Genus specificity and extensive methylation of the W chromosome-specific repetitive DNA sequences from the domestic fowl,Gallus gallus domesticus

Two female-specific repeating DNA units of 0.6 kilobase pairs (kb) and 1.1 kb, produced by digesting the genomic DNA of the White Leghorn chicken with Xho I, were cloned by inserting them into the Xho I site of an Escherichia coli plasmid vector pACYC177. Two such recombinant plasmids, pAGD0601 and pAGD1101, containing a single 0.6-kb and 1.1-kb sequence, respectively, were used as molecular probes. In situ hybridization of the ³Hprobes to the metaphase chromosomes from the female White Leghorn embryos revealed their localization in the W chromosome. Semiquantitative Southern blot hybridization with ³²P-probes in excess indicated that the 0.6-kb unit and 1.1-kb unit were repeated approximately 14,000 and 6,000 times, respectively, in the W chromosome. The two units comprised about 46% of the W chromosomal DNA. These two repeating units were found in the female genomes of every line of Gallus g. domesticus tested and in the female genomes of three jungle fowl species (G. gallus, G. sonneratii, and G. varius) but not in three species belonging to other genera in the suborder Galli. Hha I sites in the 0.6-kb and 1.1-kb repeating units were shown to be extensively methylated and a significant fraction of the Hpa II sites in the 0.6-kb repeating units were also shown to be methylated in the female genome of the White Leghorn. Methylation patterns of Hpa II sites in or around the 0.6-kb repeating units examined by the Msp I digestion were similar in the various lines of domestic fowls and the two species of jungle fowls, but G. varius (black or green jungle fowl) produced a different pattern of digestion with Msp I.     

The predominant roles of the sequence periodicity in the self‐assembly of collagen‐mimetic mini‐fibrils

Collagen fibrils represent a unique case of protein folding and self‐association. We have recently successfully developed triple‐helical peptides that can further self‐assemble into collagen‐mimetic mini‐fibrils. The 35 nm axially repeating structure of the mini‐fibrils, which is designated the d‐period, is highly reminiscent of the well‐known 67 nm D‐period of native collagens when examined using TEM and atomic force spectroscopy. We postulate that it is the pseudo‐identical repeating sequence units in the primary structure of the designed peptides that give rise to the d‐period of the quaternary structure of the mini‐fibrils. In this work, we characterize the self‐assembly of two additional designed peptides: peptide Col877 and peptide Col108rr. The triple‐helix domain of Col877 consists of three pseudo‐identical amino acid sequence units arranged in tandem, whereas that of Col108rr consists of three sequence units identical in amino acid composition but different in sequence. Both peptides form stable collagen triple helices, but only triple helices Col877 self‐associate laterally under fibril forming conditions to form mini‐fibrils having the predicted d‐period. The Co108rr triple helices, however, only form nonspecific aggregates having no identifiable structural features. These results further accentuate the critical involvement of the repeating sequence units in the self‐assembly of collagen mini‐fibrils; the actual amino acid sequence of each unit has only secondary effects. Collagen is essential for tissue development and function. This novel approach to creating collagen‐mimetic fibrils can potentially impact fundamental research and have a wide range of biomedical and industrial applications.     

To achieve self‐assembled collagen mimetic fibrils using designed peptides

It has proven challenging to obtain collagen‐mimetic fibrils by protein design. We recently reported the self‐assembly of a mini‐fibril showing a 35 nm, D‐period like, axially repeating structure using the designed triple helix Col108. Peptide Col108 was made by bacterial expression using a synthetic gene; its triple helix domain consists of three pseudo‐identical units of amino acid sequence arranged in tandem. It was postulated that the 35 nm d‐period of Col108 mini‐fibrils originates from the periodicity of the Col108 primary structure. A mutual staggering of one sequence unit of the associating Col108 triple helices can maximize the inter‐helical interactions and produce the observed 35 nm d‐period. Based on this unit‐staggered model, a triple helix consisting of only two sequence units is expected to have the potential to form the same d‐periodic mini‐fibrils. Indeed, when such a peptide, peptide 2U108, was made it was found to self‐assemble into mini‐fibrils having the same d‐period of 35 nm. In contrast, no d‐periodic mini‐fibrils were observed for peptide 1U108, which does not have long‐range repeating sequences in its primary structure. The findings of the periodic mini‐fibrils of Col108 and 2U108 suggest a way forward to create collagen‐mimetic fibrils for biomedical and industrial applications.     

Nucleotide sequence of a highly repeated DNA sequence and its chromosomal localization in Allium fistulosum

A highly repeated DNA sequence with a repeating unit of approximately 380bp was found in EcoRV digests of the total genomic DNA of Allium fistulosum. Three independent clones containing this unit were isolated, and their repeating units sequenced. These units showed more than 94% sequence homology, and the copy number was estimated to be about 2.8×10⁶ per haploid genome. In situ hybridization, with the repeating unit as a probe, and C-banding analyses indicated that the repeated DNA sequence of A. fistulosum is closely associated with the major C-heterochromatin in the terminal regions of all 16 chromosomes at mitotic metaphase. The characters of the repeating unit are similar to those of the A. cepa unit, which is taxonomically closely related to A. fistulosum.     

An effective peptide screening system using recombinant fluorescent bacterial surface display

An effective method for peptide screening of ligand-binding proteins was applied by using recombinant E. coli which is capable of expressing green fluorescent protein (GFP) and which can also express random peptides displayed on flagella of the cells. This screening method used a combination of fluorescence-activated cell sorting (FACS) and flagella display on the basis of a commercial FliTrx random peptide library for isolating the peptide-displaying clones which are able to bind Alexa 546 fluorescence-labeled cytochrome c. Flow cytometry simultaneously detected the two different fluorescence intensities, from GFP in the library and Alexa546-labeled to cytochrome c, enabling the specific clones bound to cytochrome c to be obtained from the first or second round of cell sorting. Compared with original FliTrx peptide screening system that requires repeating biopanning five times, our results suggested that detecting two different fluorescence intensities by flow cytometry is feasible for effective peptide screening.     

High-level expression of the angiotensin-converting-enzyme-inhibiting peptide, YG-1, as tandem multimers in Escherichia coli

To produce a large quantity of the angiotensin-converting-enzyme(ACE)-inhibiting peptide YG-1, which consists of ten amino acids derived from yeast glyceraldehyde-3-phosphate dehydrogenase, a high-level expression was explored with tandem multimers of the YG-1 gene in Escherichia coli. The genes encoding YG-1 were tandemly multimerized to 9-mers, 18-mers and 27-mers, in which each of the repeating units in the tandem multimers was connected to the neighboring genes by a DNA linker encoding Pro-Gly-Arg for the cleavage of multimers by clostripain. The multimers were cloned into the expression vector pET-21b, and expressed in E. coli BL21(DE3) with isopropyl β-d-thiogalactopyranoside induction. The expressed multimeric peptides encoded by the 9-mer, 18-mer and 27-mer accumulated intracellularly as inclusion bodies and comprised about 67%, 25% and 15% of the total proteins in E. coli respectively. The multimeric peptides expressed as inclusion bodies were cleaved with clostripain, and active monomers were purified to homogeneity by reversed-phase high-performance liquid chromatography. In total, 105 mg pure recombinant YG-1 was obtained from 1 l E. coli culture harboring pETYG9, which contained the 9-mer of the YG-1 gene. The recombinant YG-1 was identical to the natural YG-1 in molecular mass, amino acid sequence and ACE-inhibiting activity. Received: 6 January 1998 / Received revision: 23 February 1998 / Accepted: 24 February 1998     

<Emphasis Type="Italic">Streptomyces lividans</Emphasis> and <Emphasis Type="Italic">Brevibacterium lactofermentum</Emphasis> as heterologous hosts for the production of X<Subscript>22</Subscript> xylanase from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

The Aspergillus nidulans gene xlnA coding for the fungal xylanase X₂₂ has been cloned and expressed in two heterologous bacterial hosts: Streptomyces lividans and Brevibacterium lactofermentum. Streptomyces strains yielded 10 units/ml of xylanase when the protein was produced with its own signal peptide, and 19 units/ml when its signal peptide was replaced by the one for xylanase Xys1 from Streptomyces halstedii. B. lactofermentum was also able to produce xylanase X₂₂, affording 6 units/ml upon using either the Aspergillus xlnA signal peptide or Streptomyces xysA. These production values are higher than those previously reported for the heterologous expression of the A. nidulans xlnA gene in Saccharomyces cerevisiae (1 unit/ml). Moreover, the X₂₂ enzyme produced by Streptomyces lividans showed oenological properties, indicating that this Streptomyces recombinant strain is a good candidate for the production of this enzyme at the industrial scale.     

CbpA: a novel surface exposed adhesin of Clostridium difficile targeting human collagen

Clostridium difficile is the leading cause of antibiotic‐associated diarrhoea and pseudomembranous colitis. While the role of toxins in pathogenesis has been extensively described, the contribution of surface determinants to intestinal colonization is still poorly understood. We focused our study on a novel member of the MSCRAMM family, named CbpA (C ollagen b inding p rotein A ), for its adhesive properties towards collagen. We demonstrate that CbpA, which carries an LPXTG‐like cell wall anchoring domain, is expressed on the bacterial surface of C. difficile and that the recombinant protein binds at high affinity to collagens I and V (apparent K_d in the order of 10^?9 M). These findings were validated by confocal microscopy studies showing the colocalization of the protein with type I and V collagen fibres produced by human fibroblasts and mouse intestinal tissues. However, the collagen binding activity of the wild‐type C. difficile 630 strain was indistinguishable to the cbpA knock‐out strain. To overcome this apparent clostridial adherence redundancy, we engineered a Lactococcus lactis strain for the heterologous expression of CbpA. When exposed on the surface of L. lactis, CbpA significantly enhances the ability of the bacterium to interact with collagen and to adhere to ECM‐producing cells. The binding activity of L. lactis–CbpA strain was prevented by an antiserum raised against CbpA, demonstrating the specificity of the interaction. These results suggest that CbpA is a newsurface‐exposed adhesin contributing to the C. difficile interaction with the host.     

Investigations on Sarcosine and Isonipecotic Acid Containing Peptide Nucleic Acids

Abstract

Two different series of peptide nucleic acids (with the repeating units AlaT-Sar and AlaT-Sar-Inp) were synthesised and evaluated for potential binding to an oligoA/oligoT duplex.     

Prevention of Clostridium difficile -induced ileocecitis with Bacteriophage

A bacteriophage specific for Clostridium difficile was examined for its ability to prevent ileocecitis in a hamster model. This species- and strain-specific bacteriophage was isolated from a lysogenic strain of C. difficile . Hamsters were maintained in sterile isolation cages to prevent the acquisition of C. difficile from the environment. Bicarbonate neutralization of gastric acidity was necessary for bacteriophage survival in the hamster's gastrointestinal tract. Bacteriophage recovery from the hamster cecum was 2×10⁴plaque forming units/mL of cecal contents 24 h after orogastric challenge with 10⁸plaque forming units/mL of bacteriophage. However, there was no bacteriophage recovery 48 h post challenge, indicating dissipation of bacteriophage from the hamster intestinal tract within this time frame. Twenty-four hours after being challenged with clindamycin, one group of hamsters was challenged with C. difficile followed by a single dose of bacteriophage (10⁸plaque forming units/mL). Two additional groups of hamsters received phage doses immediately after C. difficile challenge and subsequently thereafter every 8 h up to 48 and 72 h, respectively. The gastric acidity was neutralized with bicarbonate buffer preceding every bacteriophage treatment. Control animals that received only clindamycin and C. difficile died within 96 h after challenge while the majority of bacteriophage treated hamsters survived. Two weeks after stopping bacteriophage treatment, the surviving hamsters were re-challenged with clindamycin and C. difficile . All the hamsters died within 96 h indicating susceptibility of the surviving hamsters to C. difficile disease in the absence of bacteriophage treatment.     

Expression of recombinant <Emphasis Type="Italic">Clostridium difficile</Emphasis> toxin A and B in <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Background  

Major Clostridium difficile virulence factors are the exotoxins TcdA and TcdB. Due to the large size and poor stability of the proteins, the active recombinant TcdA and TcdB have been difficult to produce.     

Investigating the candidacy of a lipoteichoic acid-based glycoconjugate as a vaccine to combat Clostridium difficile infection

A lipoteichoic acid has recently been shown to be conserved in the majority of strains from Clostridium difficile and as such is being considered as a possible vaccine antigen. In this study we examine the candidacy of the conserved lipoteichoic acid by demonstrating that it is possible to elicit antibodies against C. difficile strains following immunisation of rabbits and mice with glycoconjugates elaborating the conserved lipoteichoic acid antigen. The present study describes a conjugation strategy that utilises an amino functionality, present at approximately 33 % substitution of the N-acetyl-glucosamine residues within the LTA polymer repeating unit, as the attachment point for conjugation. A maleimide-thiol linker strategy with the maleimide linker on the carboxyl residues of the carrier protein and the thiol linker on the carbohydrate was employed. Immunisation derived antisera from rabbits and mice, recognised all strains of C. difficile vegetative cells examined, despite an immune response to the linkers also being observed. These sera recognised live cells in an immunofluorescence assay and were also able to recognise the spore form of the bacterium. This study has illustrated that the LTA polymer is a highly conserved surface polymer of C. difficile that is easily accessible to the immune system and as such merits consideration as a vaccine antigen to combat C. difficile infection.     

Construction and characterization of mutated LEA peptides in Escherichia coli to develop an efficient protein expression system

To develop an efficient protein expression system, we designed a late embryogenesis abundant (LEA) peptide by mutating the LEA peptide constructed in our previous study (LEA‐I). The peptide is based on the repeating units of an 11mer motif characteristic of LEA proteins from Polypedilum vanderplanki larvae. In the amino acid sequence of the 13mer LEA peptide, glycine at the 6th and 12th positions was replaced with other amino acids via point mutations. Glutamic acid, lysine, leucine, and asparagine in the LEA peptide at the 6th and 12th positions increased green fluorescence protein (GFP) expression. The GFP expression of the mutated LEA peptide was 1.5 to 2.0 times higher than that without LEA peptide. In contrast, the serine‐containing mutated LEA peptide has low GFP expression levels. We hypothesize that the position of amino acids and the nature of amino acid in LEA peptide are important for our coexpression system. These data suggest that the size, structure, and charge of amino acids in the LEA peptide improve the protection and expression of the target protein. The amino acid balance also plays an important role in the expression of the target protein.     

Electrophoretic analysis of the lipopolysaccharides ofBacteroides spp.

Lipopolysaccharide (LPS) extracts of reference strains and isolates ofBacteroides spp. prepared by the proteinase K method were resolved by tricine-sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis and located by silver staining. A considerable diversity of LPS profiles was evident within theBacteroides genus although profiles were essentially species-specific, with some minor interstrain variations apparent among isolates ofB. uniformis, B. ovatus, B. eggerthii andB. thetaiotaomicron. The LPS of most species consisted of a major rough LPS component of 2–5 kDa and a series of higher molecular weight bands which varied with species.B. vulgatus LPS was distinctive in showing an extensive ladder of multiple repeating oligosaccharide units with molecular weights ranging from 4 to >17 kDaB. stercoris LPS included a high molecular weight (>17 kDa) ladder of repeating oligosaccharide units.B. fragilis andB. thetaiotaomicron differed from most other species in producing a short ladder of repeating oligosaccharide units interspersing the rough LPS and a 5.6 kDa (B. fragilis) or 9 kDa (B. thetaiotaomicron) yellow-staining component. The heterogeneity of LPS profiles within theBacteroides genus may reflect the differences in pathogenicity among the species and prove useful for typing.     

Pipecolic acid pathway: the major lysine metabolic route in the rat brain.

A peptide of 6,700–8,000 daltons has been isolated from an ethanolic KOH digest of bovine ligamentum nuchae elastin by a combination of ion exchange chromatography and gel filtration techniques. The peptide is non-polar and is composed almost entirely of glycine, valine, and proline (or hydroxyproline) in the ratio 2:2:1. Edman degradation of the first 17 residues gave the sequence: NH₂-Gly-Phe-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val. These results suggest that the peptide is made up of repeating pentapeptide units with the sequence: Pro-Gly-Val-Gly-Val.     

Isolation and characterization of <Emphasis Type="Italic">Clostridium difficile</Emphasis> from shellfish and marine environments

This pilot study was carried out to evaluate the occurrence of Clostridium difficile in marine environments and in edible shellfish. Samples of seawater, sediment, and zooplankton were collected at five sampling stations in the Gulf of Naples. Six samples of edible shellfish, furthermore, were obtained: two from mussel farms and four from wholesalers. The isolation and the characterization of C. difficile strains were carried out using selective media and molecular techniques, respectively. C. difficile was isolated from nine of the 21 samples investigated. Shellfish and zooplankton showed the highest prevalence of positive samples. No C. difficile was detected in marine sediment. Majority of the C. difficile isolates were toxin A/B positive. Six known different PCR ribotypes (003, 005, 009, 010, 056, and 066) were identified, whereas one strain may represent a new PCR ribotype. C. difficile may be present in the marine environment in Southern Italy, including shellfish and zooplankton. This study is reporting the isolation of C. difficile from zooplankton, clams, and mussels and pointing out a new possible route to exposure to C. difficile of healthy individuals in the community.     

Two proposed general configurations for bacterial cell wall peptidoglycans shown by space-filling molecular models

Bacterial cell wall peptidoglycans are built from unbranched β-(1 → 4)-linked glycan chains composed of alternately repeating units of N-acetylglucosamine and N-acetylmuramic acid residues, with peptide side chains attached to the muramic acid residues. The glycan chains are interconnected by peptide bonds formed between the peptide side chains. Through the use of three-dimensional molecular models, two configurations of the glycan strands and the peptide side chains are described, which by their constancy of form reflect the fundamental constancies of the covalent structures. Each of these two models will accommodate any chemical modification that has been observed in bacteria without change in the configuration of the peptide backbone. Some alterations in the chemical structure, which have been sought in bacteria, but not found, would not be tolerated by the models. In these models, glycan strands are parallel, with their lengths and widths predominantly in the plane of the cell wall. The cross-bridging portions of the peptide side chains are at right angles to the glycan strand, in a separate, parallel plane. A compact model is presented in which the peptide side chain is closely appressed to the glycan strand and is stabilized by three hydrogen bonds per disaccharide–peptide subunit. In a second model, the peptide side chain is raised away from the glycan strand in an entirely extended configuration. The compact and extended forms are interconvertible. The thickness of a sheet of peptidoglycan would be from 10.6 to 11.1 Å for the compact model, and 19.1 Å for the extended model.     

Isolation of Clostridium difficile from the Feces and the Antibody in Sera of Young and Elderly Adults

Attempts were made to isolate Clostridium difficile from a total of 431 fecal specimens from 149 young and 213 elderly healthy adults, and 69 elderly adults with cerebrovascular disease but no gastrointestinal disease. C. difficile was isolated from 49 specimens, and the frequency of isolation was 15.4% in healthy young adults, 7.0% in healthy elderly adults, and 15.9% in elderly adults with cerebrovascular disease. Thirty-four (about 70%) of the 49 C. difficile strains isolated produced cytotoxin which was neutralized by Clostridium sordellii antitoxin in vitro; in both young and elderly adults approximately 30% of the C. difficile isolates were nontoxigenic. The mean concentration of C. difficile in feces was 10^4.1/g in young adults and 10^4.6/g in elderly adults, with a range of 10^2.0 to 10^6.9/g. Antibody against C. difficile toxin was found in most of the sera obtained from young adults carrying toxigenic C. difficile, but not in sera of elderly adults, no matter how abundant was toxigenic C. difficile in the feces.