Two DNA invertases contribute to flagellar phase variation in Salmonella enterica serovar Typhimurium strain LT2

Salmonella enterica serovar Typhimurium strain LT2 possesses two nonallelic structural genes, fliC and fljB, for flagellin, the component protein of flagellar filaments. Flagellar phase variation occurs by alternative expression of these two genes. This is controlled by the inversion of a DNA segment, called the H segment, containing the fljB promoter. H inversion occurs by site-specific recombination between inverted repetitious sequences flanking the H segment. This recombination has been shown in vivo and in vitro to be mediated by a DNA invertase, Hin, whose gene is located within the H segment. However, a search of the complete genomic sequence revealed that LT2 possesses another DNA invertase gene that is located adjacent to another invertible DNA segment within a resident prophage, Fels-2. Here, we named this gene fin. We constructed hin and fin disruption mutants from LT2 and examined their phase variation abilities. The hin disruption mutant could still undergo flagellar phase variation, indicating that Hin is not the sole DNA invertase responsible for phase variation. Although the fin disruption mutant could undergo phase variation, fin hin double mutants could not. These results clearly indicate that both Hin and Fin contribute to flagellar phase variation in LT2. We further showed that a phase-stable serovar, serovar Abortusequi, which is known to possess a naturally occurring hin mutation, lacks Fels-2, which ensures the phase stability in this serovar.     

Interaction of FimB and FimE with the fim switch that controls the phase variation of type 1 fimbriae in Escherichia coli K-12

The phase variation of type 1 fimbriae in Escherichia coli is associated with the site-specific inversion of a short DNA element. Recombination at fim requires fimB and fimE , and their products are considered to be the fim recombinases. In this study, FimB and FimE were overproduced and extracts containing the proteins were shown to (i) bind to and (ii) invert the fim switch in vitro . Phenanthroline-copper protection of DNA–protein complexes showed that both FimB and FimE bind to half-sites that flank, and overlap with, the left and right inverted repeats (IRL and IRR, respectively) of the fim switch. Alignment of the four half-sites identified a conserved 5'-CA doublet; mutation of these two bases lowers the affinity of binding of both FimB and FimE to the inverted repeats, and greatly diminishes inversion of the fim switch in vivo . The specificity of the fim recombinases observed in vivo (FimB switching in both directions; FimE switching from on-to-off only) was maintained in vitro Furthermore, the different binding affinities of FimB and FimE for the various half-site combinations suggests that the specificity of FimE could arise, in part, from the low affinity of FimE for IRL (off).     

Isolation of an integrated provirus of Moloney murine leukemia virus with long terminal repeats in inverted orientation: integration utilizing two U3 sequences.

We have previously described the construction of a mutant of Moloney murine leukemia virus, in594-2, which carries a 2-base-pair insertion in the U5 region of the genome and is partially defective in forming the integrated proviral DNA. We have now recovered a cloned copy of an unusual provirus from rat cells infected with this mutant. The viral genome is flanked by long terminal repeats in inverted orientation, with U3 sequences joined to cellular DNA at both of the outer edges. In addition, the provirus is a recombinant, containing a segment of a VL30 element in inverted orientation in place of the Moloney murine leukemia virus env region. The recovery of this provirus indicates that two U3 regions can be used for viral integration and suggests that there may be no absolute requirement in the reaction for those U5 sequences outside the 13-base-pair inverted repeats.     

Machinery to support genome segment inversion exists in a herpesvirus which does not naturally contain invertible elements

In many herpesviruses, genome segments flanked by inverted repeats invert during DNA replication. It is not known whether this inversion is a consequence of an inherently recombinagenic replicative mechanism common to all herpesviruses or whether the replication enzymes of viruses with invertible segments have specifically evolved additional enzymatic activities to drive inversion. By artificially inserting a fusion of terminal sequences into the genome of a virus which normally lacks invertible elements (murine cytomegalovirus), we created a genome composed of long and short segments flanked by 1,359- and 543-bp inverted repeats. Analysis of genomic DNA from this virus revealed that inversion of both segments generates equimolar amounts of four isomers during the viral propagation necessary to produce DNA for analysis from a single viral particle. We conclude that a herpesvirus which naturally lacks invertible elements is able to support efficient segment inversion. Thus, the potential to invert is probably inherent in the replication machinery of all herpesviruses, irrespective of genome structure, and therefore genomes with invertible elements could have evolved simply by acquisition of inverted repeats and without concomitant evolution of enzymatic activities to mediate inversion. Furthermore, the recombinagenicity of herpesvirus DNA replication must have some importance independent of genome segment inversion.     

PapB paralogues and their effect on the phase variation of type 1 fimbriae in Escherichia coli

Recent work has demonstrated that expression of type 1 fimbriae is repressed by PapB, a regulator of pyelonephritis-associated pili (P-pili). PapB belongs to family of related adhesin regulators, for which consensus residues required for DNA binding and oligomerization have been identified. Of the regulators tested in this study, PapB, SfaB (S-fimbriae) and PefB (Salmonella enterica serovar Typhimurium--plasmid-encoded fimbriae) repressed FimB-promoted off-to-on inversion of the fim switch, although complete repression was only demonstrated by PapB. DaaA, FaeB, FanA, FanB and ClpB had no effect on fim switching. In addition, only PapB stimulated FimE-promoted on-to-off inversion. Deletion analysis demonstrated that this specificity resides in the carboxy terminal of the protein, and not the amino terminal, with the central region being homologous among the family members. Exchange of Leu(82) and Ile(83) of PapB for the equivalent residues from the DaaA protein (Phe and Gln) within the carboxy terminal virtually abolished cross-talk activity. Whereas PapB can bind to a region around the left inverted repeat of the fim switch, DaaA and the PapB double mutant were effectively unable to bind this region. A previously characterized PapB DNA binding mutant also failed to bind to this region and failed to inhibit FimB activity at the fim switch. Thus, repression of fim expression appears unique to PapB and SfaB within E. coli and requires DNA binding involving amino acid residues located both within the homologous core and in the heterogeneous carboxy terminus. The variation in the carboxy terminus between the PapB family members explains their differential effects on fim. This mechanism of cross-talk seems restricted to the P and S family adhesins with type 1 fimbriae and may ensure variable and sequential expression of adhesins during urinary tract infections.     

DNA sequence heterogeneity in Fim tyrosine-integrase recombinase-binding elements and functional motif asymmetries determine the directionality of the fim genetic switch in Escherichia coli K-12

Phase-variable expression of type 1 fimbriae in Escherichia coli K-12 involves inversion by site-specific recombination of a 314 bp sequence containing the promoter for fim structural gene expression. The invertible sequence is flanked by 9 bp inverted repeats, and each repeat is in turn flanked by non-identical recombinase-binding elements (RBEs) to which the FimB or FimE site-specific recombinases bind. These proteins have distinct DNA inversion preferences: FimB inverts the switch in the ON-to-OFF and OFF-to-ON directions with similar efficiencies, whereas FimE inverts it predominantly in the ON-to-OFF direction. We have found that FimB and FimE invert the switch through a common mechanism. A genetic investigation involving base-by-base substitution combined with a biochemical study shows that the same DNA cleavage and religation sites are used within the 9 bp inverted repeats, and that each recombination involves a common 3 bp spacer region. A comprehensive programme of RBE exchanges and replacements reveals that FimB is much more tolerant of RBE sequence variation than FimE. The asymmetric location of conserved 5'-CA motifs at either side of each spacer region allows the inside and outside of the switch to be differentiated while the RBE sequence heterogeneity permits its ON and OFF forms to be distinguished by the recombinases.     

Genetic analysis of the mechanism of the Salmonella phase variation site specific recombination system

Summary Phase variation, the alternation of expression of flagellar antigens H1 and H2, in Salmonella typhimurium is mediated by site specific inversion of a 995 bp DNA segment of the chromosome. Hin, a protein encoded within the 995 bp segment, is thought to catalyze the recombination reaction between 14 bp inverted repeats flanking the 995 bp segment. By comparison of the relative rates of inversion of two different plasmids containing the H2 inversion segment flanked by different sequences, we conclude that the sequences adjacent to the inversion segment affect the rate of inversion. Homologous pairing of the repeats is important in H2 inversion since the orientation of the repeats on the host molecule(s) determines the result of the recombination reaction. The presence of the hin gene mediates the fusion of two plasmids when each contains one of the 14 bp repeat sequences. When the 14 bp sequences are direct repeats on a single molecule the sequence between them is deleted. These results support the hypothesis that the H2 inversion system functions by homologous, conservative, site specific recombination which is similar to the systems found associated with TnA transposons and temperate bacteriophage.     

Assaying chromosomal inversions by single-molecule haplotyping

Inversions are an important form of structural variation, but they are difficult to characterize, as their breakpoints often fall within inverted repeats. We have developed a method called 'haplotype fusion' in which an inversion breakpoint is genotyped by performing fusion PCR on single molecules of human genomic DNA. Fusing single-copy sequences bracketing an inversion breakpoint generates orientation-specific PCR products, exemplified by a genotyping assay for the int22 hemophilia A inversion on Xq28. Furthermore, we demonstrated that inversion events with breakpoints embedded within long (>100 kb) inverted repeats can be genotyped by haplotype-fusion PCR followed by bead-based single-molecule haplotyping on repeat-specific markers bracketing the inversion breakpoint. We illustrate this method by genotyping a Yp paracentric inversion sponsored by >300-kb-long inverted repeats. The generality of our methods to survey for, and genotype chromosomal inversions should help our understanding of the contribution of inversions to genomic variation, inherited diseases and cancer.     

Inversion within the haloalkaliphilic virus phi Ch1 DNA results in differential expression of structural proteins

The sequence of phi Ch1 contains an open reading frame (int1) in the central part of its genome that belongs to the lambda integrase family of site-specific recombinases. Sequence similarities to known integrases include the highly conserved tetrad R-H-R-Y. The flanking sequences of int1 contain several direct repeats of 30 bp in length (IR-L and IR-R), which are orientated in an inverted direction. Here, we show that a recombination active region exists in the genome of phi Ch1: the number of those repeats, non-homologous regions within the repeat clusters IR-L and IR-R and the orientation of the int1 gene vary in a given virus population. Within this study, we identified circular intermediates, composed of the int1 gene and the inwards orientated repeat regions IR-L and IR-R, which could be involved in the recombination process itself. IR-L and IR-R are embedded within ORF34 and ORF36 respectively. As a consequence of the inversion within this region of phi Ch1, the C-terminal parts of the proteins encoded by ORF34 and 36 are exchanged. Both proteins, expressed in Escherichia coli, interact with specific antisera against whole virus particles, indicating that they could be parts of phi Ch1 virions. Expression of the protein(s) in Natrialba magadii could be detected 98 h after inoculation, which is similar to other structural proteins of phi Ch1. Taken together, the data show that the genome of phi Ch1 contains an invertible region that codes for a recombinase and structural proteins. Inversion of this segment results in a variation of these structural proteins.     

A 21 kilobase-pair deletion/addition difference in the inverted repeat sequence of chloroplast DNA from Chlamydomonas eugametos and C. moewusii

Summary Our recent physical mapping of chloroplast DNA (cpDNA) from Chlamydomonas moewusii, a unicellular green alga which is interfertile with Chlamydomonas eugametos, has revealed a two-fold size difference between the inverted repeat sequences of these algae. With a size of 42 kbp, the inverted repeat of C. moewusii is the largest yet identified in any chloroplast genome. Here we have compared the arrangement of conserved sequences within the two algal inverted repeats by hybridizing cloned restriction fragments representing over 90% of these repeats to Southern blots of cpDNA digests from the two algae. We found that the size difference between the two algal inverted repeats is due to the presence of an extra DNA segment of 21 kilobase pairs (kbp) in C. moewusii. Except for this sequence, the C. moewusii inverted repeat is highly homologous to the entire C. eugametos repeat and the arrangement of conserved sequences in the two repeats is identical. Southern hybridizations with specific gene probes revealed that the conserved sequences include the rDNA region and the genes coding for the large subunit of ribulose 1,5 bisphosphate carboxylase-oxygenase (rbcL) and for the 32 kilodalton thylakoid membrane protein (psbA). With respect to the conserved sequences, the extra 21 kbp DNA segment of C. moewusii lies in the region of psbA, most probably slightly downstream from this gene.     

Natural variation in a subtelomeric region of Arabidopsis: implications for the genomic dynamics of a chromosome end

We investigated genome dynamics at a chromosome end in the model plant Arabidopsis thaliana through a study of natural variation in 35 wild accessions. We focused on the single-copy subtelomeric region of chromosome 1 north (approximately 3.5 kb), which represents the relatively simple organization of subtelomeric regions in this species. PCR fragment-length variation across the subtelomeric region indicated that the 1.4-kb distal region showed elevated structural variation relative to the centromere-proximal region. Examination of nucleotide sequences from this 1.4-kb region revealed diverse DNA rearrangements, including an inversion, several deletions, and an insertion of a retrotransposon LTR. The structures at the deletion and inversion breakpoints are characteristic of simple deletion-associated nonhomologous end-joining (NHEJ) events. There was strong linkage disequilibrium between the distal subtelomeric region and the proximal telomere, which contains degenerate and variant telomeric repeats. Variation in the proximal telomere was characterized by the expansion and deletion of blocks of repeats. Our sample of accessions documented two independent chromosome-healing events associated with terminal deletions of the subtelomeric region as well as the capture of a scrambled mitochondrial DNA segment in the proximal telomeric array. This natural variation study highlights the variety of genomic events that drive the fluidity of chromosome termini.     

Sequence of the site-specific recombinase gene cin and of its substrates serving in the inversion of the C segment of bacteriophage P1.

Inversion of the 4.2-kb C segment flanked by 0.6-kb inverted repeats on the bacteriophage P1 genome is mediated by the P1-encoded site-specific cin recombinase. The cin gene lies adjacent to the C segment and the C inversion cross-over sites cixL and cixR are at the external ends of the inverted repeats. We have sequenced the DNA containing the cin gene and these cix sites. The cin structural gene consists of 561 nucleotides and terminates at the inverted repeat end where the cixL site is located. Only two nucleotides in the cixL region differ from those in the cixR and they are within the cin TAA stop codon. The cin promoter was localized by transposon mutagenesis within a 0.1-kb segment, which contains probable promoter sequences overlapping with a 'pseudo-cix' sequence cixPp. In a particular mutant, integration of an IS1-flanked transposon into the cin control region promoted weak expression of the cin gene. The cin and cix sequences show homology with corresponding, functionally related sequences for H inversion in Salmonella and with cross-over sites for G inversion in phage Mu. Based on a comparison of the DNA sequences and of the gene organizations, a possible evolutionary relationship between these three inversion systems and the possible significance of the cixPp sequence in the cin promoter are discussed.     

Origin of adenovirus DNA replication. Role of the nuclear factor I binding site in vivo

An assay is described that detects in vivo a single round of initiation and DNA synthesis directed by a linear molecule containing an exposed single copy of an adenovirus (Ad) origin of replication. This and a previously described assay, which measures multiple rounds of DNA replication, were used to identify DNA sequences within the Ad2 and Ad4 origins of replication that are important for ori function. Linear DNA molecules containing sequences from the Ad2 or Ad4 genome termini were cotransfected with homologous and heterologous helper virus, and net amounts of DNA synthesis were compared. Linear molecules containing the Ad4 inverted terminal repeats were replicated 20-fold better in the presence of the homologous helper, whereas both Ad2 and Ad4 inverted terminal repeats were utilized efficiently by Ad4. DNA sequence analysis of the Ad2 ori and the corresponding region in Ad4 indicated that, although there are only ten variant base-pairs, eight are located within the Ad2 DNA sequence recognized by the cellular protein nuclear factor I. This protein is required to achieve the maximal rate of Ad2 DNA replication in vitro, and these differences therefore identify DNA sequences that are crucial to Ad2 ori function. The Ad4 ITR does not contain a functional nuclear factor I binding site, and deletion analysis has demonstrated that this region of the Ad4 genome is not required for ori function. In contrast to Ad2, the DNA sequences required for the initiation of Ad4 DNA replication were shown to reside entirely within the terminal 18 base-pairs of the Ad4 inverted terminal repeat.     

Nucleotide sequence analysis of the long terminal repeat of avian myeloblastosis virus and adjacent host sequences.

The nucleotide sequence of the integrated avian myeloblastosis virus long terminal repeat has been determined. The sequence is 385 base pairs long and is present at both ends of the viral DNA. The cell-virus junctions at each end consist of a 6-base-pair direct repeat of cell DNA next to the inverted repeat of viral DNA. The long terminal repeat also contains promoter-like sequences, an mRNA capping site, and polyadenylation signals. Several features of this long terminal repeat suggest a structural and functional similarity with sequences of transposable and other genetic elements. Comparison of these sequences with long terminal repeats of other avian retroviruses indicates that there is a great variation in the 3' unique sequence (U3), whereas the 5' specific sequences (U5) and the R region are highly conserved.     

The 1723 element: a long, homogeneous, highly repeated DNA unit interspersed in the genome of Xenopus laevis

We describe a highly repeated DNA element in the Xenopus laevis genome. This sequence, named the 1723 element, was first identified among sequences that are transcribed during embryonic development. The element is present in about 8500 copies per haploid genome, which together accounts for about 2.4% of the genome. Most copies of the element have highly conserved restriction maps, and are interspersed in the genome. The copies range in size from 6000 to 10,000 base-pairs due to an expandable region that contains variable numbers of a tandemly repeating 183 to 204 base-pair unit. The element is framed by an imperfect 18 base-pair inverted sequence, and inverted repeats of 180 to 185 base-pairs are nearby. Sequence analysis of DNA adjacent to three cloned elements shows that the elements are flanked by 8 base-pair direct repeats. These and other properties of 1723 suggest that it may be transposable.     

Spontaneous variation and synthesis in the U3 region of the long terminal repeat of an avian retrovirus.

Recombinant DNA clones of a viral clone of spleen necrosis virus, an avian retrovirus, were found to have long terminal repeats of different sizes. The variation was in the U3 region of the long terminal repeats, and any one clone had U3 of the same size in both long terminal repeats. The U3 regions in the 5' and 3' long terminal repeat were shown both to be derived from the 3' long terminal repeat of parental virus DNA.     

Unstable heterozygosity in a diploid region of herpes simplex virus DNA

We have examined the behavior of a herpes simplex virus strain KOS isolate in which the two inverted repeats flanking the short segment of viral DNA differ in length by approximately 60 base pairs. We find that individual viral DNA molecules exist which contain the two distinguishable repeats, demonstrating that heterology between the repeats is tolerated. However, viruses heterozygous for the two different repeats are unstable, segregating both classes of homozygotes at a high frequency. We propose that this segregation is a consequence of the high-frequency recombination events which also result in genome segment inversion.     

Repetitive DNA variation and pivotal-differential evolution of wild wheats.

Several polyploid species in the genus Triticum contain a U genome derived from the diploid T. umbellulatum. In these species, the U genome is considered to be unmodified from the diploid based on chromosome pairing analysis, and it is referred to as pivotal. The additional genome(s) are considered to be modified, and they are thus referred to as differential genomes. The M genome derived from the diploid T. comosum is found in many U genome polyploids. In this study, we cloned three repetitive DNA sequences found primarily in the U genome and two repetitive DNA sequences found primarily in the M genome. We used these to monitor variation for these sequences in a large set of species containing U and M genomes. Investigation of sympatric and allopatric accessions of polyploid species did not show repetitive DNA similarities among sympatric species. This result does not support the idea that the polyploid species are continually exchanging genetic information through introgression. However, it is also possible that repetitive DNA is not a suitable means of addressing the question of introgression. The U genomes of both diploid and polyploid U genome species were similar regarding hybridization patterns observed with U genome probes. Much more variation was found both among diploid T. comosum accessions and polyploids containing M genomes. The observed variation supports the cytogenetic evidence that the M genome is more variable than the U genome. It also raises the possibility that the differential nature of the M genome may be due to variation within the diploid T. comosum, as well as among polyploid M genome species and accessions.