Rhs elements of Escherichia coli K-12: complex composites of shared and unique components that have different evolutionary histories.

The complete sequences of the RhsB and RhsC elements of Escherichia coli K-12 have been determined. These sequence data reveal a new repeated sequence, called H-rpt (Hinc repeat), which is distinct from the Rhs core repetition that is found in all five Rhs elements. H-rpt is found in RhsB, RhsC, and RhsE. Characterization of H-rpt supports the view that the Rhs elements are composite structures assembled from components with very different evolutionary histories and that their incorporation into the E. coli genome is relatively recent. In each case, H-rpt is found downstream from the Rhs core and is separated from the core by a segment of DNA that is unique to the individual element. The H-rpt's of RhsB and RhsE are very similar, diverging by only 2.1%. They are 1,291 bp in length, and each contains an 1,134-bp open reading frame (ORF). RhsC has three tandem copies of H-rpt, all of which appear defective in that they are large deletions and/or have the reading frame interrupted. Features of H-rpt are analogous to features typical of insertion sequences; however, no associated transposition activity has been detected. A 291-bp fragment of H-rpt is found near min 5 of the E. coli K-12 map and is not associated with any Rhs core homology. The complete core sequences of RhsB and RhsC have been compared with that of RhsA. As anticipated, the three core sequences are closely related, all having identical lengths of 3,714 bp each. Like RhsA, the RhsB and RhsC cores constitute single ORFs that begin with the first core base. In each case, the core ORF extends beyond the core into the unique sequence. Of the three cores, RhsB and RhsA are the most similar, showing only 0.9% sequence divergence, while RhsB and RhsC are the least similar, diverging by 2.9%. All three cores conserve the 28 repetitions of a peptide motif noted originally for RhsA. A secondary structure is proposed for this motif, and the possibility of its having an extracellular binding function is discussed. RhsB contains one additional unique ORF, and RhsC contains two additional unique ORFs. One of these ORFs includes a signal peptide that is functional when fused to TnphoA.     

Structure of the rhsA locus from Escherichia coli K-12 and comparison of rhsA with other members of the rhs multigene family.

The complete nucleotide sequence of the rhsA locus and selected portions of other members of the rhs multigene family of Escherichia coli K-12 have been determined. A definition of the limits of the rhsA and rhsC loci was established by comparing sequences from E. coli K-12 with sequences from an independent E. coli isolate whose DNA contains no homology to the rhs core. This comparison showed that rhsA comprises 8,249 base pairs (bp) in strain K-12 and that the Rhs0 strain, instead, contains an unrelated 32-bp sequence. Similarly, the K-12 rhsC locus is 9.6 kilobases in length and a 10-bp sequence resides at its location in the Rhs0 strain. The rhsA core, the highly conserved portion shared by all rhs loci, comprises a single open reading frame (ORF) 3,714 bp in length. The nucleotide sequence of the core ORF predicts an extremely hydrophilic 141-kilodalton peptide containing 28 repeats of a motif whose consensus is GxxxRYxYDxxGRL(I or T). One of the most novel aspects of the rhs family is the extension of the core ORF into the divergent adjacent region. Core extensions of rhsA, rhsB, rhsC, and rhsD add 139, 173, 159, and 177 codons to the carboxy termini of the respective core ORFs. For rhsA, the extended core protein would have a molecular mass of 156 kilodaltons. Core extensions of rhsB and rhsD are related, exhibiting 50.3% conservation of the predicted amino acid sequence. However, comparison of the core extensions of rhsA and rhsC at both the nucleotide and the predicted amino acid level reveals that each is highly divergent from the other three rhs loci. The highly divergent portion of the core extension is joined to the highly conserved core by a nine-codon segment of intermediate conservation. The rhsA and rhsC loci both contain partial repetitions of the core downstream from their primary cores. The question of whether the rhs loci should be considered accessory genetic elements is discussed but not resolved.     

Correlation of Rhs Elements with Escherichia Coli Population Structure

The Rhs family of composite genetic elements was assessed for variation among independent Escherichia coli strains of the ECOR reference collection. The location and content of the RhsA-B-C-F subfamily correlates highly with the clonal structure of the ECOR collection. This correlation exists at several levels: the presence of Rhs core homology in the strain, the location of the Rhs elements present, and the identity of the Rhs core-extensions associated with each element. A provocative finding was that an identical 1518-bp segment, covering core-extension-b1 and its associated downstream open reading frame, is present in two distinct clonal groups, but in association with different Rhs elements. The sequence identity of this segment when contrasted with the divergence of other chromosomal segments suggests that shuffling of Rhs core extensions has been a relatively recent variation. Nevertheless the copies of core-extension-b1 were placed within the respective Rhs elements before the emergence of the clonal groups. In the course of this analysis, two new Rhs elements absent from E. coli K-12 were discovered: RhsF, a fourth member of the RhsA-B-C-F subfamily, and RhsG, the prototype of a third Rhs subfamily.     

A stationary-phase-dependent viability block governed by two different polypeptides from the RhsA genetic element of Escherichia coli K-12.

Multicopy plasmids bearing a small internal portion of the RhsA genetic element of Escherichia coli K-12 imparted a viability block on cultures grown to stationary phase in broth. Inclusion of the last 25 codons of the RhsA core open reading frame (called core-ORF) in the plasmid insert was crucial for eliciting this toxic effect. The toxic effect could be suppressed by including the adjacent Rhs component, dsORF-a1, on the multicopy plasmid. The toxic effect was enhanced in RpoS- strains.     

The barley chloroplast DNA atpBE, trnM2, and trnV1 loci.

The nucleotide sequence of a barley chloroplast DNA 3.7 kb SmaI-HindIII fragment is presented. This fragment contains atpBE, the genes for the beta and epsilon subunits of ATPase; trnM2, the gene for tRNA2met; and trnV1, the gene for tRNA1va1. The atpE-trnM2 interval is 126 bp and trnM2 is transcribed towards atpBE. The trnM2-trnV1 interval is 203 bp and trnV1 is transcribed away from trnM2. The trnV1 locus has a 597 bp intervening sequence. the organization and sequences of these genes are compared to the analogous genes from maize and tobacco chloroplast DNA. Using the latter comparisons the nature of sequence divergence between chloroplast DNAs is discussed.     

IS421, a new insertion sequence in Escherichia coli

The nucleotide sequence of a new insertion sequence (IS) in Escherichia coli, IS421, was determined. It is 1340 bp long and contains inverted repeats of 22 bp at its termini. It is flanked by 13 bp direct repeats apparently generated upon insertion. There are two ORFs longer than 200 bp in IS421. One can encode a polypeptide of 371 amino acids (aa) and the other, which is on the other strand, can encode a polypeptide of 102 aa. The C-terminal part of the 371 aa polypeptide shows some homology to that of transposases encoded in some other known IS elements. The copy number of IS421 in chromosomal DNA was 4 for E. coli K-12 and B, and 5 for E. coli C, as determined by the Southern hybridization of restriction fragments.     

Integration of the temperate phage phiU into the putative tRNA gene on the chromosome of its host Rhizobium leguminosarum biovar trifolii

The plasmid pCI6, carrying the attP site of the temperate phage phiU, integrates into the attB site on the chromosome of Rhizobium leguminosarum biovar trifolii strain 4S. The 4 kb EcoRI-HindIII region of pCI6 involved in site-specific integration was subcloned as the attP fragment of phage phiU and sequenced. The attL fragment, one of the new DNA junctions generated from the insertion of pCI6 into the chromosome of the host Rhizobium, was used as a hybridization probe for isolation of the attB fragment of strain 4S. The nucleotide sequence of the 2 kb PstI fragment of strain 4S, which hybridized with the attL fragment, was decided and compared with that of the attP fragment. A 53 bp common sequence was expected to be the core sequence of site-specific integration between phage phiU and strain 4S. One of the ORFs on the attP fragment, which was located adjacent to the core sequence, had structural homology to the integrase family. However, the attB fragment showed high homology with the tRNA genes of Agrobacterium tumefaciens and E. coli. A 47 bp sequence of the 53 bp core sequence overlapped with this tRNA-like sequence. This indicates that the target site of phage phiU integration is the putative tRNA gene on the chromosome of the Rhizobium host.     

Characterization of cryptic flagellin genes in Shigella boydii and Shigella dysenteriae.

Flagellin (fliC) genes of 12 Shigella boydii and five Shigella dysenteriae strains were characterized. Though these strains are nonmotile, the cryptic fliCSB gene, cloned from S. boydii strain C3, is functional for expression of flagellin. It consists of 1,704 bp, and encodes 568 amino acid residues (57,918 Da). The fliCSD gene from S. dysenteriae strain 16 consists of 1,650 bp encoding 549 amino acid residues (57,591 Da) and contains an IS1 element inserted in its 3' end. The two genes are composed of the 5'-constant, central variable and 3'-constant sequences, like other known fliC genes. The two genes share high homology in nucleotide and amino acid sequences with each other and also with the Escherichia coli fliCE gene, indicating that both genes are closely related to the fliCE gene. Comparison of the central variable sequences of six different fliC genes showed that the fliCSB and fliCSD genes share low homology in amino acid sequence with the other fliC genes, suggesting that they encode antigenic determinants intrinsic to respective subgroups. However, Southern blotting using as probes the central variable sequences of several fliC genes showed that four of 12 S. boydii strains have a fliC gene similar to that of Shigella flexneri, and that among five fliC genes from S. dysenteriae strains, one is similar to that of S. flexneri, two are similar to that of S. boydii, and only one is unique to S. dysenteriae. Some of these variant alleles were verified by immunoblotting with flagellins produced from cloned fliC genes. The presence of variant fliC alleles in S. boydii and S. dysenteriae indicates that subdivision into subgroups does not reflect the ancestral flagella H antigenic relationships. These data will be useful in considering the evolutionary divergence of the Shigella spp..     

rhs gene family of Escherichia coli K-12.

Two additional members of a novel Escherichia coli gene family, the rhs genes, have been cloned and characterized. The structures of these loci, rhsC and rhsD, have been compared with those of rhsA and rhsB. All four loci contain a homologous 3.7-kilobase-pair core. Sequence comparison of the first 300 nucleotides of the cores showed that rhsA, rhsB, and rhsC are closely related, with only 1 to 2% sequence divergence, whereas rhsD is 18% divergent from the others. The beginning of the core coincides with the initiation of an open reading frame that extends beyond the 300 nucleotides compared. Whether a protein product is produced from this open reading frame has not been established. However, nucleotide substitutions which differentiate the cores have highly conservative effects on the predicted protein products; this suggests that products are made from the open reading frame and are under severe selection. The four rhs loci have been placed on both the genetic and restriction maps of E. coli K-12. A fifth rhs locus remains to be characterized. In terms of size, number, and sequence conservation, the rhs genes make up one of the most significant repetitions in E. coli, comparable to the rRNA operons.     

Molecular cloning and sequence analysis of the cDNA for small proteoglycan II of bovine bone.

The cDNA for the full-length core protein of the small chondroitin sulphate proteoglycan II of bovine bone was cloned and sequenced. A 1.3 kb clone (lambda Pg28) was identified by plaque hybridization with a previously isolated 1.0 kb proteoglycan cDNA clone (lambda Pg20), positively identified previously by polyclonal and monoclonal antibody reactivity and by hybrid-selected translation in vitro [Day, Ramis, Fisher, Gehron Robey, Termine & Young (1986) Nucleic Acids Res. 14, 9861-9876]. The cDNA sequences of both clones were identical in areas of overlap. The 360-amino-acid-residue protein contains a 30-residue propeptide of which the first 15 residues are highly hydrophobic. The mature protein consists of 330 amino acid residues corresponding to an Mr of 36,383. The core protein contains three potential glycosaminoglycan-attachment sites (Ser-Gly), only one of which is within a ten-amino-acid-residue homologous sequence seen at the known attachment sites of related small proteoglycans. Comparisons of the published 24-residue N-terminal protein sequence of bovine skin proteoglycan II core protein with the corresponding region in the deduced sequence of the bovine core protein reveals complete homology. Comparison of the cDNA-derived sequences of bovine bone and human embryonic fibroblast proteoglycans shows a hypervariable region near the N-terminus. Nucleotide homology between bone and fibroblast core proteins was 87% and amino acid homology was 90%.     

Complete nucleotide sequence of the 6 kb element and conserved cytochrome b gene sequences among Indian isolates of Plasmodium falciparum.

The malaria parasite contains a nuclear genome with 14 chromosomes and two extrachromosomal DNA molecules of 6 kb and 35 kb in size. The smallest genome, known as the 6 kb element or mitochondrial DNA, has been sequenced from several Plasmodium falciparum isolates because this is a potential drug target. Here we describe the complete nucleotide sequence of this element from an Indian isolate of P. falciparum. It is 5967 bp in size and shows 99.6% homology with the 6 kb element of other isolates. The element contains three open reading frames for mitochondrial proteins-cytochrome oxidase subunit I (CoI), subunit III (CoIII) and cytochrome b (Cyb) which were found to be expressed during blood stages of the parasite. We have also sequenced the entire cyb gene from several Indian isolates of P. falciparum. The rate of mutation in this gene was very low since 12 of 14 isolates showed the identical sequence. Only one isolate showed a maximum change in five amino acids whereas the other isolate showed only one amino acid change. However, none of the Indian isolates showed any change in those amino acids of cyb which are associated with resistance to various drugs as these drugs are not yet commonly used in India.     

Nucleotide sequence of an OXA-2 beta-lactamase gene from the R-plasmid R1767 derived plasmid pBP11 and comparison to closely related resistance determinants found in R46 and Tn2603

Plasmid pBP11 contains a sequence homologous to Tn21-like element Tn2410 encoding dihydropteroate synthetase and beta-lactamase OXA-2. The nucleotide sequence of a 1.5 kb segment of this region has been determined including the bla gene. It reveals strong sequence homology with the OXA-2 operon of plasmid R46. The implications of an additional 319 bp segment in pBP11 for the different evolution of R46/pKM101 and pBP11 are discussed.     

Structure of a bacterial Rhs effector exported by the type VI secretion system

The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 Å resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise β-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs ‘cocoon’ where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal ‘plug’ domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS.     

One short well conserved region of Alu-sequences is involved in human gene rearrangements and has homology with prokaryotic chi.

Alu elements have repeatedly been found involved in gene rearrangements in humans. Although these elements have been suggested to stimulate gene rearrangements, sparse information is available for the possible mechanism(s) of these events. Here we present a compilation of Alu elements that have been involved in recombinational events leading to gene rearrangements, indicating the presence of a common 26 bp core sequence at or close to the sites of recombination. Besides the obvious possibility of retrotransposition, gene rearrangements may be induced by sequences that stimulate genetic recombination. We suggest that the core sequence stimulates recombination and may thereby cause the frequent involvement of these elements in gene rearrangements. Curiously, the core sequence contains the pentanucleotide motif CCAGC, which is also part of chi, an 8 bp sequence known to stimulate recBC mediated recombination in Escherichia coli.