Int Gene Product Research Articles

Genetic Structure and Transcriptional Analysis of a Mobilizable, Antibiotic Resistance Transposon from Bacteroides

Tn4555 is a 12.1-kb Bacteroides antibiotic resistance transposon representative of a novel class of transmissible genetic elements that can be transferred by resident conjugative tetracycline resistance transposons (Tcr-elements) but are not capable of self-transfer. Previously it was shown that Tn4555 transposes by a site-specific recombination mechanism that utilizes a circular intermediate. This circular form is induced by tetracycline and it also is the substrate for conjugation. To better understand the mechanism of transposition, the entire nucleotide sequence of Tn4555 was determined and a set of genes potentially involved in transposition was identified. The transposon was 12,105 bp including a variable 6-bp coupling sequence associated with one of the transposon termini. The element had a 44.3% G + C composition and nine potential protein coding regions were observed, eight of which were encoded on the forward strand. Two putative transposition genes were found. The int gene product had significant C-terminal homology to the lambda family of integrases and the xis gene product was similar to several excisionase proteins encoded by both plasmids and conjugative transposons. The mobA mobilization gene and cfxA β-lactamase gene of Tn4555 had been previously identified, and the remaining five open reading frames had no significant matches with sequences in the available databases. Northern hybridization analysis revealed that all Tn4555 genes except for orf-9 were expressed and two sets of genes, tnpA, int and xis, orf-5, orf-6 were organized in operons. None of the genes seemed to be induced significantly by the addition of tetracycline to cultures. Although a small 0.4-kb xis-specific transcript appeared in tetracycline-treated cultures it was not clear if this was due to an induction or if it was a specific degradation product.

Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4.

Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3' end of a tRNA(Ser) gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA(Ser) gene. In the other bacterial species where this tRNA gene is less or not conserved; secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5' end of the core in the strand exchange reaction.

Overexpression of an mRNA dependent on rare codons inhibits protein synthesis and cell growth.

lambda's int gene contains an unusually high frequency of the rare arginine codons AGA and AGG, as well as dual rare Arg codons at three positions. Related work has demonstrated that Int protein expression depends on the rare AGA tRNA. Strong transcription of the int mRNA with a highly efficient ribosome-binding site leads to inhibition of Int protein synthesis, alteration of the overall pattern of cellular protein synthesis, and cell death. Synthesis or stability of int and ampicillin resistance mRNAs is not affected, although a portion of the untranslated int mRNA appears to be modified in a site-specific fashion. These phenotypes are not due to a toxic effect of the int gene product and can be largely reversed by supplementation of the AGA tRNA in cells which bear plasmids expressing the T4 AGA tRNA gene. This indicates that depletion of the rare Arg tRNA due to ribosome stalling at multiple AGA and AGG codons on the overexpressed int mRNA underlies all of these phenomena. It is hypothesized that int mRNA's effects on protein synthesis and cell viability relate to phenomena involved in lambda phage induction and excision.

Site-specific integration of the phage phi CTX genome into the Pseudomonas aeruginosa chromosome: characterization of the functional integrase gene located close to and upstream of attP.

The site-specific integration of the phage phi CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 bp integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage phi CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates.

Identification and characterization of genes involved in excision of the Lactococcus lactis conjugative transposon Tn5276.

The 70-kb transposon Tn5276, originally detected in Lactococcus lactis NIZO R5 and carrying the genes for nisin production and sucrose fermentation, can be conjugally transferred to other L. lactis strains. Sequence analysis and complementation studies showed that the right end of Tn5276 contains two genes, designated xis and int, which are involved in excision. The 379-amino-acid int gene product shows high (up to 50%) similarity with various integrases, including that of the Tn916-related conjugative transposons. The xis gene product, like almost all known excisionase (Xis) proteins, is a small (68-residue), basic protein. Expression of both the Tn5276 int and xis genes is required for efficient excision of the ends of Tn5276 in Escherichia coli that appeared to be circularized in the excision process. Mutational analysis of the xis and int genes showed that excision efficiency is dependent on the integrity of the int gene but that an intact xis gene is also required for efficient excision.

Excisive recombination of the SLP1 element in Streptomyces lividans is mediated by Int and enhanced by Xis

The functions mediating site-specific recombination of the SLP1 element have been mapped to a 2.2-kb region that includes the site of integration (attP), a gene (int) that specifies a function both necessary and sufficient for integration of SLP1, and an open reading frame, orf61, suspected of encoding a protein, Xis, that shows limited similarity to the excisionases of other site-specific recombination systems. Here we describe experiments that investigate the respective roles of orf61 and int in the excision of SLP1. We constructed derivatives of the high-copy-number Streptomyces plasmid pIJ101 that express orf61, int, or both orf61 and int from transcriptional fusions to the Tn5 aph gene and tested the ability of these constructs to promote excision of an adventitious attP-containing plasmid that had been integrated site-specifically into the attB site of the Streptomyces lividans chromosome. Expression of the int gene product alone from an exogenous promoter was sufficient for excision of the integrated plasmid. This result indicates that the SLP1 int-encoded protein can carry out excisive, as well as integrative, recombination. The orf61 gene product, when expressed from an exogenous promoter, inhibited int-mediated integration at the chromosomal attB site. Moreover, under conditions in which excision and transfer normally occur, precise excision of SLP1 was enhanced by the orf61-encoded protein. On the basis of these findings, we here designate the orf61 gene as xis.

The spleen necrosis virus int gene product expressed in Escherichia coli has DNA binding activity and mediates att and U5-specific DNA multimer formation in Vitro

To facilitate the in vitro study of the spleen necrosis virus (SNV) int gene product, we expressed the viral int locus in an Escherichia coli expression vector. Antiserum made against the protein produced in bacteria precipitated a 44-kDa polypeptide from virus-infected chicken embryo fibroblasts. This result is consistent with the expected size of the SNV int polypeptide. In a protein blotting assay, the expressed protein binds strongly to DNA and was able to complex nonspecifically with both single- and double-stranded DNAs containing or lacking viral sequences. However, under solution conditions favoring transient DNA unwinding, DNA binding was confined to supercoiled molecules containing either the SNV att sequence (the viral cis-acting region required for integration) or the U5 region of the long terminal repeat alone. Under these conditions of specific binding, multimeric DNA species were formed by apparent intermolecular interaction between protein-DNA complexes. These data indicate that retroviral integration may require local DNA unwinding at the att site for complex formation between the int gene product and DNA. This complex may be an intermediate in the viral DNA insertion process.

Mutations of bacteriophage lambda that define independent but overlapping RNA processing and transcription termination sites

Bacteriophage λ int gene expression is regulated differentially from transcripts originated at the p L and p I promoters. Transcripts initiated at p I terminate at the site t I and express int gene product efficiently. Polymerases starting at p L do not terminate at t I, due to the antiterminating activity of λ N protein. The p L transcripts are unable to express Int protein efficiently because sib, a control site overlapping t I in the unterminated RNA, is processed by host RNase III. We have isolated λsib − mutants by their inability to inhibit int expression from p L transcripts. sib mutations were genetically mapped to the left of the λ attachment site, and do not structurally alter this site for recombination. Several sib mutations do alter the nucleotide sequence of the overlapping sib and t I sites. The λsib − mutants tested prevent RNA processing but do not affect transcription termination in vivo.

Purification and properties of Int-h, a variant protein involved in site-specific recombination of bacteriophage lambda.

Under physiological conditions, integration of lambda DNA into the Escherichia coli chromosome requires the direct participation of only two proteins, the viral int gene product and E. coli integration host factor (IHF). A variant of the int gene has been isolated that permits integrative recombination in cells mutant for one of the two subunits of IHF (Miller, H.I., Mozola, M.A., and Friedman, D.I. (1980) Cell 20, 721-729). In the present work, we have purified Int-h, the product of this variant gene. In contrast to the wild-type int gene product (Int+), which produces almost no recombinants in the absence of IHF, purified Int-h protein sponsors reduced but significant levels of integrative recombination in the absence of any E. coli supplement. This shows that the int gene encodes all the information necessary for the elementary steps in recombination and implies that IHF functions as an accessory protein. When supplemented by IHF, recombination promoted by Int-h resembles that promoted by Int+ in kinetics, stoichiometry of Int and IHF, and nature of the recombinant product. Under these conditions, Int-h uses supercoiled DNA more effectively than nonsupercoiled DNA as a substrate for recombination, as does Int+. However, in the absence of IHF, Int-h recombines supercoiled and nonsupercoiled substrates identically, indicating that IHF is an important part of the mechanism that senses the supercoiled state of the substrate DNA during recombination. A surprising difference in recombination carried out by Int-h in the presence or absence of IHF concerns the degree to which sites on the same circle recombine with one another as opposed to sites on sister molecules. In the presence of IHF, Int-h favors intramolecular recombination, as does Int+. However, in the absence of IHF, Int-h almost exclusively promotes intermolecular recombination.

Removal of a terminator structure by RNA processing regulates int gene expression

The int gene of phage λ encodes a protein involved in site-specific recombination. Its expression is regulated differentially during successive phases of the λ infective cycle. The gene is transcribed early after infection from one promoter, p L, and later from a second promoter p I. Each transcription event requires different positive activation factors, λ N and cII proteins, respectively. Transcription from the p I promoter, located adjacent to int, passes through int and terminates 277 nucleotides beyond int at t I. Polymerases initiating at p L transcribe through t I, and into the b segment of λ DNA. The read-through p L transcript is sensitive to cleavage by the endonuclease, RNase III, both in vivo and in vitro. Two specific cuts are made by RNase III in a double-stranded structure about 260 nucleotides beyond int in the location of the t I terminator. Functionally, the processed p L transcript is unable to synthesize the int gene product, whereas the terminated and unprocessed p I transcript expresses int. Interestingly, unprocessed p L transcripts made in hosts defective in RNase III ( rnc −) can express int. Thus a correlation exists between processing and negative control of int expression. The place where processing occurs, some 260 nucleotides beyond int, is called sib, and the control of int expression from this site is called retroregulation. Retroregulation by sib is not restricted just to the int gene; we show that if the sib site is cloned beyond a bacterial gene, the gene is controlled by sib and RNase III. Specific models are discussed with respect to control of gene expression by RNase III from a site beyond the controlled gene.

Posttranscriptional control of bacteriophage lambda gene expression from a site distal to the gene.

The bacteriophage lambda int gene product, integrase, recombines the phage DNA with the host DNA at specific sites on each to accomplish lysogeny. The int gene is transcribed from two promoters, PL and PI, each regulated positively by lambda proteins. The expression of integrase is also controlled from a site, sib, in the b region of the phage genome. This is a unique regulatory site because it is located distal to the structural gene in relation to the promoters. The expression of int from the PL promoter is inhibited when sib is present. This effect appears to be specific for PL because sib does not cause inhibition of PI-dependent int synthesis. lambda mutants that contain alterations in the site have been isolated. Sequence analyses of the mutations reveal single base changes, spanning 37 base pairs (bp) in the b region, some 240 bp beyond the int gene. Another mutant, hef13, which has a phenotype similar to that of sib, introduces a nucleotide change within the same 37-bp region. The sib and hef mutations cluster within a region of dyad symmetry. Regulation of int synthesis by sib occurs after transcription of the int gene. There is no difference in the rate of PL-promoted int mRNA synthesis in either sib+ or sib- phage infections, yet int mRNA is less stable in the sib+ infection. Because RNase III host mutants are defective in sib regulation, processing of the PL mRNA at sib by this endoribonuclease may cause int mRNA decay and decrease int synthesis.

Direct role of the himA gene product in phage lambda integration.

The integration of phage lambda into the Escherichia coli chromosome is accomplished by a site-specific recombination between two unique DNA sequences (attB on the bacterial genome and attP on the phage; reviewed in refs 2, 3) and requires proteins encoded by both the bacterium and the phage. Genetic and biochemical studies have shown that bacterial strains mutant in the himA gene, located at 38 min on the E. coli map, are defective in the activity of the host-encoded component. They are, moreover, defective for the growth of bacteriophage Mu, for precise excision of transposable antibiotic resistance determinants and for the synthesis of the lambda int gene product. We now show that the himA gene product (phimA) is not solely a regulator of genes involved in integration but is one of two host polypeptides required for integrative recombination.

Bacteriophage P22 proteins specified by the region between genes 9 and erf

By examining the proteins produced by bacteriophage P22 strains carrying deletions in the region between genes 9 and erf, we show that this region directs the synthesis of at least nine early proteins other than the int gene product. Analysis of these deletion strains allows us to construct a map of this nonessential region of the P22 genome.

Nicking-closing activity associated with bacteriophage lambda int gene product.

Integrative recombination of bacteriophage lambda requires the action of the protein Int, the product of the phage int gene. In this paper we show that highly purified Int relaxes supercoiled DNA. The association of this nicking-closing activity with Int is shown by: (i) the cosedimentation of nicking-closing and recombination activities of purified Int, (ii) the parallel inactivation of the two activities in purified Int by both heat and a specific antiserum, and (iii) the alteration of both activities in crude extracts of a strain expressing a mutant int gene. The nicking-closing activity of Int functions in the absence of divalent cations and in the absence of an apparent source of chemical energy. The activity displays no obvious sequence specificity and is inhibited by Mg2+, spermidine, and single-stranded DNA. Int relaxes positive as well as negative supercoils. We present a model for the mechanism of strand exchange that describes how the nicking-closing activity of Int might be used during recombination.

The bacteriophage lambda int gene product. A filter assay for genetic recombination, purification of int, and specific binding to DNA.

Bacteriophage lambda int gene is required for the integration of viral DNA into the chromosome of Escherichia coli. We have extensively purified the product of the int gene (Int) from a lysogen of E. coli that constitutively expresses this gene. Int was assayed by its ability to promote integrative recombination of supertwisted substrate DNA in vitro using a new method based on filter trapping of a recombinant product DNA. In order to catalyze integrative recombination, Int must be supplemented by other factors that can be extracted from bacterial host cells. By itself, purified Int does not demonstrate detectable endonuclease, exonuclease, or nicking-closing activities. However, Int does make stable complexes with double-stranded lambda-DNA containing an attachment site, the region at which recombination takes place. No stable complexes are observed between Int and lambda-DNA without an attachment site or between Int and DNA containing the bacterial site of integration. Int, therefore, appears to be a specificity element that relies on additional factor(s) to provide or activate the catalytic functions required for recombination.

On the role of the bacteriophage λ int gene product in site specific recombination

Integrative recombination of phage λ DNA occurs in extracts made from cells synthesizing int protein. In this paper we show that extracts of cells containing temperature-sensitive int protein are inactivated more rapidly by incubation at 38 °C than are wild-type extracts. This indicates that the int protein is directly involved in the recombination reaction.

Site-specific recombination in bacteriophage lambda

Lambda attB-attP is a derivative of bacteriophage lambda that contains both attB and attP, two sites required for integrative recombination. Lambda attB-attP can undergo int-mediated recombination to yield progeny phages whose DNA is 15% shorter than that of the parental phage. We have studied intracellular phage DNA from an infection of lysogenic bacteria with λ attB-attP in the presence of int gene product, rifampicin and chloramphenicol. The majority of the intracellular phage DNA consisted of circles with lengths of 17.51, 15.09 and 2.38 μm. Partial denaturation mapping confirmed that the 15.09 and the 2.38-μm molecules arose by an int-mediated intramolecular recombination reaction of the type predicted by the Campbell (1962) model. A minor proportion of the circles (3%) were much larger (33.9, 30.2 and 4.7 μm); in these cases denaturation mapping indicated that both intra- and intermoleeular recombination could take place.

Integrative recombination of bacteriophage lambda DNA in vitro.

An in vitro system for the production of integrative recombinant DNA of bacteriophage lambda is described. The in vitro recombination mimics the in vivo integration of viral DNA into host DNA in its requirement for int gene product, for the presence of a thermolabile component, and for the limitation of the recombination to a pair of specialized sites (attachment sites) on the DNA. The enzymes are extracted from Escherichia coli containing phage lambda gene products. The substrate is the DNA from lambda-attB-attP, a phage variant that contains two attachment sites on the same chromosome. The product is a recombinant phage chromosome that has lost the DNA between the attachment sites. The parental and recombinant DNA are distinguished following transfection to mature phage in spheroplasts. The reaction requires ATP, Mg++, spermidine, and a monovalent cation. Recombination occurs preferentially between attachment sites on the same molecule. The enzymatic activity is completely inhibited by extracts containing xis gene product.

Requirements for insertion of bacteriophage λ DNA into the DNA of Escherichia coli

Insertion of phage λ DNA into the normal attachment site of the DNA of the host Escherichia coli has been studied by ultracentrifugation analysis of the conversion of covalent circles of F′450 (F′ gal att λ bio ) to F′450(λ) circles. We have found that integration proceeds at the normal rate if, in addition to the int gene product and a proper combination of phage and bacterial attachment sites, a large pool of λ DNA and some activity of the excision gene xis are present. In addition, turnoff of both phage DNA synthesis and xis gene activity are required.

Purification of bacteriophage lambda Int protein.

BACTERIOPHAGE λ can direct the formation of small amounts of several λ proteins in a host whose endogenous protein synthesis has been decreased by ultraviolet irradiation before phage infection1. The phage protein can be labelled by the incorporation of radioactive amino acids after infection. Many of these proteins can be resolved by sodium dodecyl sulphate (SDS) gel electrophoresis and tentative gene identifications can be made by comparing the products of wild type phage infection with those of phage carrying mutation(s) which selectively remove certain gene product(s)2. I have purified the int gene product using differential label techniques to detect the Int polypeptide. This gene product functions in vivo to direct recombination between specific sites in DNA; the radiochemically pure product has been used to investigate the in vitro interaction between Int and DNA.