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I S Roberts

Publications and source records attributed to I S Roberts.

At least 73 records · Page 4Linked to original sources
PubMed1995

Expression and characterization of UDPGlc dehydrogenase (KfiD), which is encoded in the type-specific region 2 of the Escherichia coli K5 capsule genes.

Region 2 of the Escherichia coli K5 capsule gene cluster contains four genes (kfiA through -D) which encode proteins involved in the synthesis of the K5 polysaccharide. A DNA fragment containing kfiD was amplified by PCR and cloned into the gene fusion vector pGEX-2T to generate a GST-KfiD fusion protein. The fusion protein was isolated from the cytoplasms of IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced recombinant bacteria by affinity chromatography and cleaved with thrombin. The N-terminal amino acid sequence of the cleavage product KfiD' corresponded to the predicted amino acid sequence of KfiD with an N-terminal glycyl-seryl extension from the cleavage site of the fusion protein. Anti-KfiD antibodies obtained with KfiD' were used to isolate the intact KfiD protein from the cytoplasms of E. coli organisms overexpressing the kfiD gene. The fusion protein, its cleavage product (KfiD'), and overexpressed KfiD converted UDPGlc to UDPGlcA. The KfiD protein could thus be characterized as a UDPglucose dehydrogenase.

Bacterial Capsules↗
PubMed1995

Cloning, expression, and characterization of the lon gene of Erwinia amylovora: evidence for a heat shock response.

The gene encoding the Lon protease of Erwinia amylovora has been cloned by complementation of an Escherichia coli lon mutant. Analysis of the determined nucleotide sequence of the lon gene revealed extensive homology to the nucleotide sequences of cloned lon genes from E. coli, Myxococcus xanthus, and Bacillus brevis. The predicted amino acid sequence of the E. amylovora Lon protease was 94, 59, and 54% identical to the predicted amino acid sequences of the Lon proteases of E. coli, M. xanthus, and B. brevis, respectively. The -10 and -35 promoter regions of the cloned lon gene had extensive homology to the respective consensus sequences of E. coli heat shock promoters. Promoter mapping of the lon gene located the start site 7 bases downstream of the -10 region. Cloning of the lon promoter upstream of a cat reporter gene demonstrated that expression of the E. amylovora lon gene was inducible by a heat shock. This is the first demonstration of a heat shock-regulated gene in E. amylovora. Site-directed mutagenesis of the -10 region of the lon promoter confirmed that the heat shock expression of the E. amylovora lon gene may be mediated by a sigma 32-like factor. Insertional inactivation of the E. amylovora chromosomal lon gene confirmed that the lon gene was not essential for either vegetative growth or infection of apple seedlings. E. amylovora lon mutants had increased sensitivity to UV irradiation and elevated levels of extracellular polysaccharide, suggesting comparable roles for the Lon proteases in both E. amylovora and E. coli.

ATP-Dependent Proteases↗
PubMed1995

Characterization and localization of the KpsE protein of Escherichia coli K5, which is involved in polysaccharide export.

In Escherichia coli with group II capsules, the synthesis and cellular expression of capsular polysaccharide are encoded by the kps gene cluster. This gene cluster is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of the K5 polysaccharide, which is a group II capsular polysaccharide, have been cloned and sequenced. Region 1 contains the kpsE, -D, -U, -C, and -S genes. In this communication we describe the KpsE protein, the product of the kpsE gene. A truncated kpsE gene was fused with a truncated beta-galactosidase gene to generate a fusion protein containing the first 375 amino acids of beta-galactosidase and amino acids 67 to 382 of KpsE (KpsE'). This fusion protein was isolated and cleaved with factor Xa, and the purified KpsE' was used to immunize rabbits. Intact KpsE was extracted from the membranes of a KpsE-overexpressing recombinant strain with octyl-beta-glucoside. It was purified by affinity chromatography with immobilized anti-KpsE antibodies. Cytofluorometric analysis using the anti-KpsE antibodies with whole cells and spheroplasts, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of proteins from spheroplasts and membranes before and after treatment with proteinase K, indicated that the KpsE protein is associated with the cytoplasmic membrane and has an exposed periplasmic domain. By TnphoA mutagenesis and by constructing beta-lactamase fusions to the KpseE protein, it was possible to determine the topology of the KpsE protein within the cytoplasmic membrane.

Amino Acid Sequence↗
PubMed1994

Regulation of Escherichia coli K5 capsular polysaccharide expression: evidence for involvement of RfaH in the expression of group II capsules.

Expression of the Escherichia coli K5 antigen was used as a model system to study the role of known regulators of gene expression on production of group II capsules in E. coli. Only mutations in the rfaH gene had an effect on production of the K5 antigen, abolishing the expression of any detectable capsule at 37 degrees C. None of the mutations studied induced capsule expression at 18 degrees C. A sequence, termed JUMPstart, found in group II capsule gene clusters and upstream of a number of polysaccharide biosynthesis genes in enteric bacteria is homologous to sequences found in RfaH regulated operons. This may indicate a common mode of regulation of these polysaccharide biosynthesis genes by RfaH.

Antigens, Bacterial↗
PubMed1994

Characterisation of IS1126 from Porphyromonas gingivalis W83: a new member of the IS4 family of insertion sequence elements.

The nucleotide sequence of IS1126, the only insertion sequence so far isolated from the oral pathogen Porphyromonas gingivalis, has been determined. It had a nucleotide sequence of 1338 base pair (bp) flanked by 12 bp perfect inverted repeats and generated a 5 bp target site duplication. The single major open reading frame encoded a predicted protein of 361 amino acids and molecular mass of 41 kDa. The gene encoding the transposase was subcloned into pUC18 and the transposase expressed in Escherichia coli minicells. The predicted amino acid sequence of the transposase had homology to putative transposases of IS1106 and IS1186 both of which belong to the IS5 group within the IS4 super-family of insertion elements. On the basis of this homology we propose that IS1126 should also be included in the IS5 group. Southern-blot analysis of a number of P. gingivalis strains using IS1126 as a probe revealed a unique pattern of hybridisation for each strain and the absence of IS1126 from other closely related Porphyromonas species. This should allow IS1126 to be used as a rapid epidemiological tool in studying oral infections by P. gingivalis.

Amino Acid Sequence↗
PubMed1994

Cloning and expression in Escherichia coli of the nahA gene from Porphyromonas gingivalis indicates that beta-N-acetylhexosaminidase is an outer-membrane-associated lipoprotein.

Porphyromonas gingivalis has been implicated in human periodontal diseases. It expresses a number of exoglycosidase enzymes capable of hydrolysing host proteoglycan residues. As a first stage to explore the role of these enzymes in periodontal tissue damage, the nahA gene of P. gingivalis W83, which encodes beta-N-acetylhexosaminidase (beta-Nahase), was cloned. The gene was expressed poorly in Escherichia coli, but increased expression was achieved by cloning the nahA gene downstream of the tac promoter. Southern blot analysis revealed that nahA was present as a single copy, and it was found in all the other P. gingivalis strains tested. In contrast, sequences homologous to nahA were not detected in either P. endodontalis or P. asaccharolytica. The nahA gene was 2331 bp long and encoded a beta-Nahase enzyme of 777 amino acids with a predicted molecular mass of 87 kDa. A characteristic signal peptide for an acylated lipoprotein was present at the amino-terminus, suggesting that the mature beta-Nahase is a lipoprotein. The predicted amino acid sequence of the P. gingivalis beta-Nahase shared homology with the catalytic domains of the human beta-Nahase enzyme and the chitinase of Vibrio harveyi, suggesting a common catalytic mechanism.

Amino Acid Sequence↗
PubMed1994

The application of luciferase as a reporter of environmental regulation of gene expression in mycobacteria.

This paper describes the construction of pSG10, the first mycobacterial promoter probe shuttle vector to use the structural gene of a bacterial luciferase as a reporter gene. To examine the utility of using bacterial luciferase to measure gene expression in mycobacteria, the authors have used this vector to monitor the induction of the acetamidase gene promoter of Mycobacterium smegmatis. Luciferase proved to be a rapid, sensitive and easily assayable reporter of changes in gene activity in response to environment in mycobacteria.

Acetamides↗
PubMed1994

Molecular cloning and analysis of genes for sialic acid synthesis in Neisseria meningitidis group B and purification of the meningococcal CMP-NeuNAc synthetase enzyme.

The gene encoding for the CMP-NeuNAc synthetase enzyme of Neisseria meningitidis group B was cloned by complementation of a mutant of Escherichia coli defective for this enzyme. The gene (neuA) was isolated on a 4.1-kb fragment of meningococcal chromosomal DNA. Determination of the nucleotide sequence of this fragment revealed the presence of three genes, termed neuA, neuB, and neuC, organized in a single operon. The presence of a truncated ctrA gene at one end of the cloned DNA and a truncated gene encoding for the meningococcal sialyltransferase at the other confirmed that the cloned DNA corresponded to region A and part of region C of the meningococcal capsule gene cluster. The predicted amino acid sequence of the meningococcal NeuA protein was 57% homologous to that of NeuA, the CMP-NeuNAc synthetase encoded by E. coli K1. The predicted molecular mass of meningococcal NeuA protein was 24.8 kDa, which was 6 kDa larger than that formerly predicted (U. Edwards and M. Frosch, FEMS Microbiol. Lett. 96:161-166, 1992). Purification of the recombinant meningococcal NeuA protein together with determination of the N-terminal amino acid sequence confirmed that this 24.8-kDa protein was indeed the meningococcal CMP-NeuNAc synthetase. The predicted amino acid sequences of the two other encoded proteins were homologous to those of the NeuC and NeuB proteins of E. coli K1, two proteins involved in the synthesis of NeuNAc. These results indicate that common steps exist in the biosynthesis of NeuNAc in these two microorganisms.

Amino Acid Sequence↗
PubMed1993

Utilization of transferrin-bound iron by Listeria monocytogenes.

It has been demonstrated that under iron-restricted conditions, Listeria monocytogenes can utilize iron-loaded transferrin (Tf) from a range of species as its sole source of iron for growth. Human transferrin conjugated to horseradish-peroxidase (HRP-Tf) bound directly to whole cells of L. monocytogenes. This binding was blocked by apotransferrin indicating that the receptor can bind transferrin in either the iron-bound or iron-free form. Transferrin-binding was not host specific because both bovine and equine transferrin inhibited the binding of HRP-conjugated human transferrin. SDS-PAGE and Western blotting of bacterial surface extracts revealed the presence of a transferrin-binding protein of approximately 126 kDa.

Animals↗
PubMed1993

Ulcer-associated cell lineage ('pyloric metaplasia') in Crohn's disease: a lectin histochemical study.

Chronic intestinal ulceration in Crohn's disease is associated with the development of an epidermal growth factor-secreting cell lineage, or 'ulcer-associated cell lineage' (UACL). Expression of oligosaccharides by UACL was studied using a panel of 25 biotinylated lectins with an avidin peroxidase revealing system and compared with that of adult and fetal Brunner's glands, gastric antral mucosa, and 'gastric metaplasia' within the duodenum, in order to clarify further the interrelationships of these lineages. UACL was obtained from ileal resections performed for Crohn's disease. Lectin binding of the glandular component of UACL closely resembled that of antral mucosal glands and also that of fetal and adult Brunner's glands. Lectin binding of the ductal component of immature UACL, in which a surface component had not developed, resembled that of the gland. The surface and ductal components of mature UACL showed a distinct lectin-binding profile, which was very different from that of the gland, but closely resembled that of antral foveolar epithelium and 'gastric metaplasia' within the duodenum. It is concluded that there is differentiation of UACL from the glandular to surface components and that oligosaccharide expression of the lineage reflects that of normal Brunner's gland and gastric antral mucosa.

Brunner Glands↗
PubMed1993

The Escherichia coli serA-linked capsule locus and its flanking sequences are polymorphic, genetic evidence for the existence of more than two groups of capsule gene clusters.

Two families of Escherichia coli capsules, termed groups I and II, have been defined previously on the basis of a number of biochemical and genetic criteria. Recently, a third group of capsules, termed I/II has been suggested on the basis of chemical structure and mode of expression. In this paper, we show that group I capsule-producing strains lack the serA-linked group II capsule genes. In addition, group I/II capsule-producing strains lack the group II capsule genes despite the former genes also mapping near to serA. Therefore, the genetic data presented in this paper support the existence of three groups of capsule gene clusters, two of which are linked to serA. Sequences flanking the K4 capsule genes were found in the chromosome of all E. coli strains examined and were sometimes present in multiple copies at different loci, indicating that this chromosomal region is highly polymorphic.

Bacterial Capsules↗
PubMed1993

The nature of atypical multinucleated stromal cells: a study of 37 cases from different sites.

The morphology, immunophenotype and ultrastructure of atypical multinucleated stromal cells in 37 specimens from different anatomical sites were compared. Overall, cellular morphology was similar between sites but nuclear features, immunophenotype and ultrastructure varied and reflected that of adjacent normal mononuclear stromal cells. It is concluded that the atypical multinucleated stromal cells represent a reactive change in the indigenous stromal cells. In all the cases mast cells were seen as a prominent component of the inflammatory infiltrate and many of them were intimately related to the atypical stromal cells. It is suggested that reactive stromal cell changes may relate to an interaction with mast cells.

Cell Nucleus↗
PubMed1993

Characterization of an aromatic amino acid-dependent Listeria monocytogenes mutant: attenuation, persistence, and ability to induce protective immunity in mice.

A transposon insertion mutant of Listeria monocytogenes was shown to be deficient in prephenate dehydratase, an enzyme acting late in the pathway for biosynthesis of phenylalanine. This mutant had reduced virulence in mice. The mutant and parent strains persisted to the same extent in the tissues of infected mice and elicited similar degrees of splenomegaly. Mice vaccinated with the mutant were protected significantly from subsequent challenge with virulent L. monocytogenes.

Animals↗
PubMed1993

Molecular analysis of region 1 of the Escherichia coli K5 antigen gene cluster: a region encoding proteins involved in cell surface expression of capsular polysaccharide.

The nucleotide sequence of region 1 of the K5 antigen gene cluster of Escherichia coli was determined. This region is postulated to encode functions which, at least in part, participate in translocation of polysaccharide across the periplasmic space and onto the cell surface. Analysis of the nucleotide sequence revealed five genes that encode proteins with predicted molecular masses of 75.7, 60.5, 44, 43, and 27 kDa. The 27-kDa protein was 70.7% homologous to the CMP-2-keto-3-deoxyoctulosonic acid synthetase enzyme encoded by the E. coli kdsB gene, indicating the presence of a structural gene for a similar enzyme within the region 1 operon. The 43-kDa protein was homologous to both the Ctrb and BexC proteins encoded by the Neisseria meningitidis and Haemophilus influenzae capsule gene clusters, respectively, indicating common stages in the expression of capsules in these gram-negative bacteria. However, no homology was detected between the 75.7, 60.5-, and 44-kDa proteins and any of the proteins so far described for the H. influenzae and N. meningitidis capsule gene clusters.

Amino Acid Sequence↗
PubMed1993

Expression of the capsular K5 polysaccharide of Escherichia coli: biochemical and electron microscopic analyses of mutants with defects in region 1 of the K5 gene cluster.

The gene cluster of the capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three regions responsible for polymerization and surface expression have been defined (I.S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1330, 1988). Region 1 has now been sequenced, and five open reading frames (kpsEDUCS) have been defined (C. Pazzani, C. Rosenow, G. J. Boulnois, D. Bronner, K. Jann, and I. S. Roberts, J. Bacteriol. 175:5978-5983, 1993). In this study, we characterized region 1 mutants by immunoelectron microscopy, membrane-associated polymerization activity, cytoplasmic CMP-2-keto-3-deoxyoctonate (KDO) synthetase activity, and chemical analysis of their K5 polysaccharides. Certain mutations within region 1 not only effected polysaccharide transport (lack of region 1 gene products) but also impaired the polymerization capacity of the respective membranes, reflected in reduced amounts of polysaccharide but not in its chain length. KDO and phosphatidic acid (phosphatidyl-KDO) substitution was found with extracellular and periplasmic polysaccharide and not with cytoplasmic polysaccharide. This and the fact that the K5 polysaccharide is formed in a kpsU mutant (defective in capsule-specific K-CMP-KDO synthetase) showed that CMP-KDO is engaged not in initiation of polymerization but in translocation of the polysaccharide.

Antigens, Bacterial↗
PubMed1993

Penicillin-binding proteins from Erwinia amylovora: mutants lacking PBP2 are avirulent.

Radiolabelled penicillin G was used to examine penicillin-binding proteins (PBPs) from Erwinia amylovora (OT1). This procedure identified seven PBPs with molecular masses ranging from 22 to 83 kDa. E. amylovora PBPs were compared with those from Escherichia coli (JM101) and from two spherical, avirulent TnphoA mutants derived from OT1. Radiolabelled penicillin G bound to only six proteins from the spherical mutants which lacked a 69-kDa PBP. The spherical mutants could be complemented by the cloned E. coli pbpA-rodA operon, which restored both cell shape and virulence to apple seedlings. This suggested that the E. amylovora 69-kDa PBP is probably the functional equivalent of the E. coli PBP2 protein. Southern blot analysis using the E. coli rodA and pbpA genes as radiolabelled probes showed that TnphoA had inserted into the E. amylovora equivalent of the E. coli rodA-pbpA operon. Southern blots to chromosomal DNAs of the two spherical mutants, using the cloned hrp and dsp genes from E. amylovora as radiolabelled probes, confirmed that the TnphoA insertions were not located in the region of the E. amylovora chromosome postulated to encode known virulence factors. Both of the spherical TnphoA mutants synthesized amounts of extracellular polysaccharide equivalent to those synthesized by the wild-type strain (OT1), were resistant to lysis in distilled water and to lysozyme, and elicited the hypersensitive response on nonhost plants. These results indicate a possible role for cell shape in the virulence of this plant pathogen.

Bacterial Proteins↗