Characterization of ISRgn1, a Novel Insertion Sequence of the IS3 Family Isolated from a Bacteriocin-Negative Mutant of Ruminococcus gnavus E1

ISRgn1, an insertion sequence of the IS3 family, has been identified in the genome of a bacteriocin-negative mutant of Ruminococcus gnavus E1. The copy number of ISRgn1 in R. gnavus E1, as well as its distribution among phylogenetically E1-related strains, has been determined. Results obtained suggest that ISRgn1 is not indigenous to the R. gnavus phylogenetic group but that it can transpose in this bacterium.     

Characterization of IS1474, an insertion sequence of the IS21 family isolated from Pseudomonas alcaligenes NCIB 9867

A new insertion sequence (IS) designated IS1474 was isolated from Pseudomonas alcaligenes NCIB 9867 (P25X). IS1474 is a 2632 bp element which showed a characteristic IS structure with 12 bp inverted repeats (IRs) flanking a 2608 bp central region. IS1474 contained four open reading frames (ORF1–ORF4), two in each orientation. Similarities were detected between ORF1 and ORF2 and the putative transposases of the IS21 family. Sequences upstream from IS1474 were found to display up to 89% homology with IS53 from Pseudomonas syringae suggesting that IS1474 had inserted into another related IS element designated IS1475. An open reading frame, ORF5, located at the junction of IS1474 and IS1475, showed similarities with the IstB protein of IS21 and could possibly be the transposase subunit of IS1475. Transposition assays showed that IS1474 transposed at a relatively low frequency leading to cointegration with target plasmids. Hybridization studies showed that IS1474 is present in at least 13 copies in the chromosome of P25X and one copy on its endogenous plasmid.     

Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family

A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment-length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30-bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).     

Characterization of IS1389, a new member of the IS3 family of insertion sequences isolated from Xanthomonas campestris pv. amaranthicola

IS1389, a new insertion sequence belonging to the IS3 family, has been identified in Xanthomonas campestris pv. amaranthicola. The genome of this bacterium contains at least 11 copies of the element, whereas no hybridizing sequences were detected in other Xanthomonas species [X. axonopodis, X. fragaridae, X. phaseoli, and X. (Stenotrophomonas) maltophila]. Two nearly identical copies of the element (IS1389-A and IS1389-B) were characterized. According to analysis of sequence alignments and similar structural features, IS1389 belongs to the IS407 subgroup of the IS3 family, which duplicates 4 bp of target DNA upon insertion. IS1389-A was found in the proximity of the modification gene of the XamI restriction-modification system. Received: 17 November 1998 / Accepted: 22 April 1999     

Description and characterization of IS994, a putative IS3 family insertion sequence from the salmon pathogen, Renibacterium salmoninarum

The motor properties of myosin reside in the globular S1 region of the myosin heavy chain (MHC) subunit. All vertebrates express a family of MHC isoforms in skeletal muscle that have a major influence on the mechanical properties of the various fiber types. Differences in molecular composition of S1 among MHC isoforms within a species have not been studied to any great detail. Presently, we have isolated, cloned and sequenced the S1 subunit of four MHC isoforms from skeletal muscle in Rana pipiens that are specifically expressed in four mechanically divergent fiber types. Paired analysis showed that the overall amino acid identity was higher between the three S1 isoforms expressed in twitch fibers than between the twitch and tonic isoforms. Relatedness in amino acid composition was evaluated in regions reported to govern cross-bridge kinetics. Surface loops 1 and 2, thought to influence motor velocity and ATPase, respectively, were both highly divergent between isoforms. However, the divergence in the loops was roughly equal to that of the amino-terminal region, a domain considered less important for motor function. We tested the hypothesis that the loops are more conserved in pairs of isoforms with more similar kinetics. Comparisons including other vertebrate species showed no tendency for loops from pairs with similar kinetics to be more conserved. These data suggest that the overall structure of loops 1 and 2 is not critical in regulating the kinetic properties of R. pipiens S1 isoforms. Cloning of this family of frog S1 isoforms will facilitate future structure/function studies of the molecular basis of variability in myosin cross-bridge kinetics.     

Characterization of a Mycobacterium bovis BCG insertion sequence related to the IS21 family

The structure and distribution of a Mycobacterium bovis BCG insertion element of the IS21 family were investigated. Several IS21-like elements found in mycobacterial genomes were separated in four types, following their nucleic acid similarities. The M. bovis BCG IS21 element is highly similar to IS1533 (class I), 70% similar to IS1534 (class II), 52% similar to IS1532 (class III) of Mycobacterium tuberculosis, and 54% similar to both an Mycobacterium avium serovar 2 and an M. avium silvaticum IS (class IV). The M. bovis BCG IS21 element of the class I appears to be present in a single copy in the genome of M. bovis BCG, M. bovis, M. tuberculosis and Mycobacterium africanum and to be absent from all other tested species of the Corynebacteria-Mycobacteria-Nocardia group.     

Ruminococcin A, a new lantibiotic produced by a Ruminococcus gnavus strain isolated from human feces

When cultivated in the presence of trypsin, the Ruminococcus gnavus E1 strain, isolated from a human fecal sample, was able to produce an antibacterial substance that accumulated in the supernatant. This substance, called ruminococcin A, was purified to homogeneity by reverse-phase chromatography. It was shown to be a 2,675-Da bacteriocin harboring a lanthionine structure. The utilization of Edman degradation and tandem mass spectrometry techniques, followed by DNA sequencing of part of the structural gene, allowed the identification of 21 amino acid residues. Similarity to other bacteriocins present in sequence libraries strongly suggested that ruminococcin A belonged to class IIA of the lantibiotics. The purified ruminococcin A was active against various pathogenic clostridia and bacteria phylogenetically related to R. gnavus. This is the first report on the characterization of a bacteriocin produced by a strictly anaerobic bacterium from human fecal microbiota.     

Characterization of insertion sequence IS946, an Iso-ISS1 element, isolated from the conjugative lactococcal plasmid pTR2030.

The self-transmissible plasmid pTR2030 mobilized nonconjugative heterologous cloning vectors pGK12 (Cmr Emr) and pSA3 (Emr) at frequencies of 10(-5) to 10(-6) per input donor. Transconjugants harbored a 51- or 58-kilobase (kb) plasmid not found in the parental strains that cotransferred at high frequency with Cmr Emr and pTR2030-encoded phage resistance (Hsp+) in second-round matings (10(-1) per input donor). Restriction endonuclease mapping and DNA-DNA hybridization identified the 51- to 58-kb plasmids as pTR2030::vector cointegrates. Examination of four cointegrates indicated that pGK12 and pSA3 had inserted within two locations on pTR2030. Resolution of the cointegrates generated vector derivatives containing a 0.8-kb insert of pTR2030 DNA. Restriction analyses of several resolution plasmids indicated that the 0.8-kb element had inserted into various positions within pGK12 and pSA3 and in certain cases had inactivated the Cmr or Emr marker of pGK12. A conjugative mobilization assay demonstrated that the 0.8-kb element, designated IS946, mediated transpositional recombination. Nucleotide sequence determination identified IS946 as an 808-base-pair (bp) insertion sequence sharing ca. 96% homology with lactococcal insertion sequence ISS1. IS946 differed by 27 and 31 bp from ISS1S and ISS1T, respectively, and in 2 of 226 amino acids in the deduced sequence of the putative transposase. IS946 has perfect 18-bp terminal inverted repeats, identical to ISS1, and similarly generated 8-bp direct repeats of the target site upon insertion.     

Characterization of an insertion sequence, IS12528, from Gluconobacter suboxydans.

A novel insertion sequence element, IS12528, was found to be associated with inactivation of the alcohol dehydrogenase by insertion in the adhA gene, which encodes the primary dehydrogenase subunit of the three-component membrane-bound alcohol dehydrogenase complex in Gluconobacter suboxydans. Cloning and sequencing analyses revealed that IS12528 was 905 bp in length and had a terminal inverted repeat of 18 bp. In addition, IS12528 was found to generate a 3-bp duplication (TMA, where M represents C or A) at the inserted site upon transposition. IS12528 encoded one long product of 274 amino acids that was rich in basic amino acids. This protein showed significant homology with putative transposases of the IS1031 family isolated from Acetobacter xylinum, which belongs to another genus of acetic acid bacteria. IS12528-like sequences were distributed in a wide variety of acetic acid bacteria, as determined by Southern hybridization and PCR. These observations suggest that IS12528 is one of the insertion sequences that are responsible for genetic instability leading to deficiencies in various physiological properties in a variety of acetic acid bacteria.     

Ruminococcin C, a new anti-Clostridium perfringens bacteriocin produced in the gut by the commensal bacterium Ruminococcus gnavus E1

When colonizing the digestive tract of mono-associated rats, Ruminococcus gnavus E1 - a bacterium isolated from human faeces - produced a trypsin-dependent anti-Clostridium perfringens substance collectively named Ruminococcin C (RumC). RumC was isolated from the caecal contents of E1-monocontaminated rats and found to consist of two antimicrobial fractions: a single peptide (RumCsp) of 4235 Da, and a mixture of two other peptides (RumCdp) with distinct molecular masses of 4324 Da and 4456 Da. Both RumCsp and RumCdp were as effective as metronidazole in combating C. perfringens and their activity spectra against different pathogens were established. Even if devoid of synergistic activity, the combination of RumCsp and RumCdp was observed to be much more resistant to acidic pH and high temperature than each fraction tested individually. N-terminal sequence analysis showed that the primary structures of these three peptides shared a high degree of homology, but were clearly distinct from previously reported amino acid sequences. Amino acid composition of the three RumC peptides did not highlight the presence of any Lanthionine residue. However, Edman degradation could not run beyond the 11th amino acid residue. Five genes encoding putative pre-RumC-like peptides were identified in the genome of strain E1, confirming that RumC was a bacteriocin. This is the first time that a bacteriocin produced in vivo by a human commensal bacterium was purified and characterized.     

IS801, an insertion sequence element isolated from Pseudomonas syringae pathovar phaseolicola

A transposable element, designated IS801, was isolated from strain LR781 of Pseudomonas syringae pathovar phaseolicola in two independent events using the entrapment plasmid, pUCD800. IS801 is 1517 base pairs in length and contains open reading frames that potentially encode proteins of 311 and 172 amino acids, as well as smaller proteins. Unlike most other prokaryotic transposable elements, IS801 lacks terminal repeats. Sequence analysis revealed two target pentamers for IS801 insertion that differ by one base pair. One copy of IS801 generated a perfect duplication of its target, TGAAC. The second copy of IS801 was flanked by the target, TGGAC, at one end, and TGAAC at the other end. A third copy of IS801 was cloned from pMMC7105, an indigenous plasmid of strain LR781, and it was flanked by copies of the pentamer TGAAC.     

ISWpi1 from Wolbachia pipientis defines a novel group of insertion sequences within the IS5 family

Insertion sequences are transposable elements that can represent substantial proportions of prokaryotic genomes and play a substantial role in shaping host genome evolution. As such, evaluating and understanding insertion sequence diversity is an important task to fulfill, because it is expected to yield new insight into the evolution of bacterial transposable elements and contribute to improve genome annotations. Here, I characterized an insertion sequence, termed ISWpi1, for which the taxonomic distribution appears to be restricted to the obligate intracellular alpha-Proteobacterium Wolbachia pipientis. ISWpi1 exhibits approximately 46% identity at the amino acid level with members of the IS1031 group of insertion sequences from the IS5 family. However, the IS1031 group is characterized by a transposase gene encoded by a single open reading frame, whereas the ISWpi1 transposase gene consists of two overlapping open reading frames presumably translated as a single protein via programmed translational frameshifting. Such structure suggests that ISWpi1 may instead be related to the IS427 group of insertion sequences from the IS5 family. Altogether, these data indicate that ISWpi1 extends the known spectrum of diversity of the IS5 family, and I propose to define a novel group of insertion sequences within the IS5 family typified by ISWpi1. Probable transpositional activity, relevant insertion site preferences and taxonomic specificity make ISWpi1 a promising tool for experimentally manipulating W. pipientis bacteria, especially in light of the increasing interest in developing these bacteria as tools for controlling insect disease vectors and agricultural pests.     

Characterisation of IS153, an IS3-family insertion sequence isolated from Lactobacillus sanfranciscensis and its use for strain differentiation

An insertion sequence has been identified in the genome of Lactobacillus sanfranciscensis DSM 20451T as segment of 1351 nucleotides containing 37-bp imperfect terminal inverted repeats. The sequence of this element encodes two out of phase, overlapping open reading frames, orfA and orfB, from which three putative proteins are produced. OrfAB is a transframe protein produced by -1 translational frame shifting between orf A and orf B that is presumed to be the transposase. The large orfAB of this element encodes a 342 amino acid protein that displays similarities with transposases encoded by bacterial insertion sequences belonging to the IS3 family. In L. sanfranciscensis type strain DSM 20451T multiple truncated IS elements were identified. Inverse PCR was used to analyze target sites of four of these elements, but except of their highly AT rich character not any sequence specificity was identified so far. Moreover, no flanking direct repeats were identified. Multiple copies of IS153 were detected by hybridization in other strains of L. sanfranciscensis. Resulting hybridization patterns were shown to differentiate between organisms at strain level rather than a probe targeted against the 16S rDNA. With a PCR based approach IS153 or highly similar sequences were detected in L. acidophilus, L. casei, L. malefermentans, L. plantarum, L. hilgardii, L. collinoides L. farciminis L. sakei and L. salivarius, L. reuteri as well as in Enterococcus faecium, Pediococcus acidilactici and P. pentosaceus.     

Sequence and distribution of IS866, a novel T region-associated insertion sequence from Agrobacterium tumefaciens

We have identified a new insertion sequence, IS866, located in the auxin synthesis gene TA iaaH of Tm4, a wide host range biotype III octopine/cucumopine type Agrobacterium tumefaciens strain with two T regions on its tumor-inducing (Ti) plasmid, TA, and TB. IS866 is 2716 bp long, has inverted repeats of 27 bp with three mismatches, and generates 8-bp direct repeats upon integration. In addition to IS866, pTiTm4 carries two copies of a related element, IS867, associated with TA and TB, respectively. A systematic study of 92 virulent Agrobacterium strains has shown that among the three biotypes all octopine/cucumopine and vitopine biotype III isolates contain IS866-like elements. The various octopine/cucumopine Ti plasmids always carry IS866 and IS867 at the same position as in pTiTm4. The chromosomes of the bacteria which contain these Ti plasmids also carry IS866 and IS867 copies but in varying numbers and locations.     

Identification and characterisation of IS1383, a new insertion sequence isolated from Pseudomonas putida strain H

A new insertion sequence (IS1383) was identified on plasmids from Pseudomonas putida strain H and its nucleotide sequence was determined. IS1383 contains perfect terminal inverted repeats of 13-bp flanking a 1.4-kb internal sequence. A single significant open reading frame was identified that can encode a 342-amino acid polypeptide which was predicted to be highly basic and to have homology to polypeptides known from several other bacterial insertion sequences. At least six copies of IS1383 are present on the plasmids pPGH1 and pPGH2, whereas no copy could be detected on the chromosome of P. putida strain H. Target duplications did not flank the inverted repeats of any of the six IS1383 copies examined. Analysis of the integration sites of IS1383 revealed hints for a target specificity. Multiple sequence alignments of the transposases, the inverted repeats and the integration sites pointed to the assignment of IS1383 into a putative new family of insertion sequences defined as the IS1111 family.     

Identification of IS 1206, a Corynebacterium glutamicum IS3-related insertion sequence and phylogenetic analysis

Integration of plasmid pCGL320 into a Corynebacterium glutamicum ATCC21086 derivative led to tandem amplification of the inserted plasmid (Labarre et a/., 1993). One amplification event was associated with integration of an insertion sequence that we have named IS 1206. Hybridizing sequences were only found in C. glutamicum strains and at various copy numbers. IS1206 is 1290bp long, carries 32 bp imperfect inverted repeats and generates a 3bp duplication of the target DNA upon insertion. IS1206 presents the features characteristic of the IS3 family and part of the DNA sequence centering on the putative transposase region (orfB) is similar to those of IS3 and some other related elements. Phylogenetic analysis of orfB deduced protein sequences from IS 1206 and IS3-related elements contradicts the phylogeny of the species, suggesting that evolution of these elements might be complex. Horizontal transfer could be invoked but other alternatives like ancestral polymorphism or/and different rates of evolution could also be involved.     

Characterization of a novel,defense-related Arabidopsis mutant,cir1, isolated by luciferase imaging

In order to identify components of the defense signaling network engaged following attempted pathogen invasion, we generated a novel PR-1::luciferase (LUC) transgenic line that was deployed in an imaging-based screen to uncover defense-related mutants. The recessive mutant designated cir1 exhibited constitutive expression of salicylic acid (SA), jasmonic acid (JA)/ethylene, and reactive oxygen intermediate-dependent genes. Moreover, this mutation conferred resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and a virulent oomycete pathogen Peronospora parasitica Noco2. Epistasis analyses were undertaken between cir1 and mutants that disrupt the SA (nprl, nahG), JA (jar1), and ethylene (ET) (ein2) signaling pathways. While resistance against both P. syringae pv. tomato DC3000 and Peronospora parasitica Noco2 was partially reduced by npr1, resistance against both of these pathogens was lost in an nahG genetic background. Hence, cirl-mediated resistance is established via NPR1-dependent and -independent signaling pathways and SA accumulation is essential for the function of both pathways. While jar1 and ein2 reduced resistance against P. syringae pv. tomato DC3000, these mutations appeared not to impact cir1-mediated resistance against Peronospora parasitica Noco2. Thus, JA and ET sensitivity are required for cir1-mediated resistance against P. syringae pv. tomato DC3000 but not Peronospora parasitica Noco2. Therefore, the cir1 mutation may define a negative regulator of disease resistance that operates upstream of SA, JA, and ET accumulation.     

Characterisation of IS1126 from Porphyromonas gingivalis W83: a new member of the IS4 family of insertion sequence elements

Abstract The nucleotide sequence of IS 1126 , the only insertion sequence so far isolated from the oral pathogen Porphyromonas gingivalis , has been determined. It had a nucleotide sequence of 1338 base pair (bp) flanked by 12 bp perfect inverted repeats and generated a 5 bp target site duplication. The single major open reading frame encoded a predicted protein of 361 amino acids and molecular mass of 41 kDa. The gene encoding the transpsosase was subcloned into pUC18 and the transposase expressed in Escherichia coli minicells. The predicted amino acid sequence of the transposashad homology to putative transposases of IS 1106 and IS 1186 both of which belong to the IS 5 group within the IS₄ super-family of insertion elements. On the basis of this homology we propose that IS 1126 should also be included in the IS 5 group. Southern-blot analysis of a number of P. gingivalis strains using IS 1126 as a probe revealed a unique pattern of hybridisation for each strain and the absence of IS 1126 from other closely related Porphyromonas species. This should allow IS 1126 to be used as a rapid epidemiological tool in studying oral infections by P. gingivalis .