NKBOR, a mini-Tn10-based transposon for random insertion in the chromosome of Gram-negative bacteria and the rapid recovery of sequences flanking the insertion sites in Escherichia coli.

We have constructed an R6K-based suicide vector that permits the random insertion of a mini-transposon named NKBOR into the chromosome of Gram-negative bacteria and the subsequent rapid cloning of sequences flanking the insertion site in Escherichia coli. This mini-transposon contains a conditional R6K plasmid origin of replication, a kanamycin resistance gene and unique restriction sites between the IS10 inverted repeats. NKBOR can be propagated by replication in an E. coli strain containing the R6K replicase pi protein. Alternatively the mini-transposon can be replicated in a pSC 101 derivative that is thermosensitive for its replication so that the mini-transposon acts as a suicide plasmid at nonpermissive temperatures. Efficient NKBOR transposition is ensured by expression of an adjacent transposase gene and has been demonstrated in E. coli, Klebsiella pneumoniae, and Erwinia carotovora. Sequences flanking the insertion sites in these strains can be rapidly recovered and identified in E. coli strains expressing the R6K pi protein.

Transcription attenuation associated with bacterial repetitive extragenic BIME elements.

Transcription attenuation comprises several processes that affect transcript elongation and transcription termination, and has an important role in regulating gene expression. In most cases, transcription attenuation is used as a regulatory mechanism that allows the cell to adjust protein synthesis levels in response to a specific signal. Here, by using a tRNA gene as a transcriptional reporter, we characterize a new type of transcription attenuation mechanism in Escherichia coli that involves bacterial interspersed mosaic elements (BIMEs), the main family of repetitive extragenic elements. The transcription termination factor Rho is required for attenuation in association with BIMEs, thus revealing a new role for Rho as a BIMEs-dependent global regulator of gene expression. By mutational analyses, we identified nucleotide determinants of BIMEs that are required for attenuation and showed that this process relies on a sequence-specific mechanism. Our data are consistent with a model in which BIMEs provoke a pause in RNA polymerase movement and Rho acts ultimately to terminate transcription. BIME-dependent transcription attenuation may be used as a means to differentially regulate expression of adjacent genes belonging to a single operon. BIMEs are dispersed in more than 250 operons such that attenuation can simultaneously affect expression of a large number of genes encoding unrelated proteins. This attenuation phenomenon, together with the known ability of BIMEs to stabilize upstream mRNA, reveals how dispersion of these abundant repetitive elements may affect gene regulation at the genome level.

The phytopathogenic bacteria Erwinia carotovora infects Drosophila and activates an immune response.

Although Drosophila possesses potent immune responses, little is known about the microbial pathogens that infect Drosophila. We have identified members of the bacterial genus Erwinia that induce the systemic expression of genes encoding antimicrobial peptides in Drosophila larvae after ingestion. These Erwinia strains are phytopathogens and use flies as vectors; our data suggest that these strains have also evolved mechanisms for exploiting their insect vectors as hosts. Erwinia infections induce an antimicrobial response in Drosophila larvae with a preferential expression of antibacterial versus antifungal peptide-encoding genes. Antibacterial peptide gene expression after Erwinia infection is reduced in two Drosophila mutants that have reduced numbers of hemocytes, suggesting that blood cells play a role in regulating Drosophila antimicrobial responses and also illustrating that this Drosophila-Erwinia interaction provides a powerful model for dissecting host-pathogen relationships.

A quantitative UV laser footprinting analysis of the interaction of IHF with specific binding sites: re-evaluation of the effective concentration of IHF in the cell.

The integration host factor (IHF) of Escherichia coli is a major nucleoid-associated protein that binds to specific sites on DNA. Using gel retardation and competition experiments we have estimated that in vitro IHF binds specific sites 1000-10,000 times more tightly than non-specific, chromosomal DNA. We have analyzed the in vitro and in vivo interaction of IHF with three specific binding sites using UV laser footprinting. Because there is a strict correspondence between the intensity of the footprinting signal and the occupancy of a site, we can correlate in vitro association constants with in vivo site occupancy. From the fractional occupancy of various ihf sites in vivo, we then estimate the amount of free IHF in the cell. Exponentially growing cells contain only about 0.7 nM of free IHF, a value 20-fold smaller than the one previously deduced from DMS footprinting. As a consequence low affinity sites are only partially occupied and strong binding sites reach semi-saturation. In stationary phase the concentration of free IHF in the cell increases about sevenfold. These results show that only a very small fraction of total IHF is free in solution. Given the affinity of IHF for non-specific DNA our data imply that a large part of chromosomal DNA is accessible to IHF, and that IHF is a major contributor to chromosomal DNA condensation. The in vivo UV-laser footprinting method is of general interest, because it allows the measurement and the comparison of DNA-protein interactions in vitro and in vivo.

In vivo cleavage of Escherichia coli BIME-2 repeats by DNA gyrase: genetic characterization of the target and identification of the cut site.

The Escherichia coli chromosome contains about 300 bacterial interspersed mosaic elements (BIMEs). These elements, located at the 3' end of genes, are composed of three types of alternating repetitive extragenic palindromes (REPs). Based on the type of REP they contain and on their ability to interact with the integration host factor (IHF), BIMEs are subdivided into two families: BIME-1 elements contain an IHF binding site flanked by converging Y and Z1 REPs, whereas BIME-2 elements contain a variable number of alternating Y and Z2 REPs without an IHF site. Although some BIMEs have been implicated in the protection of mRNA against 3' exonucleolytic degradation, the main role of elements belonging to both families remains to be elucidated. In this paper, we used oxolinic acid, a drug that reveals potential sites of DNA gyrase action, to demonstrate that DNA gyrase interacts in vivo with BIME-2 elements. The frequency of cleavage varied from one element to another, and the cleavage pattern observed in elements containing several REPs indicated that DNA gyrase cut DNA every two REPs. A single cleavage site has been identified in the Y REP in six out of seven instances, and the nucleotide sequence of a 44 bp fragment containing the scission point displayed conserved residues at six positions. The lack of one of the conserved residues accounted for the absence of cleavage in most of the Z2 REPs. Our results also showed that cleaved REPs were always associated with another REP, suggesting that a pair of diverging REPs constitutes the target of DNA gyrase. DNA gyrase cleavage at repetitive BIME-2 elements may have consequences for DNA topology and genomic rearrangements.

Phage HK022 Roi protein inhibits phage lytic growth in Escherichia coli integration host factor mutants.

Temperate coliphage HK022 requires integration host factor (IHF) for lytic growth. The determinant responsible for this requirement was identified as a new gene (roi) located between genes P and Q. This gene encodes a DNA-binding protein (Roi) containing a helix-turn-helix motif. We have shown that Roi binds a site within its own gene that is closely linked to an IHF binding site. By gel retardation experiments, we have found that IHF binding stabilizes the interaction of Roi with its gene. We have isolated three independent phage mutants that are able to grow on an IHF- host. They carry different mutations scattered in the roi gene and specifying single amino-acid changes. The interactions of all three Roi mutant proteins with the Roi binding site differed from that of the wild type. Roi displays strong similarities, in its C-terminal half, to two putative DNA-binding proteins of bacteriophage P1: Ant1 and KilA. The mode of action of the Roi protein and the possibility that IHF is modulating the expression and/or the action of Roi are discussed.

In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell.

In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell.

Identification of a gene encoding the replication initiator protein of the Streptomyces integrating element, pSAM2.

pSAM2 is an 11-kilobase integrating element from Streptomyces ambofaciens which was previously shown to generate single-stranded DNA during replication, indicating that it probably replicates by a rolling-circle replication (RCR) mechanism. Two separate regions are involved in its replication, one of which was shown to contain the plus origin of replication (ds origin). We report here the study of the second region. Its nucleotide sequence was determined and analysed for open reading frames (ORFs). Three putative ORFs were identified: orf183 (183 amino acids (aa)), orf50 (50 aa), and repSA (459 aa). orf183 is not necessary for replication. The function of orf50 is unknown. repSA is essential for pSAM2 replication; it could encode a protein, RepSA, presenting similarities to the replication initiator proteins (Rep) of elements that replicate by an RCR mechanism. A derivative consisting of repSA, the region containing ds origin, a Streptomyces antibiotic resistance marker, and pBR322, could replicate in Streptomyces, further demonstrating that this ORF encodes the major replication protein of pSAM2. repSA might be co-transcribed with the genes involved in integration and excision of pSAM2.

Specific interaction of IHF with RIBs, a class of bacterial repetitive DNA elements located at the 3' end of transcription units.

The prokaryotic REP (repetitive extragenic palindromes) or PU (palindromic units) sequences are often associated with other repetitive elements, forming arrangements which have been called 'BIMEs' (bacterial interspersed mosaic elements). It is estimated that the Escherichia coli chromosome carries approximately 300-500 BIMEs, whose biological role is at present unknown. We have identified a family of BIMEs consisting of two converging REP sequences flanking a 35 bp conserved segment which carries a static DNA bend and a binding site for IHF, the integration host factor of E.coli. We estimate that the E.coli genome harbors approximately 100 copies of this module, which we name 'RIB' (reiterative ihf BIME). We have analyzed by gel retardation and by footprinting the in vitro interaction of IHF with individual RIBs, and shown that the protein binds strongly and specifically to their center. We have also demonstrated binding of IHF to the chromosomal population of RIBs, using a new approach which combines two-dimensional bandshift and Southern blotting. RIB elements are at the end of transcription units, and thus define a new class of ihf sites. Possible implications for genome structure and DNA topology are discussed.

Mutational analysis of cis-acting sequences and gene function in RNA3 of cucumber mosaic virus.

RNA3 of the Kin strain of the tripartite (+)-strand cucumber mosaic virus has 2199 nucleotide residues. Two open reading frames encoding 3a protein (a putative movement protein) and coat protein (CP) are separated by a 286-nucleotide inter-cistronic region (IR). This IR contains a subgenomic promoter for production of a subgenomic RNA (RNA4), from which CP is synthesized. Using transcripts generated from mutant forms of a cDNA clone of RNA3, we have characterized the cis-acting sequences necessary for RNA3 accumulation and RNA4 synthesis and analyzed the role of the RNA3-encoded proteins. Efficient accumulation of RNA3 derivatives in tobacco protoplasts required 92 nucleotides at the 5' end, 250 nucleotides in the IR, and 275 nucleotides at the 3' end of the RNA molecule. The 250-nucleotide IR includes a 90-nucleotide sequence which is necessary for subgenomic promoter activity. Although common regions are involved in RNA3 accumulation and RNA4 synthesis, the modes of action of IR for these two phenomena are different. The analysis of forms of RNA3 with internal duplications demonstrated that RNA3 accumulation depended on the context of the IR. Subgenomic promoter activity was more position dependent and was always stronger from the promoter closer to the 3' end of the (+)-strand RNA. A mutation in IR specifically affected (+)-strand RNA accumulation, indicating a role for that region in (+)-strand synthesis. The role of the RNA3-encoded proteins was analyzed by mutation and inoculation either to plants or to protoplasts. Mutation of the 3a protein had no effect on RNA3 accumulation in protoplasts, whereas CP mutations caused reduced CMV RNA accumulation. This reduction was more pronounced for (+)- than for (-)-strand accumulation. RNA of CP mutations was undetectable in inoculated leaves, whereas RNA of 3a protein mutants accumulated, albeit at levels several orders of magnitude lower than with wild-type CMV. The conclusion from these data is that both proteins are required for efficient spread of CMV from the site of infection.

Extreme resistance to potato virus X infection in plants expressing a modified component of the putative viral replicase.

Three types of mutation were introduced into the sequence encoding the GDD motif of the putative replicase component of potato virus X (PVX). All three mutations rendered the viral genome completely noninfectious when inoculated into Nicotiana clevelandii or into protoplasts of Nicotiana tabacum (cv. Samsun NN). In order to test whether these negative mutations could inactivate the viral genome in trans, the mutant genes were expressed in transformed N.tabacum (cv. Samsun NN) under control of the 35S RNA promoter of cauliflower mosaic virus and the transformed lines were inoculated with PVX. In 10 lines tested in which the GDD motif was expressed as GAD or GED there was no effect on susceptibility to PVX. In two of four lines transformed to express the ADD form of the conserved motif, the F1 and F2 progeny plants were highly resistant to infection by PVX, although only to strains closely related to the source of the transgene. The resistance was associated with suppression of PVX accumulation in the inoculated and systemic leaves and in protoplasts of the transformed plants, although some low level viral RNA production was observed in the inoculated but not the systemic leaves when the inoculum was as high as 100 or 250 micrograms/ml PVX RNA. These results suggest for a plant virus, as reported previously for Q beta phage, that virus resistance may be engineered by expression of dominant negative mutant forms of viral genes in transformed cells.

Infectious in vitro transcripts from amplified cDNAs of the Y and Kin strains of cucumber mosaic virus.

Using a method based on the polymerase chain reaction (PCR) with primers that include the phi 10 promoter of bacteriophage T7, we obtained cDNA clones of the three RNA genomes of two different strains of cucumber mosaic virus (CMV; Kin and Y strains) from which infectious in vitro transcripts were generated, and demonstrated that the same primers could be used for amplification of at least two other strains of CMV (O and Py). This method is rapid and requires only limited nucleotide (nt) sequence data (16-18 nt) from the termini of the RNA species. Either viral RNA or unpurified RNA samples from infected plants can be used as template for first-strand cDNA synthesis. For cDNAs of RNA1 and RNA2 of the Y strain, the transcription efficiency was substantially lower than with the Kin strain, unless the primer sequence included transcribed G residues on the 5' side of the viral cDNA, so that the promoter for T7 RNA polymerase resembled more closely the canonical sequence from the bacteriophage T7 phi 10 promoter. The lower specific infectivity of transcripts of the modified cDNAs was more than compensated for by increased transcription efficiency. The possibility that the PCR process may introduce deleterious mutations into the viral cDNA was investigated by re-amplification of a functional cloned cDNA of RNA2: all six cDNA clones of the re-amplified cDNA produced transcripts as infectious as those from the progenitor cDNA.

Functional analysis of the Streptomyces ambofaciens element pSAM2.

pSAM2 is an 11-kb element integrated in the Streptomyces ambofaciens ATCC23877 genome and found additionally as a free replicon present at several copies per chromosome in strain JI3212, the derivative of ATCC23877 isolated after uv irradiation. In spite of its small size, this element specifies numerous functions including maintenance, site-specific integration, self-transmissibility, pock formation, and mobilization of chromosomal markers. After transfer of the free form of pSAM2 to Streptomyces lividans, the free and the integrated forms coexist. A functional map of pSAM2 was deduced from phenotypes exhibited in S. lividans by numerous deletion or insertion derivatives. In addition to the previously characterized regions sufficient for site-specific integration we have shown that separate regions are involved in either plasmid maintenance as a free molecule, plasmid transfer, and pock formation. Transfer of pSAM2 could depend on its ability to be maintained in a free form, since plasmids deficient in this function are transferred at very low frequency. Deletions of some regions of the plasmid are lethal for the plasmid or the host, but if some other regions are deleted simultaneously, transformants can be obtained.

Construction of a series of pSAM2-based integrative vectors for use in actinomycetes.

We have developed vectors which allowed integration of cloned DNA at a single site in the chromosome of Streptomyces lividans 66. These vectors made use of (1) an Escherichia coli replicon, (2) a thiostrepton (Th)- and a streptomycin/spectinomycin-resistance gene for selection in Streptomyces, (3) a 3.5-kb fragment of the Streptomyces integrative plasmid pSAM2 containing its xis and int genes as well as its attachment site, attP, to direct the integration of the vectors at the chromosomal pSAM2 attachment site attB, (4) the origin of transfer of the IncP broad-host-range plasmid RK2 which allowed the mobilization of the vectors from E. coli to S. lividans, and (5) the Th-inducible tipA promoter to permit regulated transcription of cloned genes. We demonstrated that pPM927, a plasmid which contained all of these elements, was able to transfer cloned fragments from E. coli to S. lividans by conjugation, stably integrate into the chromosome, and express cloned genes from the tipA promoter. Furthermore, since pPM927 contained the pBR322 replicon, cloned fragments could be conveniently recovered from the S. lividans chromosome for analysis in E. coli by cleavage of genomic DNA isolated from transformed strains, intramolecular ligation and transformation. Since we have shown that the pSAM2 attB site forms part of a conserved prokaryotic tRNA gene, these integrative vectors are potentially useful tools for analysis and expression of genes in diverse bacteria.

The chromosomal integration site of the Streptomyces element pSAM2 overlaps a putative tRNA gene conserved among actinomycetes.

The pSAM2 element of Streptomyces ambofaciens integrates site-specifically in the genome of different Streptomyces species by recombination between a 58 bp sequence common to the plasmid (attP) and the chromosome (attB). Southern hybridization analysis showed that sequences similar to the pSAM2 attB site were found in other actinomycetes (Mycobacterium, Nocardia, Micromonospora) as well as unrelated bacteria (Bacillus circulans, Escherichia coli, Clostridium botulinum, Bordetella pertussis, and Legionella pneumophila). Hybridizing fragments from B. circulans and Mycobacterium tuberculosis were cloned and sequenced. Comparison of these sequences with the sequence of the integration zone of S. ambofaciens revealed a conserved region of 76 bp which overlapped with the attB site. This conserved sequence was similar to the Salmonella typhimurium and E. coli tRNA(pro1) genes as well as a number of eucaryotic tRNA genes and had a proline-tRNA-like cloverleaf structure. Furthermore, the Streptomyces lividans attB site of the Streptomyces glaucescens element pIJ408 was also found to overlap a potential tRNA gene (tRNA(thr)). We note here that these two putative tRNA genes as well as those which overlap the attB site of the elements SLP1 of Streptomyces coelicolor and pMEA100 of Nocardia mediterranei all contain the site where integrative recombination takes place. These presumptive actinomycete tRNA genes lack the 3' terminal CCA sequence found in most procaryotic tRNA genes.

Organization and nucleotide sequence analysis of a ribosomal RNA gene cluster from Streptomyces ambofaciens.

The Streptomyces ambofaciens genome contains four rRNA gene clusters. These copies are called rrnA, B, C and D. The complete nucleotide (nt) sequence of rrnD has been determined. These genes possess striking similarity with other eubacterial rRNA genes. Comparison with other rRNA sequences allowed the putative localization of the sequences encoding mature rRNAs. The structural genes are arranged in the order 16S-23S-5S and are tightly linked. The mature rRNAs are predicted to contain 1528, 3120 and 120 nt, for the 16S, 23S and 5S rRNAs, respectively. The 23S rRNA is, to our knowledge, the longest of all sequenced prokaryotic 23S rRNAs. When compared to other large rRNAs it shows insertions at positions where they are also present in archaebacterial and in eukaryotic large rRNAs. Secondary structure models of S. ambofaciens rRNAs are proposed, based upon those existing for other bacterial rRNAs. Positions of putative transcription start points and of a termination signal are suggested. The corresponding putative primary transcript, containing the 16S, 23S and 5S rRNAs plus flanking regions, was folded into a secondary structure, and sequences possibly involved in rRNA maturation are described. The G + C content of the rRNA gene cluster is low (57%) compared with the overall G + C content of Streptomyces DNA (73%).

The integrated conjugative plasmid pSAM2 of Streptomyces ambofaciens is related to temperate bacteriophages.

Streptomyces ambofaciens ATCC23877 and derivatives contain the 11-kb element pSAM2 present in an integrated state or as a free and integrated plasmid. This element, able to integrate site-specifically in the genome of different Streptomyces species, is conjugative and mobilizes chromosomal markers. Besides these plasmid functions, we have shown that the site-specific recombination system of pSAM2 presents strong similarities with that of several temperate phages. The integration event is promoted by a site-specific recombinase of the integrase family. The int gene encoding this integrase is closely linked to the plasmid attachment site (attP). A small open reading frame (ORF) overlaps the int gene and the predicted protein exhibits similarities with Xis proteins involved in phages excision. The integrated copy of pSAM2 in strain ATCC23877 is flanked by att sequences (attL and attR). Another att sequence (attX) is present in this strain and attX and attL are the boundaries of a 42-kb fragment (xSAM1) absent, as well as pSAM2, from S.ambofaciens DSM40697. Sequences partially similar to pSAM2 int gene are found near the chromosomal integration zone in both S.ambofaciens strains. The possible origin of pSAM2, an element carrying plasmid as well as phage features, is discussed.

Structural analysis of loci involved in pSAM2 site-specific integration in Streptomyces.

pSAM2 is an 11-kb plasmid integrated in the Streptomyces ambofaciens ATCC23877 and ATCC15154 genomes and found additionally as a free replicon in an uv derivative. After transfer into S. ambofaciens DSM40697 (devoid of pSAM2) or into Streptomyces lividans, specific integration of pSAM2 occurred very efficiently. A 58-bp sequence (att) present in both pSAM2 (attP) and S. ambofaciens strain DSM40697 (attB) attachment regions is found at the boundaries (attL and attR) of integrated pSAM2 in S. ambofaciens strain ATCC23877. The S. lividans chromosomal integration zone contained an imperfectly conserved att sequence (attB), and the integration event of pSAM2 was located within a 49-bp sequence of attB. Only one primary functional attB sequence was present in the S. lividans or S. ambofaciens DSM40697 total DNA. The integration zone of S. lividans hybridized with the integration zone of S. ambofaciens DSM40697. The two integration zones were homologous only to the right side of the att sequence. The conserved region contained an open reading frame (ORF A) with a stop codon located 99 bp from the attB sequence in both strains. S. ambofaciens DSM40697 contained DNA sequences related to pSAM2 on the left side of the att site. The att sequence was included in a region conserved in Streptomyces antibioticus, Streptomyces actuosus, Streptomyces bikiniensis, Streptomyces coelicolor, Streptomyces glaucescens, and Streptomyces parvulus. Site-specific integration of a pSAM2 derivative was characterized in another unrelated strain, Streptomyces griseofuscus. This strain contained an imperfectly conserved 58-bp attB sequence, and the integration event took place within a 45-bp sequence of attB. Site-specific integration of pSAM2 in three nonrelated Streptomyces strains suggests the wide host range of pSAM2 integration in Streptomyces.