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The eib genes of Escherichia coli encode surface-exposed proteins which bind immunoglobulins (Ig) such as the Fc fragment of human IgG (IgG Fc) in a nonimmune manner. The Eib proteins belong to a family which includes YadA of Yersinia, UspA2 of Moraxella, and DsrA of Haemophilus ducreyi. This family of surface-exposed proteins shares several features, such as the ability to impart resistance to human serum complement and a tendency to exist as stable multimers. Four genes, eibA, eibC, eibD and eibE, were previously identified and cloned from ECOR-9, a strain from the E. coli reference collection. EibC, -D, and -E bind human serum IgA in addition to IgG, but no IgA binding has been observed for EibA. Here, we report the cloning of a new eib gene, eibF, from a second strain of E. coli, ECOR-2. The product, EibF, has a relatively strong preference for IgA. Like the other eib genes, eibF attenuates serum sensitivity, occurs as a stable multimer, and is associated with a prophage. By subcloning portions of the eibA and eibF genes, we have identified distinct sequence segments sufficient to cause Ig binding, multimerization, and discrimination between IgA and IgG. The ability to multimerize is associated with a sequence close to the C terminus that is homologous to other family members such as YadA. Binding of IgG Fc is associated with a sequence that is highly conserved among all Eib proteins but otherwise unique. Binding of IgA is associated with a sequence of EibF that is not similar to any EibA sequence.

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Certain Escherichia coli strains bind the Fc fragment of immunoglobulin G (IgG) at the bacterial cell surface. Previous work established that this nonimmune Ig binding depends on several large proteins with apparent molecular masses that can exceed 200 kDa. For E. coli strain ECOR-9, four distinct genes (designated eibA, eibC, eibD, and eibE) are responsible for Ig binding. Two eib genes are linked to eaa genes, which are homologous to genes for the autotransporter family of secreted proteins. With reference to the E. coli K-12 chromosome, the eibA-eaaA cluster is adjacent to trpA (min 28.3) while the eibC-eaaC cluster is adjacent to aspS (min 42. 0). Sequence adjacent to the eibA-eaaA cluster converges with that of strain K-12 precisely as observed for the Atlas family of prophages, suggesting that eibA is part of one of these. All four eib genes, when cloned into plasmid vectors, impart IgG binding to E. coli K-12 strains, and three impart IgA binding also. The IgG binding occurs at the bacterial cell surface, and its expression increases survival in serum by up to 3 orders of magnitude. The eib sequences predict a C-terminal peptide motif that is characteristic of outer membrane proteins, and the protein sequences show significant similarity near the C terminus to both the YadA virulence factor of Yersinia species and the universal surface protein A II of Moraxella catarrhalis. The sizes predicted for Eib proteins from DNA sequence are much smaller than their apparent sizes on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, possibly reflecting stable oligomerization.

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The rrn operons and Rhs elements provide starkly contrasting examples of the evolution and interaction of large sequence repetitions in bacteria. Genomic sequencing of different species as well as comparative sequencing of independent isolates is providing provocative insights into previously obscure issues.

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The Rhs elements are complex genetic composites widely spread among Escherichia coli isolates. One of their components, a 3.7-kb, GC-rich core, maintains a single open reading frame that extends the full length of the core and then 400 to 600 bp beyond into an AT-rich region. Whereas Rhs cores are homologous, core extensions from different elements are dissimilar. Two new Rhs elements from strains of the ECOR reference collection have been characterized. RhsG (from strain ECOR-11) maps to min 5.3, and RhsH (from strain ECOR-45) maps to min 32.8, where it lies in tandem with RhsE. Comparison of strain K-12 to ECOR-11 indicates that RhsG was once present in but has been largely deleted from an ancestor of K-12. Phylogenetic analysis shows that the cores from eight known elements fall into three subfamilies, RhsA-B-C-F, RhsD-E, and RhsG-H. Cores from different subfamilies diverge 22 to 29%. Analysis of substitutions that distinguish between subfamilies shows that the origin of the ancestral core as well as the process of subfamily separation occurred in a GC-rich background. Furthermore, each subfamily independently passed from the GC-rich background to a less GC-rich background such as E. coli. A new example of core-extension shuffling provides the first example of exchange between cores of different subfamilies. A novel component of RhsE and RhsG, vgr, encodes a large protein distinguished by 18 to 19 repetitions of a Val-Gly dipeptide occurring with a eight-residue periodicity.

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We have identified several strains of Escherichia coli which contain immunoglobulin-binding activity on the cell surface. Affinity-purified antibodies ordinarily used as secondary antibodies in immunodetection protocols were bound by 6 of 72 strains of the ECOR reference collection of E. coli. The Fc fragments of both human and sheep immunoglobulin G (IgG) were also bound, demonstrating the nonimmune nature of the phenomenon. Binding of conjugated IgG Fc directly to unfixed cells was observed by fluorescence microscopy. Western blots showed that the immunoglobulin-binding material occurs in the form of multiple bands, with the apparent molecular masses of the most prominent bands exceeding 100 kDa. No two of the strains have the same pattern of bands. The binding activity in extracts was sensitive to proteinase K. The binding activity of intact cells was reduced preferentially by trypsin digestion, demonstrating exposure at the cell surface. Expression of binding activity in Luria-Bertani broth cultures was favored by a temperature of 37 degrees C and entry into stationary phase of growth.

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The distribution of plasmids related to the fertility factor F was examined in the ECOR reference collection of Escherichia coli. Probes specific for four F-related genes were isolated and used to survey the collection by DNA hybridization. To estimate the genetic diversity of genes in F-like plasmids, DNA sequences were obtained for four plasmid genes. The phylogenetic relationships among the plasmids in the ECOR strains is very different from that of the strains themselves. This finding supports the view that plasmid transfer has been frequent within and between the major groups of ECOR. Furthermore, the sequences indicate that recombination between genes in plasmids takes place at a considerably higher frequency than that observed for chromosomal genes. The plasmid genes, and by inference the plasmids themselves, are mosaic in structure with different regions acquired from different sources. Comparison of gene sequences from a variety of naturally occurring plasmids suggested a plausible donor of some of the recombinant regions as well as implicating a chi site in the mechanism of genetic exchange. The relatively high rate of recombination in F-plasmid genes suggests that conjugational gene transfer may play a greater role in bacterial population structure than previously appreciated.

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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.

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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.

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RhsF has been identified as the fourth member of the RhsABCF subfamily of genetic elements. This new element is found in Escherichia coli ECOR-50 and several other strains but not in strain K-12. A novel feature of RhsF is that it represents a new arrangement of components previously uniquely associated with RhsA and RhsC of strain K-12.

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The Rhs family comprises a set of composite elements found in the chromosomes of many natural Escherichia coli strains. Five Rhs elements occur in strain K-12. The most prominent Rhs component is a giant core open reading frame (core ORF) whose features are suggestive of a cell surface ligand-binding protein. This hypothetical protein contains a peptide motif, xxGxxxRYxYDxxGRL(I or T)xxxx, that is repeated 28 times. A similar repeated motif is found in a Bacillus subtilis wall-associated protein. The Rhs core ORFs consist of two distinct parts: a large N-terminal core that is conserved in all Rhs elements, and a smaller C-terminus that is highly variable. Distinctive G+C contents of Rhs components indicate that the elements have a recent origin outside the E. coli species, and that they are composites assembled from segments with very different evolutionary histories. The Rhs cores fall into three sub-families that are mutually more than 20% divergent. Downstream of the core ORF is a second, much shorter ORF. Like the adjacent core extension, these are highly variable. In most examples, the hypothetical product of this ORF has a candidate signal sequence for transport across the cytoplasmic membrane. Another Rhs component, the 1.3 kb H-rpt, has features typical of insertion sequences. Structures homologous to H-rpt have been detected in other bacterial genera, such as Vibrio and Salmonella, where they are associated with loci that determine O-antigen variation.

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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.

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The Escherichia coli K-12 chromosome contains a family of five large, unlinked sequences known as the Rhs elements. They share several complex homologies, the most prominent being a 3.7 kb Rhs core. The elements are divided into two subfamilies, RhsA-B-C and RhsD-E, according to the sequence similarities of the cores. The RhsD core is 3747 bp long compared to 3714 bp for RhsA. Despite a 22% sequence divergence, the RhsD core conserves features previously noted for RhsA. Similar to RhsA, the RhsD core maintains a single ORF, the start codon coinciding with the first nucleotide of the homology. The RhsD core-ORF continues 177 codons beyond the homology, resulting in a carboxy terminal extension unrelated to that of RhsA. The RhsD core retains all 28 copies of the repeated motif GxxxRYxYDxxGRL(I/T) seen in RhsA. The other member of the RhsD-E subfamily, RhsE, has been mapped to minute 32 of the E. coli map. It appears defective in that it contains only the last 1550 bp of the 3.7 kb core. Its sequence is more closely related to that of RhsD than RhsA. In addition, RhsE and RhsB share a 1.3 kb homology, known as the H-repeat. The H-repeats from RhsE and RhsB are more closely related than their cores, showing only 1% nucleotide divergence.

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The Escherichia coli rRNA operons each have one of two types of spacer separating the 16S and 23S coding regions. The spacers of four operons encode tRNA(Glu2) and the other three encode both tRNA(Ile) and tRNA(Ala1B). We have prepared a series of mutants in which the spacer region of a particular rrn operon has been replaced by the opposite type. Included among these were a mutant retaining only a single copy of the tRNA(Glu2) spacer (at rrnG) and another retaining only a single copy of the tRNA(Ile)-tRNA(Ala1B) spacer (at rrnA). While both mutants grew more slowly than controls, the mutant deficient in tRNA(Glu2) spacers was more severely affected. At a frequency of 6 X 10(-5), these mutants phenotypically reverted to faster growing types by increasing the copy number of the deficient spacer. In most of these phenotypic revertants, the deficient spacer type appeared in a rrn operon which previously contained the surplus type, bringing the ratio of spacer types closer to normal. In a few cases, these spacer changes were accompanied by an inversion of the chromosomal material between the donor and recipient rrn operons. Two examples of inversion of one-half of the E. coli chromosome between rrnG and rrnH were observed. The correlation of spacer change with inversion indicated that, in these particular cases, the change was due to an intrachromatid gene conversion event accompanied by a reciprocal crossover rather than reciprocal exchange between sister chromatids.

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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.

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The e14 element appears to be integrated into the Escherichia coli K-12 isocitrate dehydrogenase structural gene (icd). In being integrated, it replaced the last 52 codons of the gene with a closely related sequence. The two versions of the icd gene produce proteins of the same length but differ by 12 base substitutions that would cause two conservative amino acid replacements.

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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.

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In an effort to learn what factors might mitigate the establishment of Escherichia coli variants bearing major chromosomal rearrangements, we have examined the effects on cell growth of two inversions between rRNA operons. One of these inversions, IN(rrnD-rrnE), had been propagated in a commonly used subline of E. coli K-12 for approximately 30 yr before its discovery, a fact that illustrates the absence of obvious detrimental effects associated with the inversion. We found that culturing under conditions requiring repeated transition from stationary phase to rapid growth led to the replacement of IN(rrnD-rrnE) cells by cells that had undergone either of two types of additional chromosomal inversion: one type fully restored the wild-type order, while the other partially restored it. The partial reinversion was also between rrn operons, but it left a small transposition. The tendency for overgrowth by these revertants persisted through several rounds of periodic selection. In contrast, the other inversion, IN(rrnG-rrnE), was associated with severe, detrimental effects. The effects of IN(rrnG-rrnE) were also alleviated by full or partial reinversion. The probable relationship between the severity of the effects caused by the inversions and the degree of displacement of the replication origin is discussed. Spontaneous inversion events between rrn operons separated by 18% of the chromosome were estimated to occur at a frequency of roughly 10(-5). If extended to natural situations, the growth disadvantage together with the relatively high frequency of reinversion suggest that clones of cells with an inversion between these rrn operons would be readily overgrown by revertants.

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The Escherichia coli K-12 genetic element, e14, contains a 216-base-pair region that is homologous to a portion of the host chromosome. This region serves as the integration site for the element. The 216-base-pair homology is interrupted by 28 mismatches distributed through the sequence. The actual integrative crossover occurs within the first 11 base pairs from one end of the region. To test factors which affect e14 site-specific recombination, we cloned the attachment sites of free e14 and the host chromosome into the same plasmid. The cloned attachment sites recombined intramolecularly in a process that required the presence of a chromosomal copy of e14 in the host cell as well as the induction of SOS. Recombination events that mimicked both integration and excision occurred under the same conditions and to roughly the same extent.

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A polymorphism affecting the spacer region of the rrnB rRNA operon is described. Strains from a major Escherichia coli K-12 subbranch are missing a 106-nucleotide portion of the rrnB 16S-to-23S spacer, and a 20-nucleotide sequence is found in its place. We have called this mutant operon rrnB2. The rrnB2 spacer was most probably derived from either rrnC or rrnE. This alteration of rrnB may have occurred by a recombinational exchange or by gene conversion. In the genealogy of E. coli K-12 strains, the appearance of rrnB2 is associated with the spontaneous occurrence of the first relaxed mutation, but attempts to show a selective relationship between the two mutational events have had negative results. The sequences of the rrnG and rrnC 16S-to-23S spacers have also been determined and their comparisons to the other rrn operons encoding tRNAGlu2 are presented.

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