequence in Streptococcus pneumoniae

We describe the characterization of a new insertion sequence, IS1515, identified in the genome of Streptococcus pneumoniae I41R, an unencapsulated mutant isolated many years ago (R. Austrian, H. P. Bernheimer, E. E. B. Smith, and G. T. Mills, J. Exp. Med. 110:585–602, 1959). A copy of this element located in the cap1E_I41R gene was sequenced. The 871-bp-long IS1515 element possesses 12-bp perfect inverted repeats and generates a 3-bp target duplication upon insertion. The IS encodes a protein of 271 amino acid residues similar to the putative transposases of other insertion sequences, namely IS1381 from S. pneumoniae, ISL2 from Lactobacillus helveticus, IS702 from the cyanobacterium Calothrix sp. strain PCC 7601, and IS112 from Streptomyces albus G. IS1515 appears to be present in the genome of most type 1 pneumococci in a maximum of 13 copies, although it has also been found in the chromosome of pneumococcal isolates belonging to other serotypes. We have found that the unencapsulated phenotype of strain I41R is the result of both the presence of an IS1515 copy and a frameshift mutation in the cap1E_I41R gene. Precise excision of the IS was observed in the type 1 encapsulated transformants isolated in experiments designed to repair the frameshift. These results reveal that IS1515 behaves quite differently from other previously described pneumococcal insertion sequences. Several copies of IS1515 were also able to excise and move to another locations in the chromosome of S. pneumoniae. To our knowledge, this is the first report of a functional IS in pneumococcus.     

ISCce1 and ISCce2, two novel insertion sequences in Clostridium cellulolyticum

Two new insertion sequences, ISCce1 and ISCce2, were found to be inserted into the cipC gene of spontaneous mutants of Clostridium cellulolyticum. In these insertional mutants, the cipC gene was disrupted either by ISCce1 alone or by both ISCce1 and ISCce2. ISCce1 is 1,292 bp long and has one open reading frame. The open reading frame encodes a putative 348-amino-acid protein with significant levels of identity with putative proteins having unknown functions and with some transposases belonging to the IS481 and IS3 families. Imperfect 23-bp inverted repeats were found near the extremities of ISCce1. ISCce2 is 1,359 bp long, carries one open reading frame, and has imperfect 35-bp inverted repeats at its termini. The open reading frame encodes a putative 398-amino-acid protein. This protein shows significant levels of identity with transposases belonging to the IS256 family. Upon transposition, both ISCce1 and ISCce2 generate 8-bp direct repeats of the target sequence, but no consensus sequences could be identified at either insertion site. ISCce1 is copied at least 20 times in the genome, as assessed by Southern blot analysis. ISCce2 was found to be mostly inserted into ISCce1. In addition, as neither of the elements was detected in seven other Clostridium species, we concluded that they may be specific to the C. cellulolyticum strain used.     

The participation of AtXPB1, the XPB/RAD25 homologue gene from Arabidopsis thaliana, in DNA repair and plant development

Nucleotide excision repair in Arabidopsis thaliana differs from other eukaryotes as it contains two paralogous copies of the corresponding XPB/RAD25 gene. In this work, the functional characterization of one copy, AtXPB1, is presented. The plant gene was able to partially complement the UV sensitivity of a yeast rad25 mutant strain, thus confirming its involvement in nucleotide excision repair. The biological role of AtXPB1 protein in A. thaliana was further ascertained by obtaining a homozygous mutant plant containing the AtXPB1 genomic sequence interrupted by a T-DNA insertion. The 3' end of the mutant gene is disrupted, generating the expression of a truncated mRNA molecule. Despite the normal morphology, the mutant plants presented developmental delay, lower seed viability and a loss of germination synchrony. These plants also manifested increased sensitivity to continuous exposure to the alkylating agent MMS, thus suggesting inefficient DNA damage removal. These results indicate that, although the duplication seems to be recent, the features described for the mutant plant imply some functional or timing expression divergence between the paralogous AtXPB genes. The AtXPB1 protein function in nucleotide excision repair is probably required for the removal of lesions during seed storage, germination and early plant development.