Charles W. Hill

Rhs elements of Escherichia coli: a family of genetic composites each encoding a large mosaic protein

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 motff, 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 fail 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.

Four Different Genes Responsible for Nonimmune Immunoglobulin-Binding Activities within a Single Strain of Escherichia coli

ABSTRACT 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. ForE. coli strain ECOR-9, four distinct genes (designatedeibA, eibC, eibD, andeibE) are responsible for Ig binding. Two eibgenes 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, theeibA-eaaA cluster is adjacent to trpA (min 28.3) while the eibC-eaaC cluster is adjacent toaspS (min 42.0). Sequence adjacent to theeibA-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 eibgenes, 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. Theeib 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.

Transcription of herpes simplex type 1 DNA in nuclei isolated from infected HEp-2 and KB cells

Abstract Viral RNA synthesis in nuclei isolated from cells infected with Herpes simplex virus type 1 (HSV-1) has been examined for sensitivity to the fungal toxin α-amanitin. In both HEp-2 and KB cell lines, the production of HSV-1 specific RNA is inhibited 90% or greater by the administration of the toxin at 0.32 μg/ml. These results suggest that an α-amanitin sensitive, DNA-dependent RNA polymerase, possibly the host cell nucleoplasmic polymerase, is responsible for HSV-1 RNA synthesis in the infected cell.

Instability of a missense suppressor resulting from a duplication of genetic material

Abstract A number of spontaneously arising suppressor mutations of the trpA36§ mutation (an arginine for glycine replacement in the tryptophan synthetase A protein of Escherichia coli) have been isolated. All map at or near the same genetic locus (linked to argH) as the trpA36 suppressors described by Brody & Yanofsky (1965); they differ from the Brody-Yanofsky suppressor isolates by their slower growth rates in minimal medium, their death following a shift-up from minimal medium to Tryptone, and their failure to segregate Trp− cells. Our experiments suggest that these new suppressed isolates are haploid for the suppressor gene and that the Brody-Yanofsky suppressed strains are partially heterozygous for at least the suppressor locus i.e. (su+/ su−). Transduction of the new suppressors, by phage P1, occasionally yield fast-growing, Tryptoneinsensitive and genetically unstable suppressed clones. This is due to formation of a partial heterozygote analogous to the Brody-Yanofsky suppressors. In one strain the partial heterozygosity included the linked metB, argH and thi loci. Interrupted mating experiments established that both copies of the duplication are integrated within the bacterial chromosome: the segregation patterns of the duplicated markers suggest that the duplications are in tandem and that segregation usually occurs by a single crossover between copies of the duplication, thereby causing elimination of one set of the duplicated genes. The slow growth and sensitivity to enriched media of the haploid suppressor strains may be due to the loss of the wild-type suppressor gene function.

Tandem duplications resulting from recombination between ribosomal RNA genes in Escherichia coli.

Abstract A set of Escherichia coli K12 mutants, which carry a tandem duplication of the glyT purD region, have been analyzed. Three types of duplications have occurred repeatedly, and we show that they were generated by recombination between the ribosomal RNA gene, rrnE , which lies to one side of the glyT purD region and one of three rrn genes which occur as direct repetitions on the other side of this region. Characterization of these duplication mutants has involved the isolation of the duplicated material in the form of a DNA circle. Class I duplications, which extend from rrnE to rrnE , are 39,500 base-pairs long, class II duplications, which extend from rrnA to rrnE , are 164,000 base-pairs long, and class III duplications, which extend from rrnC to rrnE , are 258,000 base-pairs long.

Genetic duplications induced at very high frequency by ultraviolet irradiation in Escherichia coli

SummaryWe have found that mild ultraviolet irradiation of Escherichia coli leads to a duplication of the glyT purD region of the chromosome to the extent that 3–5% of all surviving chromosomes carry a genetic duplication of this material. The duplications vary in size from less than one to more than five minutes of the chromosome. While the endpoints of the duplications vary, seven of ten characterized have one end between the purD and metA loci, and five of these seven have the other endpoint near the argH locus. Consequently, the region between purD and metA (only 0.1 minutes) seems to be particularly prone to participating in abnormal recombinational events. The UV-induced damage leading to the genetic duplications is subject to dark repair, suggesting the involvment of pyrimidine dimers. Other mutagens such as nitrous acid, ethyl methanesulfonate and nitrosoguanidine are also effective in generating these duplications at high frequency. Evidence is discussed which indicates that some and probably most of the duplications are tandem duplications. However, at least one example was found that is more readily explained by a translocation.

Nonimmune Binding of Human Immunoglobulin A (IgA) and IgG Fc by Distinct Sequence Segments of the EibF Cell Surface Protein of Escherichia coli

ABSTRACT The eib genes of Escherichia coliencode 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 ofYersinia, UspA2 of Moraxella, and DsrA ofHaemophilus 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. colireference 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 othereib genes, eibF attenuates serum sensitivity, occurs as a stable multimer, and is associated with a prophage. By subcloning portions of the eibA andeibF 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.

Large genomic sequence repetitions in bacteria: lessons from rRNA operons and Rhs elements

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.

Glycine transfer RNA of Escherichia coli: II. Impaired GGA-recognition in strains containing a genetically altered transfer RNA; Reversal by a secondary suppressor mutation☆☆☆

Abstract The glycine-specific tRNA from Escherichia coli is separable into three components (tRNAIGly, tRNAIIGly and tRNAIIIGly) by chromatography over BD-ellulose. ‡ Triplet binding studies reveal that tRNAIGly recognizes predominantly GpGpG, tRNAIIGly recognizes both GpGpG and GpGpA, and tRNAIIIGly recognizes GpGpU and GpGpC. Strains carrying a suppressor (glyTsu) of the trpA36 mutation (Gly → Arg, GGA → AGA) lack a functional tRNAIIGly and, as a result, exhibit severe pleiotropic effects. As expected, the binding of [14C]glycyl-tRNA from glyTsu strains to ribosomes is only weakly stimulated by GpGpA (8% of the control), while binding in the presence of GpGpG, GpGpU and GpGpC is relatively normal. On the other hand, suppressed strains (glyUsu) lacking a functional tRNAIGly do not show these adverse effects, and tRNA from these strains displays a nearly normal codon recognition pattern. The pleiotropic effects seen in glyTsu strains lacking a normal tRNAIIGly are reversed either by (a) the presence of a normal glyT+ gene in the same organism, or (b) by secondary mutations (ins) unlinked to the glyT region. The GpGpA-stimulated binding of glyTsu ins tRNA to ribosomes is partially restored to normal. Transfer RNA from ins strains contains a new tRNAGly species (tRNAIIIGly), easily detectable by BD-cellulose chromatography. This new tRNAIIIGly, binds to ribosomes in the presence of GpGpA or GpGpG, but not GpGpU or GpGpC. The results indicate that tRNAIIIGly is formed by a genetically induced alteration of a portion of tRNAIIIGly. It seems likely that the ins mutation results in a GGU/C → GGA/G change in a redundant tRNAIIIGly species, thus relieving the pleiotropy induced by the loss of a normal tRNAIIGly.

Glycine transfer RNA of Escherichia coli. I. Structural genes for two glycine tRNA species.

Abstract Glycyl transfer RNA of Escherichia coli is separated into three distinct fractions by chromatography on BD-cellulose † . Studies of two distinct missense suppressor mutations have led to the identification of the structural genes for the components of two of these fractions. One of these suppressor mutations occurs in the glyT gene which is linked to the argH locus. The other occurs in the glyU gene which is linked to the lysA locus. Both suppressors result in tRNA species which insert glycine into polypeptides in response to the arginine codon, AGA. The glyUsu mutation alters the first BD-cellulose fraction (tRNAIGly), while the glyTsu mutation alters the second (tRNAIIGly). Gene dosage and dominance effects observed in merodiploid strains indicate that the glyT and glyU genes are the structural genes for the affected glycine tRNA species. Furthermore, each of the species is completely altered by single mutations, showing that there is only one structural gene per genome for each of these species. The results indicate that the genes for the various species of glycine tRNA are not clustered on the E. coli chromosome. Mutagenesis specificity and frequency measurements suggest that glyT suppressors can be obtained by point mutations, quite likely in the portion of the DNA specifying the anticodon. On the other hand, conversion of the glyU tRNA to an AGA suppressor tRNA appears to require a complex mutation.