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PubMed2000

Characterization of Porphyromonas gingivalis insertion sequence-like element ISPg5.

Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is found in periodontitis lesions, and its presence in subgingival plaque significantly increases the risk for periodontitis. In contrast to many bacterial pathogens, P. gingivalis strains display considerable variability, which is likely due to genetic exchange and intragenomic changes. To explore the latter possibility, we have studied the occurrence of insertion sequence (IS)-like elements in P. gingivalis W83 by utilizing a convenient and rapid method of capturing IS-like sequences and through analysis of the genome sequence of P. gingivalis strain W83. We adapted the method of Matsutani et al. (S. Matsutani, H. Ohtsubo, Y. Maeda, and E. Ohtsubo, J. Mol. Biol. 196:445-455, 1987) to isolate and clone rapidly annealing DNA sequences characteristic of repetitive regions within a genome. We show that in P. gingivalis strain W83, such sequences include (i) nucleotide sequence with homology to tRNA genes, (ii) a previously described IS element, and (iii) a novel IS-like element. Analysis of the P. gingivalis genome sequence for the distribution of the least used tetranucleotide, CTAG, identified regions in many of the initial 218 contigs which contained CTAG clusters. Examination of these CTAG clusters led to the discovery of 11 copies of the same novel IS-like element identified by the repeated sequence capture method of Matsutani et al. This new 1,512-bp IS-like element, designated ISPg5, has features of the IS3 family of IS elements. When a recombinant plasmid containing much of ISPg5 was used in Southern analysis of several P. gingivalis strains, including clinical isolates, diversity among strains was apparent. This suggests that ISPg5 and other IS elements may contribute to strain diversity and can be used for strain fingerprinting.

Amino Acid Sequence↗

PubMed1993

440-kD ankyrinB: structure of the major developmentally regulated domain and selective localization in unmyelinated axons.

440-kD ankyrinB is an alternatively spliced variant of 220-kD ankyrinB, with a predicted 220-kD sequence inserted between the membrane/spectrin binding domains and COOH-terminal domain (Kunimoto, M., E. Otto, and V. Bennett. 1991. J. Cell Biol. 236:1372-1379). This paper presents the sequence of 2085 amino acids comprising the alternatively spliced portion of 440-kD ankyrinB, and provides evidence that much of the inserted sequence has the configuration of an extended random coil. Notable features of the inserted sequence include a hydrophilicity profile that contains few hydrophobic regions, and 220 predicted sites for phosphorylation by protein kinases (casein kinase 2, protein kinase C, and proline-directed protein kinase). Secondary structure and folding of the inserted amino acid residues were deduced from properties of recombinant polypeptides. Frictional ratios of 1.9-2.4 were calculated from Stokes radii and sedimentation coefficients, for polypeptides comprising 70% of the inserted sequence, indicating a highly asymmetric shape. Circular dichroism spectra of these polypeptides indicate a nonglobular structure with negligible alpha-helix or beta sheet folding. These results suggest a ball-and-chain model for 440-kD ankyrinB with a membrane-associated globular head domain and an extended filamentous tail domain encoded by the inserted sequence. Immunofluorescence and immunoblot studies of developing neonatal rat optic nerve indicate that 440-kD ankyrinB is selectively targeted to premyelinated axons, and that 440-kD ankyrinB disappears from these axons coincident with myelination. Hypomyelinated nerve tracts of the myelin-deficient Shiverer mice exhibit elevated levels of 440-kD ankyrinB. 440-kD ankyrinB thus is a specific component of unmyelinated axons and expression of 440-kD ankyrinB may be downregulated as a consequence of myelination.

Amino Acid Sequence↗

PubMed2001

ISMh2, a novel insertion sequence-like element associated with nifA from Mesorhizobium huakuii.

Sequence analysis of the nifA gene, including its 5' flanking region, of Mesorhizobium huakuii revealed the presence of a novel IS-like element called ISMh2. It is 828 bp in length and possesses two imperfect terminal inverted repeats of 14 bp with only one mismatch. The putative transposase encoded by ISMh2 is composed of 204 amino acids. In comparison with other insertion sequences ISMh2 likely belongs to the IS6 family. Multiple copies of ISMh2 were detected in the genome of M. huakuii 159 by Southern hybridization. RT-PCR analysis showed that nifA and ISMh2 cotranscribed. Attempts to detect the transposition ability of ISMh2 were unsuccessful.

Alphaproteobacteria↗

PubMed2001

Identification and structural analysis of SINE elements in the Arabidopsis thaliana genome.

An insertion sequence was found in a Mu homologue in the genome of Arabidopsis thaliana. The insertion sequence had poly(A) at the 3' end, and promoter motifs (A- and B-boxes) recognized by RNA polymerase III. The sequence was flanked by direct repeats of a 15-bp sequence of the Mu homologue, which appears to be a target-site sequence duplicated upon insertion. These findings indicate that the insertion sequence is a retroposon SINE, and it was therefore named AtSN (A. thaliana SINE). Many members of the AtSN family were identified through a computer-aided homology search of databases and classified into two subfamilies, AtSN1 and AtSN2, having consensus sequences 159 and 149 bp in length, respectively. These had no homology to SINEs in other organisms. About half of AtSN members were truncated through loss of a region at either end of the element. Most of them were truncated at the 5' end, and had a duplication of the target-site sequence. This suggests that the ones with 5' truncation retroposed by the same mechanism as those without truncation. Members of the AtSN1 or AtSN2 subfamilies had many base substitutions when compared with the consensus sequence. All of the members examined were present in three different ecotypes of A. thaliana (Columbia, Landsberg erecta, and Wassilewskija). These findings suggest that AtSN members had proliferatedbefore the A. thaliana ecotype strains diverged.

Arabidopsis↗

PubMed1997

Cloning and sequence analysis of a novel insertion element from plasmids harbored by the carbofuran-degrading bacterium, Sphingomonas sp. CFO6.

Sphingomonas sp. CFO6 (a member of the alpha group of Proteobacteria) was isolated from a Washington soil by enrichment on the insecticide carbofuran as a sole source of carbon and energy. This strain has been shown to harbor five plasmids, at least some of which are required for catabolism of carbofuran. Rearrangements, deletions, and loss of individual plasmids resulting in the loss of the carbofuran-degrading phenotype were observed following treatment with heat or introduction of Tn5. Several putative insertion sequence elements of different sizes were cloned from these plasmids by trapping in pUCD800, a positive selection vector for isolation of transposable elements. Three of the most common putative IS elements (designated IS1412, IS1487, and IS1488) in the clone library were of different sizes and cross-hybridize with each other. An element hybridizing with IS1412, IS1487, and IS1488 was mobilized during growth of CFO6 at 42 degrees C and inserted into one of CFO6's plasmids (pCFO4), corresponding to a deletion in the plasmid and a loss of catabolic function. IS1412 was completely sequenced and its sequence analyzed. IS1412 is 1656 bp in length and possesses terminal partially matched inverted repeats of unequal length (17 and 18 bp). In addition, IS1412 contains an open reading frame which encodes a putative transposase with significant homology to the putative transposases of IS1380 from Acetobacter pasteurianus, HRS1 from Bradyrhizobium japonicum, and IS1247 from Xanthobacter autotrophicus. These related IS elements form part of a family of common IS elements distributed among members of the alpha group of the Proteobacteria.

Amino Acid Sequence↗

PubMed1996

Integration of narrow-host-range vectors from Escherichia coli into the genomes of amino acid-producing corynebacteria after intergeneric conjugation.

Conjugative transfer of mobilizable derivatives of the Escherichia coli narrow-host-range plasmids pBR322, pBR325, pACYC177, and pACYC184 from E. coli to species of the gram-positive genera Corynebacterium and Brevibacterium resulted in the integration of the plasmids into the genomes of the recipient bacteria. Transconjugants appeared at low frequencies and reproducibly with a delay of 2 to 3 days compared with matings with replicative vectors. Southern analysis of corynebacterial transconjugants and nucleotide sequences from insertion sites revealed that integration occurs at different locations and that different parts of the vector are involved in the process. Integration is not dependent on indigenous insertion sequence elements but results from recombination between very short homologous DNA segments (8 to 12 bp) present in the vector and in the host DNA. In the majority of the cases (90%), integration led to cointegrate formation, and in some cases, deletions or rearrangements occurred during the recombination event. Insertions were found to be quite stable even in the absence of selective pressure.

Amino Acids↗

PubMed1999

Distribution of insertion sequence-like element IS1272 and its position relative to methicillin resistance genes in clinically important Staphylococci.

The distribution of insertion sequence-like element IS1272 was analyzed for clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus haemolyticus. In each of the staphylococcal species, IS1272 was detected in both methicillin-resistant (MR) and methicillin-susceptible strains of different genetic types. In MR isolates, IS1272 was generally located downstream of the truncated mecR1 gene (DeltamecR1), with an identical junction sequence occurring between DeltamecR1 and IS1272, although insertion of an additional gene sequence in the junction sequence was detected in one S. epidermidis isolate. These findings suggest that the mec element with the rearranged form of mecR1 (DeltamecR1-IS1272) has been transmitted to multiple clones of staphylococci.

Bacterial Proteins↗

PubMed1989

New plasmid vectors for high level synthesis of eukaryotic fusion proteins in Escherichia coli.

Production of eukaryotic proteins in Escherichia coli has become rather simple since commercially available bacteriophage and plasmid vector systems allow investigators to select the optimal system for their particular problem. A common question is which system to use to produce the largest quantity of soluble recombinant protein with minimal, if any, bacterial protein fused to it. We have constructed a new set of plasmid vectors that produce large amounts of a fusion proteins that contain less than 25 amino acids of bacterial protein. We started with pATH-1, a plasmid expression vector comprised of the trpEp promoter and 37 kDa of the TrpE protein followed by a M13mp13 multiple cloning site for insertion of sequences to be expressed. We deleted the majority of the eukaryotic trpE sequence to produce a multiple frame, multiple enzyme cloning site, plasmid expression vector set called pRX. Transformation of E. coli CAG-456 (Baker et al., 1984) with this vector with an Acanthamoeba myosin tail sequence inserted in the correct frame yields a fusion protein that represents 45% of the total soluble protein. We have produced and purified 100 mg of this Acanthamoeba myosin-II fusion protein per liter of cell suspension.

Acanthamoeba↗

PubMed1996

Analysis of CRP-CytR interactions at the Escherichia coli udp promoter.

Multiprotein complexes regulate the transcription of certain bacterial genes in a sensitive, physiologically responsive manner. In particular, the transcription of genes needed for utilization of nucleosides in Escherichia coli is regulated by a repressor protein, CytR, in concert with the cyclic AMP (cAMP) activated form of cAMP receptor protein (CRP). We studied this regulation by selecting and characterizing spontaneous constitutive mutations in the promoter of the udp (uridine phosphorylase) gene, one of the genes most strongly regulated by CytR. We found deletions, duplications, and point mutations that affect key regulatory sites in the udp promoter, insertion sequence element insertions that activated cryptic internal promoters or provided new promoters, and large duplications that may have increased expression by udp gene amplification. Unusual duplications and deletions that resulted in constitutive udp expression that depended on the presence of CytR were also found. Our results support the model in which repression normally involves the binding of CytR to cAMP-CRP to form a complex which binds to specific sites in the udp promoter, without direct interaction between CytR protein and a specific operator DNA sequence, and in which induction by specific inducer cytidine involves dissociation of CytR from cAMP-CRP and the RNA polymerase interaction with cAMP-CRP bound to a site upstream of then transcription start point. The stimulation of udp expression by CytR in certain mutants may reflect its stabilization of cAMP-CRP binding to target DNA and illustrates that only modest evolutionary changes could allow particular multiprotein complexes to serve as either repressors or transcriptional activators.

Bacterial Proteins↗

PubMed1978

Rat alpha-crystallin A chain with an insertion of 22 residues.

Rat lens alpha-crystallin contains, besides the usual alphaA and alphaB subunits, an additional minor chain. This subunit was purified by ion-exchange chromatography and its primary structure studied. It appeared to be an elongated alphaA-like chain, having an insertion of 22 residues between position 63 and 64 of an otherwise normal alphaA2 chain. Therefore this subunit was called alphaAIns, i.e. an alphaA chain with an inserted sequence. This inserted region, which contains three methionyl, five basic and no acidic residues, apparently results in an adequately functioning alphaAIns chain. The alphaAIns chain may be the product of a gene which has originated, after duplication of the alphaA gene, by insertion in one of the copies of a stretch of 66 nucleotides of unknown origin, or alternatively be the result of unusual transcription or processing of precursor mRNA (pre-mRNA), leaving an extra 66 nucleotides internally in the mRNA to be translated.

Amino Acid Sequence↗

PubMed2003

A fluoroquinolone induces a novel mitogen-encoding bacteriophage in Streptococcus canis.

This study investigated whether the recently recognized emergence of canine streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) might be partly attributed to the use of fluoroquinolones to treat Streptococcus canis infections in dogs. Both mitomycin and the fluoroquinolone enrofloxacin caused bacteriophage-induced lysis of S. canis strain 34, an isolate from a case of canine STSS and NF. Fluoroquinolone-evoked, bacteriophage-induced lysis occurred over a range of concentrations similar to those that would occur after treatment of dogs with these agents. To search for a possible bacteriophage-encoded streptococcal superantigen gene(s), a library of the 36.5 (+/-1.1)-kb bacteriophage, designated phisc1, was made by ligating 3- to 7-kb Tsp5091-digested phisc1 fragments into an EcoRI-digested lambdaZapII vector. Recombinants were screened for mitogenic activity by using canine peripheral blood lymphocytes. Of 800 recombinants screened, 11 recombinants with mitogenic effects were identified, and their inserts were sequenced. The highest homology of 11.6 kb of sequenced phisc1 DNA was to the completely sequenced Streptococcus pneumoniae bacteriophage MM1. Seven of the 11 phisc1 sequenced inserts contained a 552-bp open reading frame, scm, with 27% amino acid similarity to pokeweed (Phytolacca americana) mitogen. PCR showed this gene to be present in 22 of 23 S. canis isolates tested. Quantitative reverse transcription-PCR showed that bacteriophage induction was associated with a 58-fold enhancement of expression of this gene relative to that in a noninduced culture of a similar age. The presence of this gene on a fluoroquinolone-induced bacteriophage may explain the association observed between fluoroquinolone use in dogs and the development of canine STTS and NF.

Amino Acid Sequence↗

PubMed2006

Genetic rearrangement of the atzAB atrazine-degrading gene cassette from pADP1::Tn5 to the chromosome of Variovorax sp. MD1 and MD2.

We report the characterization of the rearrangement phenomena responsible for the movement of the atrazine-degrading atzA and B genes from pADP1::Tn5 to the chromosome of Variovorax sp. MD1 and MD2. Long PCRs and Southern blot analyses revealed that the two genes forming a gene cassette moved in a unique rearrangement event. It also revealed that the boundaries of the plasmid sequence inserted in the chromosome correspond to IS1071or to sequences close to IS1071. It suggests that this genetic rearrangement could result from the transposition of the composite transposon delimited by IS1071 insertion sequences and containing atzA and atzB genes. In addition, for MD1 and MD2 strains the sequencing of the remaining sequence on pADP1::Tn5 indicated that the deletion of the atzA and B genes from the plasmid might be the result of a recombination event that occurred between the IS1071 insertion sequences surrounding the atzAB gene cassette.

Agrobacterium tumefaciens↗

PubMed2000

Phage display mapping for peptide 11 sensitive sequences binding to laminin-1.

We utilized a 9-mer random phage display library to identify sequences which bind to laminin-1 and elute with heparan sulfate or peptide 11 (CDPGYIGSR). Laminin-1 derivatized plates were used for biopanning. Three consecutive rounds of low pH elutions were carried out, followed by three rounds of specific elutions, each consisting of a heparan sulfate elution followed by a peptide 11 elution. The random sequence inserts were sequenced for phage populations eluted at low pH, by heparan sulfate and by peptide 11. Specifically eluted phage populations exhibited three classes of mimotopes for different regions in the cDNA derived amino acid sequence of the 67 kDa laminin binding protein (LBP). These regions were (1) a palindromic sequence known as peptide G, (2) a predicted helical domain corresponding to LBP residues 205-229, and (3) TEDWS-containing C-terminal repeats. All elution conditions also yielded phage with putative heparin binding sequences. We modeled the LBP(205-229) domain, which is strongly predicted to have a helical secondary structure, and determined that this region likely possesses heparin-binding characteristics located to one side of the helix, while the opposite side appears to contain a hydrophobic patch where peptide 11 could bind. Using ELISA plate assays, we demonstrated that peptide 11 and heparan sulfate individually bound to synthetic LBP(205-229) peptide. We also demonstrated that the QPATEDWSA peptide could inhibit tumor cell adhesion to laminin-1. These data support the proposal that the 67 kDa LBP can bind the beta-1 laminin chain at the peptide 11 region, and suggest that heparan sulfate is a likely alternate ligand for the binding interactions. Our results also confirm previous data suggesting that the most C-terminal region of the LBP, which contains the TEDWS repeats, is involved in cell adhesion to laminin-1, and we specifically implicate the repeat sequence in that activity.

Amino Acid Sequence↗

PubMed1994

The nylon oligomer biodegradation system of Flavobacterium and Pseudomonas.

This review article is a compendium of the available information on the degradation of a man-made compound, 6-aminohexanoate-oligomer, in Flavobacterium and Pseudomonas strains, and discusses the molecular basis for adaptation of microorganisms toward these xenobiotic compounds. Three plasmid-encoded enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (EI), 6-aminohexanoate-dimer hydrolase (EII), and endo-type 6-aminohexanoate-oligomer hydrolase (EIII) are responsible for the degradation of the oligomers. Two repeated sequences, designated RS-I and RS-II, are found on plasmid pOAD2, which is involved in 6-aminohexanoate degradation in Flavobacterium. RS-I appears 5 times on the pOAD2, and all copies have the same sequences as insertion sequence IS6100. RS-II appears twice on the plasmid. RS-IIA contains the gene encoding EII, while RS-IIB contains the gene for the analogous EII' protein. Both EII and EII' are polypeptides of 392 amino acids, which differ by 46 amino acid residues. The specific activity of the EII enzyme is 200-fold higher than that of EII'. Construction of various hybrid genes demonstrated that only the combination of two amino acid residues in the EII' enzyme can enhance the activity of the EII' to the same level as that of EII enzyme.

Amino Acid Sequence↗

PubMed2006

Genetic structures at the origin of acquisition and expression of the carbapenem-hydrolyzing oxacillinase gene blaOXA-58 in Acinetobacter baumannii.

Genetic structures surrounding the carbapenem-hydrolyzing oxacillinase gene blaOXA-58 were characterized in a series of OXA-58-positive Acinetobacter baumannii strains isolated from different countries. We showed that in most of the cases, acquisitions of the blaOXA-58-containing overall structure, including insertion sequence elements, may be likely the results of recombination events. In type strain A. baumannii MAD, the genetic structure surrounding the blaOXA-58 gene was bracketed by two 27-bp repeated sequences. The isolation of a clonally related OXA-58-negative A. baumannii isolate that possessed the same plasmid backbone as A. baumannii MAD but lacked this genetic structure indicated that the mechanism of acquisition could be reversible. Parts of the structure identified in A. baumannii MAD were conserved in other blaOXA-58-positive isolates from various European countries. Primer extension experiments showed that blaOXA-58 expression was related to promoter sequences brought by different insertion sequence elements, such as an ISAba3-like element, ISAba1, ISAba2, and IS18. This work identified novel structures at the origin of acquisition and expression of a carbapenem-hydrolyzing beta-lactamase identified in non-clonally related A. baumannii isolates.

Acinetobacter baumannii↗

PubMed1997

Structure and evolution of NGRRS-1, a complex, repeated element in the genome of Rhizobium sp. strain NGR234.

Much of the remarkable ability of Rhizobium sp. strain NGR234 to nodulate at least 110 genera of legumes, as well as the nonlegume Parasponia andersonii, stems from the more than 80 different Nod factors it secretes. Except for nodE, nodG, and nodPQ, which are on the chromosome, most Nod factor biosynthesis genes are dispersed over the 536,165-bp symbiotic plasmid, pNGR234a. Mosaic sequences and insertion sequences (ISs) comprise 18% of pNGR234a. Many of them are clustered, and these IS islands divide the replicon into large blocks of functionally related genes. At 6 kb, NGRRS-1 is a striking example: there is one copy on pNGR234a and three others on the chromosome. DNA sequence comparisons of two NGRRS-1 elements identified three types of IS, NGRIS-2, NGRIS-4, and NGRIS-10. Here we show that all four copies of NGRRS-1 probably originated from transposition of NGRIS-4 into a more ancient IS-like sequence, NGRIS-10. Remarkably, all nine copies of NGRIS-4 have transposed into other ISs. It is unclear whether the accumulation of potentially mutagenic sequences in large clusters is due to the nature of the IS involved or to some selection process. Nevertheless, a direct consequence of the preferential targeting of transposons into such IS islands is to minimize the likelihood of disrupting vital functions.

Base Sequence↗

PubMed1997

Transposition without transposase: a spontaneous mutation in bacteria.

Transposition mutations are typically associated with the activities of transposable elements such as transposons and insertion sequences, whose mobility is dependent upon transposase enzymes that catalyze exchanges between element ends and target sites. We describe a single transposition event in which a block of donor sequence is inserted at a target site without the involvement of any known transposase or the ends of any known transposable element. We propose that this is a new type of spontaneous mutation which may be difficult to detect in standard mutant hunts but may be of evolutionary importance.

DNA Nucleotidyltransferases↗

PubMed1984

The population genetics of Escherichia coli.

E. coli is a successful and diverse group of organisms, well defined by DNA hybridization within the Enterobacteriacae and including the closely related organisms Shigella and the Alkalescens-Dispar biogroup. The primary habitat of E. coli is the lower intestinal tract of warm-blooded animals, which is colonized shortly after birth. At any one time, most normal individuals carry several strains of E. coli in their intestinal tract, including a small number of resident clones exhibiting a rate of replacement measured in weeks or months and a much larger number of transient clones that are replaced in a matter of days or weeks. The secondary habitats of E. coli are soil, sediment, and water, where its half life is thought to be only a few days. Pathogenic forms of E. coli are associated with diarrheal diseases, urinary tract infections, neonatal meningitis, nosocomial infections, and in infections of domesticated animals. E. coli populations contain much genetic diversity, more than is found in most eukaryotes. Genetic diversity has been studied from the standpoint of (a) serology with respect to surface antigens, (b) biogrouping with respect to variable characters such as nutritional versatility, antibiotic resistance, and bacteriophage susceptibility, (c) electrophoresis of enzymes of intermediary metabolism or outer membrane proteins, (d) DNA hybridization, (e) restriction-fragment length polymorphisms, (f) DNA sequences, (g) insertion sequences, and (h) plasmids. However identified, strains of E. coli appear to have a wide, but not totally indiscriminate, host range. Aside from genes directly associated with virulence, genetic divergence between pathogenic and nonpathogenic strains, although statistically significant, is not pronounced. Electrophoretic studies indicate that, while some serotypes may represent a single genetic clone almost exclusively, other serotypes may represent two or more genetically unrelated clones. Unrelated clones may therefore converge to the same or very similar serotypes. Electrophoresis has also been used to define three groups of clones among natural isolates, perhaps corresponding to subspecies of E. coli. These groups are worldwide in distribution and have a wide host range. E. coli populations exhibit great linkage disequilibrium, which occurs as highly nonrandom combinations of alleles at different loci. Reproduction is evidently largely asexual, with insufficient genetic recombination to dissipate linkage disequilibrium.(ABSTRACT TRUNCATED AT 400 WORDS)

Animal Diseases↗