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R K Holmes

Publications and source records attributed to R K Holmes.

At least 73 records · Page 4Linked to original sources
PubMed1991

Activation of Escherichia coli heat-labile enterotoxins by native and recombinant adenosine diphosphate-ribosylation factors, 20-kD guanine nucleotide-binding proteins.

Escherichia coli heat-labile enterotoxins (LT) are responsible in part for "traveler's diarrhea" and related diarrheal illnesses. The family of LTs comprises two serogroups termed LT-I and LT-II; each serogroup includes two or more antigenic variants. The effects of LTs result from ADP ribosylation of Gs alpha, a stimulatory component of adenylyl cyclase; the mechanism of action is identical to that of cholera toxin (CT). The ADP-ribosyltransferase activity of CT is enhanced by 20-kD guanine nucleotide-binding proteins, known as ADP-ribosylation factors or ARFs. These proteins directly activate the CTA1 catalytic unit and stimulate its ADP ribosylation of Gs alpha, other proteins, and simple guanidino compounds (e.g., agmatine). Because of the similarities between CT and LTs, we investigated the effects of purified bovine brain ARF and a recombinant form of bovine ARF synthesized in Escherichia coli on LT activity. ARF enhanced the LT-I-, LT-IIa-, and LT-IIb-catalyzed ADP ribosylation of agmatine, as well as the auto-ADP ribosylation of the toxin catalytic unit. Stimulation of ADP-ribosylagmatine formation by LTs and CT in the presence of ARF was GTP dependent and enhanced by sodium dodecyl sulfate. With agmatine as substrate, LT-IIa and LT-IIb exhibited less than 1% the activity of CT and LT-Ih. CT and LTs catalyzed ADP-ribosyl-Gs alpha formation in a reaction dependent on ARF, GTP, and dimyristoyl phosphatidylcholine/cholate. With Gs alpha as substrate, the ADP-ribosyltransferase activities of the toxins were similar, although CT and LT-Ih appeared to be slightly more active than LT-IIa and LT-IIb. Thus, LT-IIa and LT-IIb appear to differ somewhat from CT and LT-Ih in substrate specificity. Responsiveness to stimulation by ARF, GTP, and phospholipid/detergent as well as the specificity of ADP-ribosyltransferase activity are functions of LTs from serogroups LT-I and LT-II that are shared with CT.

ADP-Ribosylation Factors↗
PubMed1990

Effects of DNase production, plasmid size, and restriction barriers on transformation of Vibrio cholerae by electroporation and osmotic shock.

Attempts to transform wild type strains of V. cholerae with plasmid DNA by traditional osmotic shock methods were not successful. A mutant of V. cholerae that was deficient in extracellular DNase was transformed with plasmid DNA by osmotic shock, demonstrating directly that extracellular DNase is a major barrier to transformation of V. cholerae. Transformation of wild type and DNase-negative strains of V. cholerae was accomplished by electroporation. Efficiency of transformation by electroporation increased with field strength, decreased with plasmid size, and was relatively insensitive to changes in the electrolyte composition of the buffer as long as isotonic sucrose was present. Host-controlled modification/restriction systems also affected transformation efficiency in V. cholerae.

Cell Membrane Permeability↗
PubMed1990

Coordinate regulation of siderophore and diphtheria toxin production by iron in Corynebacterium diphtheriae.

Iron is an environmental signal which regulates the coordinate expression of genes associated with virulence in many pathogenic bacteria. In response to iron-deprivation, lysogenic Corynebacterium diphtheriae C7 (beta) synthesizes and secretes diphtheria toxin and siderophore and induces a high-affinity iron uptake system. Diphtheria toxin is encoded by beta phage, but genes for siderophore production are encoded on the bacterial chromosome. Diphtheria toxin and siderophore production were shown to be coordinately induced during late logarithmic phase growth of wild-type C7(beta) in iron-limited medium. C. diphtheriae mutant C7hm723 produced siderophore and toxin constitutively under low-iron and high-iron conditions, but in mutants HC1, HC3, HC4, and HC5 their synthesis was partially repressed under high-iron conditions. The phenotypes of HC1, HC3, HC4, and HC5 are consistent with their severe defects in iron uptake, but the phenotype of C7hm723 is more likely to be explained by inactivation of the repressor for the iron regulon of C. diphtheriae.

Cetrimonium↗
PubMed1990

Transcription of the Shiga-like toxin type II and Shiga-like toxin type II variant operons of Escherichia coli.

Shiga-like toxin type II (SLT-II) and Shiga-like toxin type II variant (SLT-IIv) are cytotoxins produced by certain strains of Escherichia coli. Nucleotide sequence analyses had revealed that the structural genes for the A subunit and B subunit of SLT-II or SLT-IIv are arranged in an operon. Primer extension and S1 nuclease protection analyses identified a promoter for the slt-II operon 118 bases upstream of the slt-IIA gene. The slt-IIv promoter was demonstrated to be identical to the slt-II promoter. The slt-II and slt-IIv promoters differed significantly from the previously characterized Shiga toxin (stx) and Shiga-like toxin type 1 (slt-I) promoters. The transcriptional efficiencies of the stx and slt-II promoters were compared in fusions to the chloramphenicol acetyltransferase gene, and constitutive expression of the slt-II promoter was found to be equivalent to derepressed expression of the stx promoter. In contrast to the stx and slt-I promoters, the slt-II and slt-IIv promoters did not contain sequences for binding of the Fur repressor protein, and SLT-II production was not determined by iron levels in the media in various E. coli strains with wild-type or mutant ferric uptake regulation (fur) alleles. Northern (RNA) blot analysis demonstrated a single mRNA transcript for the slt-II operon, and further analysis of the slt-II operon by primer extension did not reveal an independent promoter for the B subunit gene. A putative rho-independent transcription terminator was identified 274 bases downstream of slt-IIB. These data indicated that the slt-II and slt-IIv operons differ from the stx/slt-I operon in regulation of their transcription by iron. Whether these regulatory differences enable the type I and type II groups of Shiga-like toxins to perform different roles in the pathogenesis of infectious diseases remains to be established.

Animals↗
PubMed1990

Functional analysis of the Shiga toxin and Shiga-like toxin type II variant binding subunits by using site-directed mutagenesis.

The B subunit of Shiga toxin and the Shiga-like toxins (SLTs) mediates receptor binding, cytotoxic specificity, and extracellular localization of the holotoxin. While the functional receptor for Shiga toxin, SLT type I (SLT-I), and SLT-II is the glycolipid designated Gb3, SLT-II variant (SLT-IIv) may use a different glycolipid receptor. To identify the domains responsible for receptor binding, localization in Escherichia coli, and recognition by neutralizing monoclonal antibodies, oligonucleotide-directed site-specific mutagenesis was used to alter amino acid residues in the B subunits of Shiga toxin and SLT-IIv. Mutagenesis of a well-conserved hydrophilic region near the amino terminus of the Shiga toxin B subunit rendered the molecule nontoxic but did not affect immunoreactivity or holotoxin assembly. In addition, elimination of one cysteine residue, as well as truncation of the B polypeptide by 5 amino acids, caused a total loss of activity. Changing a glutamate to a glutamine at the carboxyl terminus of the Shiga toxin B subunit resulted in the loss of receptor binding and immunoreactivity. However, the corresponding mutation in the SLT-IIv B subunit (glutamine to glutamate) did not reduce the levels of cytotoxicity but did affect extracellular localization of the holotoxin in E. coli.

Amino Acid Sequence↗
PubMed1989

Zeta-crystallin, a novel protein from the guinea pig lens is related to alcohol dehydrogenases.

zeta-Crystallin is a major component of the water-soluble proteins of the guinea pig lens. We have constructed a lens cDNA library from one- to seven-day-old guinea pigs in the plasmid Bluescript KS+ and used the 16 amino acid (aa) sequence of a CNBr peptide to design an oligodeoxyribonucleotide probe. Analysis of two positive clones and direct sequence of the 5' end of the RNA resulted in the completion of a most probably full-length mRNA comprising 1842 nucleotides (nt). The ATG start codon occurs 83 nt downstream from the 5' end. The open reading frame, ending with a stop codon at nt position 1070, predicts a protein of 328 aa with a calculated Mr of 35,071. Comparison of the amino acid sequence with the National Biomedical Research Foundation protein data base reveals a significant similarity of zeta-crystallin with the enzyme of the alcohol dehydrogenase family.

Alcohol Dehydrogenase↗
PubMed1989

Shiga-like toxin converting phage of enterohemorrhagic Escherichia coli strain 933.

Production of Shiga-like toxin (SLT) by enterohemorrhagic Escherichia coli (EHEC) is controlled by phage conversion, and specific phages carry either the SLT-I or SLT-II operon. EHEC strain 933 produces both SLT-I and SLT-II. Previous studies demonstrated that the vast majority of phages recovered from strain 993 have hexagonal heads with short tails and encode SLT-II. However, conflicting results were obtained concerning the properties of SLT-I converting phages from strain 933. The present study reexamined the recovery of phages from 933 by various methods and characterized the restriction fragments from strain 933 DNA that hybridized with radiolabeled DNA from the SLT-I converting phage 933J, which has an elongated head with a long tail, and the SLT-II converting phage 933W. In the present study, only SLT-II converting phages like 933W were recovered from strain 933. A set of restriction fragments that hybridized with DNA from phage 933J but not 933W was present both in wild type strain 933 and in the variant 933D, which produces only SLT-I and was shown here to be cured of phage 933W. The sizes of the restriction fragments in strain 933 that were homologous with phage 933J differed, however, from those of phage 933J. These data indicate that the phage we isolated and named 933J probably did not originate from strain 933 as we originally reported. The present evidence demonstrates that strain 933 contains both the SLT-II converting phage 933W and other sequences of DNA homologous with phage 933J that probably represent a defective SLT-I converting phage.

Bacterial Toxins↗
PubMed1989

In vivo formation of hybrid toxins comprising Shiga toxin and the Shiga-like toxins and role of the B subunit in localization and cytotoxic activity.

Shiga toxin, Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) are cell-associated cytotoxins that kill both Vero cells and HeLa cells, whereas Shiga-like toxin II variant (SLT-IIv) is an extracellular cytotoxin that is more cytotoxic for Vero cells than for HeLa cells. The basis for these differences in cytotoxin localization and host cell specificity were examined in this study. The A and B subunit genes of Shiga toxin and the SLTs were recombined by two methods so that hybrid toxins would be formed in vivo. Complementation of heterologous subunits was accomplished by cloning the individual A and B subunit genes of SLT-I, SLT-II, and SLT-IIv on plasmid vectors of different incompatibility groups so that they could be maintained in double transformants of Escherichia coli. In addition, six operon fusions were constructed so that the A and B subunit genes of Shiga toxin, SLT-II, and SLT-IIv could be expressed as a single operon. The activities of the hybrid cytotoxins were assessed in three ways: (i) level of cytotoxicity, (ii) ratio of HeLa to Vero cell cytotoxicity, and (iii) ratio of extracellular to cell-associated cytotoxicity. Neither the A subunit of Shiga toxin nor SLT-I associated with a heterologous B subunit to form an active cytotoxin. However, in all other cases the hybrid molecules formed by subunit complementation or operon fusion were cytotoxic. Furthermore, the cytotoxic specificity and localization of the hybrid cytotoxins always corresponded to the activities of the native toxin possessing the same B subunit.

Bacterial Toxins↗
PubMed1989

Cloning, nucleotide sequence, and hybridization studies of the type IIb heat-labile enterotoxin gene of Escherichia coli.

Type IIb heat-labile enterotoxin (LT-IIb) is produced by Escherichia coli 41. Restriction fragments of total cell DNA from strain 41 were cloned into a cosmid vector, and one cosmid clone that encoded LT-IIb was identified. The genes for LT-IIb were subcloned into a variety of plasmids, expressed in minicells, sequenced, and compared with the structural genes for other members of the Vibrio cholerae-E. coli enterotoxin family. The A subunits of these toxins all have similar ADP-ribosyltransferase activity. The A genes of LT-IIa and LT-IIb exhibited 71% DNA sequence homology with each other and 55 to 57% homology with the A genes of cholera toxin (CT) and the type I enterotoxins of E. coli (LTh-I and LTp-I). The A subunits of the heat-labile enterotoxins also have limited homology with other ADP-ribosylating toxins, including pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A. The B subunits of LT-IIa and LT-IIb differ from each other and from type I enterotoxins in their carbohydrate-binding specificities. The B genes of LT-IIa and LT-IIb were 66% homologous, but neither had significant homology with the B genes of CT, LTh-I, and LTp-I. The A subunit genes for the type I and type II enterotoxins represent distinct branches of an evolutionary tree, and the divergence between the A subunit genes of LT-IIa and LT-IIb is greater than that between CT and LT-I. In contrast, it has not yet been possible to demonstrate an evolutionary relationship between the B subunits of type I and type II heat-labile enterotoxins. Hybridization studies with DNA from independently isolated LT-II producing strains of E. coli also suggested that additional variants of LT-II exist.

Amino Acid Sequence↗
PubMed1988

Effects of iron and temperature on Shiga-like toxin I production by Escherichia coli.

Iron is known to depress Shiga toxin production by Shigella dysenteriae 1, and temperature has been shown to regulate several genes required for Shigella invasiveness. In this study, the influence of iron and temperature on regulation of a highly related toxin, Shiga-like toxin I (SLT-I) of enterohemorrhagic Escherichia coli, was examined in strains lysogenic for the toxin-converting coliphage 933J and in strains carrying the cloned slt-I genes on a high-copy-number plasmid vector. For comparison, S. dysenteriae 1 was included in these studies. As expected, iron suppressed Shiga toxin synthesis, and reduced growth temperature was also found to decrease Shiga toxin production. Iron also suppressed SLT-I synthesis in E. coli lysogenized with phage 933J but did not demonstrably repress toxin synthesis in E. coli strains carrying the cloned slt-I genes. Temperature had no effect on SLT-I synthesis. Mini-Mu lac operon fusions were then isolated in the cloned slt-I genes and used to test for regulation of beta-galactosidase by iron. Iron did not decrease beta-galactosidase production in strains that harbored these operon fusion plasmids. Taken together, these results indicate that iron but not temperature represses SLT-I synthesis when the slt-I genes are phage associated but this suppression is not easily demonstrated when the slt-I genes are cloned on a high-copy-number plasmid.

Bacterial Toxins↗
PubMed1988

Comparison of the carbohydrate-binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-I, LT-IIa, and LT-IIb.

The heat-labile enterotoxins of Vibrio cholerae and Escherichia coli are related in structure and function. They are oligomers consisting of A and B polypeptide subunits. They bind to gangliosides, and they activate adenylate cyclase. The toxins form two antigenically distinct groups; members of each group cross-react but are not necessarily identical. Serogroup I includes cholera toxin (CT) and type I heat-labile enterotoxin (LT-I) of E. coli. LTh-I and LTp-I are antigenic variants of LT-I produced by strains of E. coli from humans and pigs, respectively. Serogroup II contains the type II heat-labile enterotoxin (LT-II) of E. coli. Two antigenic variants designated LT-IIa and LT-IIb have been described. The binding of CT, LTh-I, LT-IIa, and LT-IIb to gangliosides was analyzed by immunostaining thin-layer chromatograms and by solid-phase radioimmunoassay. The four toxins have different glycolipid-binding specificities. LTh-I and CT bind strongly to ganglioside GM1 and less strongly to ganglioside GD1b. However, LTh-I, unlike CT, also binds weakly to GM2 and asialo GM1. LTh-I, like CT, probably binds to the terminal sugar sequence Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal . . ., where GalNAc is N-acetylgalactosamine and NeuAc is N-acetylneuraminic acid. LT-IIa probably binds to the same sugar sequence to which CT and LTh-I bind, with the additional contribution to binding of a second NeuAc as in GD1b and GD2. Also, LT-IIa must bind the Gal beta 1-3GalNAc . . . sequence in such a way that its binding is relatively unaffected by attachment of NeuAc to the terminal galactose residue as in GD1a, GT1b, and GQ1b. LT-IIb probably binds to the terminal sugar sequence NeuAc alpha 2-3Gal beta 1-4GalNAc . . ., as it binds to gangliosides GD1a and GT1b but not to GM1.

Adrenal Glands↗
PubMed1988

Iron regulation of the cloned diphtheria toxin promoter in Escherichia coli.

Regulation of the diphtheria toxin promoter by iron was studied in Escherichia coli by using a galK transcriptional fusion. A fragment of the toxin (tox) operon containing the regulatory region was cloned from corynephage beta into a galK transcription vector such that expression of galK activity was controlled by the tox promoter. When E. coli N100 (a galK mutant) harboring this tox-galK fusion plasmid was grown in Luria broth, the specific activity of galactokinase remained constant throughout the exponential phase of growth. When bacteria were shifted from such high-iron medium into low-iron Luria broth, the specific activity of galactokinase increased rapidly, but induction of galactokinase was prevented by the addition of iron to the medium. Measurement of tox-specific mRNA by dot blot hybridization showed that this regulation occurred at the level of transcription. When the plasmid containing the tox-galK fusion was introduced into a fur mutant of E. coli, expression of galK was maximal in both high-iron and low-iron media; but repressibility of galK by iron in this strain was restored by complementation with the fur+ allele. The tox promoter has significant homology with the consensus sequence for other iron-regulated promoters of E. coli that are controlled by fur. These data indicate that the product of the fur gene can function in E. coli as an iron-dependent repressor for the tox promoter from corynephage beta.

Base Sequence↗
PubMed1988

Cloning and sequencing of the genes for Shiga toxin from Shigella dysenteriae type 1.

The structural genes for Shiga toxin, designated stx A and stx B, were cloned from Shigella dysenteriae type 1 3818T, and a nucleotide sequence analysis was performed. Both stx A and stx B were present on a single transcriptional unit, with stx A preceding stx B. The molecular weight calculated for the processed A subunit was 32,225, while the molecular weight of the processed B subunit was 7,691. Comparison of the nucleotide sequences for Shiga toxin and Shiga-like toxin I (SLT-I) from Escherichia coli revealed that the genes for Shiga toxin and SLT-I were greater than 99% homologous; three nucleotide changes were detected in three separate codons of the A subunits. Only one of these codon differences resulted in a change in the amino acid sequence: a threonine in Shiga toxin at position 45 of the A subunit compared with a serine in the corresponding position in SLT-I. Furthermore, Shiga toxin and SLT-I had identical signal peptides for the A and B subunits, as well as identical ribosome-binding sites, a putative promoter, and iron-regulated operator sequences. These findings indicate that Shiga and SLT-I are essentially the same toxin. Southern hybridization studies with total cellular DNA from several Shigella strains and internal toxin probes for SLT-I and its antigenic variant SLT-II showed that a single fragment in S. dysenteriae type 1 hybridized strongly with the internal SLT-I probe. Fragments with weaker homology to the SLT-I probe were detected in S. flexneri type 2a but no other shigellae. No homology between the Shiga-like toxin II (SLT-II) probe and any of the Shigella DNAs was detected. Whereas SLT-I and SLT-II are phage encoded, no phage could be induced from S. dysenteriae type 1 or other Shigella spp. tested. These results suggest that the Shiga (SLT-I) toxin genes responsible for high toxin production are present in a single copy in S. dysenteriae type 1 but not in other shigellae. The findings further suggest that SLT-II genes are absent in shigellae, as are toxin-converting phages.

Amino Acid Sequence↗
PubMed1988

Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine.

A Shiga-like toxin type II variant (SLT-IIv) is produced by strains of Escherichia coli responsible for edema disease of swine and is antigenically related to Shiga-like toxin type II (SLT-II) of enterohemorrhagic E. coli. However, SLT-IIv is only active against Vero cells, whereas SLT-II is active against both Vero and HeLa cells. The structural genes for SLT-IIv were cloned from E. coli S1191, and the nucleotide sequence was determined and compared with those of other members of the Shiga toxin family. The A subunit genes for SLT-IIv and SLT-II were highly homologous (94%), whereas the B subunit genes were less homologous (79%). The SLT-IIv genes were more distantly related (55 to 60% overall homology) to the genes for Shiga toxin of Shigella dysenteriae type 1 and the nearly identical Shiga-like toxin type I (SLT-I) of enterohemorrhagic E. coli. (These toxins are referred to together as Shiga toxin/SLT-I.) The A subunit of SLT-IIv, like those of other members of this toxin family, had regions of homology with the plant lectin ricin. SLT-IIv did not bind to galactose-alpha 1-4-galactose conjugated to bovine serum albumin, which is an analog of the eucaryotic cell receptor for Shiga toxin/SLT-I and SLT-II. These findings support the hypothesis that SLT-IIv binds to a different cellular receptor than do other members of the Shiga toxin family but has a similar mode of intracellular action. The organization of the SLT-IIv operon was similar to that of other members of the Shiga toxin family. Iron did not suppress SLT-IIv or SLT-II production, in contrast with its effect on Shiga toxin/SLT-I. Therefore, the regulation of synthesis of SLT-IIv and SLT-II differs from that of Shiga toxin/SLT-I.

Amino Acid Sequence↗
PubMed1987

Single mutation in the A domain of diphtheria toxin results in a protein with altered membrane insertion behavior.

The insertion of the A domain of diphtheria toxin into model membranes has been shown to be both pH- and temperature-dependent (Hu and Holmes (1984) J. Biol. Chem. 259, 12226-12233). In this report, the insertion behavior of two mutant proteins of diphtheria toxin, CRM197 and CRM9, was studied and compared to that of wild-type toxin. Results indicated that both CRM197 and CRM9 resembled toxin with respect to the pH-dependence of binding to negatively-charged liposomes at room temperature. However, CRM197 differed from toxin with respect to both the pH- and temperature-dependence of fragment A insertion; fragment A197 inserts more readily into the bilayer at 0 degrees C and low pH or at neutral pH and room temperature than does wild type fragment A under these same conditions. This result indicates that the single amino acid substitution in the A domain of CRM197 facilitates entry of fragment A197 into the membrane, suggesting that CRM197 may be conformationally distinct from native toxin. In fact, the fluorescence spectra of CRM197 and wild-type toxin as well as their respective tryptic peptide patterns indicate that, at pH 7, CRM197 more closely resembles the acid form of wild-type toxin than the native form of toxin. These data suggest that CRM197 may be naturally in a more 'insertion-competent' conformation. In contrast, the mutation in the B domain of CRM9 which results in a 1000-fold decrease in binding affinity for plasma membrane receptors apparently does not cause a change in either the insertion of fragment A9 or the lipid-binding properties of CRM9 relative to toxin.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence analysis of the structural genes for Shiga-like toxin I encoded by bacteriophage 933J from Escherichia coli.

A nucleotide sequence analysis was performed on the Shiga-like toxin I genes previously cloned from the Escherichia coli bacteriophage 933J. Two structural genes designated slt-I A and slt-I B were found to be oriented on a single transcriptional unit with slt-I A preceding slt-I B. A 12 base pair gap separated the in-phase open reading frames of slt-I A and slt-I B. Putative ribosome binding sites were identified 5' to both the slt-I A and slt-I B genes. Translation of the SLT-I nucleotide sequence revealed that both the A and B subunits were synthesized with signal peptides. The molecular weight calculated for the mature A subunit was 32,211 while the molecular weight of the mature B subunit was 7690.

Amino Acid Sequence↗
PubMed1987

Transcription of cholera toxin operon in wild-type and mutant strains of Vibrio cholerae.

mRNA for cholera toxin (CT) was measured in wild-type, hypertoxinogenic, and hypotoxinogenic strains of the classical biotype. The amount of CT-specific mRNA was directly related to the amount of CT that the strains could produce. Estimates of the size of the mRNA for CT in individual experiments varied between approximately 900 and 1,100 nucleotides.

Cholera Toxin↗