Tetsuhiro Moriya

Construction of a ferritin-deficient mutant of Campylobacter jejuni: contribution of ferritin to iron storage and protection against oxidative stress

The ferritin‐encoding gene (cft) of Campylobacter jejuni was cloned and sequenced. The nucleotide sequence of cft had a 501 bp open reading frame for a protein with 167 amino acids and a predicted molecular mass of 19180 Da, and showed a high similarity to that of Helicobacter pylori and Escherichia coli ferritin genes. To determine the biological function of ferritin in C. jejuni, a ferritin‐deficient mutant was constructed. The growth of ferritin‐deficient strain SNA1 was clearly inhibited under iron deprivation. The ferritin‐deficient mutant was more sensitive to killing by H2O2 and paraquat than the isogenic parent strain. These findings demonstrate that ferritin in C. jejuni makes a significant contribution to both iron storage and protection from intracellular iron overload, and resulting iron‐mediated oxidative stress.

Bactericidal Activities of Rat Defensins and Synthetic Rabbit Defensins on Staphylococci, Klebsiella pneumoniae (Chedid, 277, and 8N3), Pseudomonas aeruginosa (Mucoid and Nonmucoid Strains), Salmonella typhimurium (Ra, Rc, Rd, and Re of LPS Mutants) and Escherichia coli

Rat defensins were purified and tested for in vitro bactericidal assay against gram‐positive and gram‐negative bacteria. Staphylococcus aureus (209P, Cowan I, Smith diffuse and Smith compact) were resistant to defensins, whereas Staphylococcus epidermidis, Staphylococcus saprophyticus, Micrococcus lysodeikticus and Bacillus subtilis were less sensitive. Gram‐negative bacteria, such as Pseudomonas aeruginosa (mucoid and K) and Klebsiella pneumoniae (Chedid, 277, and 8N3 which were heavily capsulated, moderately capsulated and noncapsulated, respectively) were all very sensitive to defensins and killed within 20 min. Escherichia coli was moderately sensitive and the rough mutants of lipopolysaccharide (LPS) of Salmonella typhimurium LT2, such as Ra, Rc, Rd, and Re were equally sensitive to defensins, being killed within 40 min. Lysozyme did not show any bactericidal activity except against M. lysodeikticus and B. subtilis, whereas it enhanced the bactericidal activity of defensins against P. aeruginosa, E. coli, and K. pneumoniae and suppressed the killing activity of defensins against S. typhimurium and S. aureus. With regard to the three synthetic rabbit defensins, NP1, NP4, and NP5, NP1 showed strong bactericidal activity against K. pneumoniae 277, comparable to that of rat defensins. Neither NP4 nor NP5 showed any bactericidal activity, while NP5 rather enhanced the bactericidal activity of NP1 against K. pneumoniae 277.

Southern blotting analyses of strains ofCampylobacter fetus using the conserved region ofsapA

Chromosomal DNA of 27 strains ofCampylobacter fetus was analyzed by Southern blotting with a probe of the conserved region ofsapA. The probe hybridized with 23 strains that produced type A lipopolysaccharide. These strains had more than sixsapA homologs. In Southern blots ofSalI-digested chromosomal DNA separated by pulsed-field gel electrophoresis, one fragment from 19 strains and two fragments from 4 strains hybridized. These data indicate that multiplesapA homologs are localized to a limited region on the chromosomal DNA ofC. fetus and thus suggest the possibility of developing a typing system using this method.

Variability of Haemolysin(s) Produced by Vibrio vulnficus

The peptide composition and antigenic cross-reactivity of partially purified and concentrated haemolysins of 16 strains of Vibrio vulnificus were examined by SDS-PAGE and immunoblotting analysis, using a monoclonal antibody (MAb), 6F8D, raised against the haemolysin. All strains produced a common peptide of 36 kDa and the MAb reacted with this peptide. In some strains, larger molecules, including a 56 kDa peptide, were produced, but the MAb did not react with this peptide. The haemolytic activity of the strains was effectively neutralized by the MAb, except in the case of strains producing the 56 kDa peptide. These findings indicate that the 36 kDa haemolysin is common to all 16 strains and that V. vulnificus can produce a second haemolysin which differs in molecular mass and antigenicity.

A deletion in the sapA homologue cluster is responsible for the loss of the S-layer in Campylobacter fetus strain TK

Abstract The surface array protein (SAP) of Campylobacter fetus strain TK is encoded by seven homologous sapA genes clustered on the chromosomal DNA. The spontaneously arising variant strain TK(SAP–) produces no SAP and carries an approximately 10-kb chromosomal deletion. To elucidate the mechanism underlying the loss of SAP synthesis, we analyzed the region containing the sapA homologues and the deletion. We constructed a physical map of the sapA cluster region by aligning the clones that contain sapA homologues. These analyses demonstrated that all sapA homologues were located within a limited region of about 50 kb of chromosomal DNA of strain TK. The TK(SAP–) deletion was located within this cluster and was 13.3 kb in size. The deletion occurred between two sapA homologues and resulted in the formation of a chimeric sapA homologue in the variant strain. Sequence analysis of the upstream regions and the conserved regions of all sapA homologues revealed a high degree of similarity. However, only one sapA homologue contained a putative promoter sequence. This promoter sequence was located in the deleted region. Thus, the deletion of the promoter appears to be responsible for the loss of SAP expression in TK(SAP–).

Studies on Escherichia coli chromosome proteins I. Analysis of the proteins by two-dimensional gel electrophoresis

As an approach for studying the function of chromosome proteins in DNA replication and gene expression, proteins remaining attached to Escherichia coli nucleoids were analyzed by two-dimensional polyacrylamide slab gel electrophoresis. Nucleoids were isolated by gentle lysis of the cells in the presence of a DNA counter-ion such as 1 M NaCl or 5 mM spermidine. In exponentially growing cells, about 100 proteins have been found to exist in the nucleoids. Kinetic studies indicated that the number of chromosome proteins remaining attached varied with time after synchronization. Based on the pattern of the variation, appearance, increase or disappearance of the 29 major proteins, nucleoid proteins were shown to be classified in six different groups (groups A--F). A strong correlation was observed between the variation of proteins belonging to group D and initiation of DNA synthesis or cell division.

Purification and Characterization of a Protein Cryoprotective for Vibrio cholerae Extracted from the Prawn Shell Surface

A substance cryoprotective for Vibrio cholerae on the prawn shell surface was purified by ammonium sulfate precipitation and gel filtration. It was a protein of 81 kDa and called cryoprotective protein (CPP). The cryoprotective activity of this protein for V. cholerae was sensitive to heat at 100 C and trypsin treatment. In the presence of Mg ion the protein can bind to the bacterial cell surface. V. cholerae can adhere to the shell surface of the prawn. The number of adhered bacteria was reduced by treating the shell with anti‐CPP serum, heat or by trypsin. The presence of Mg ion promoted the adherence. These results suggest that the CPP could serve as an adherence site for V. cholerae on the shell surface.

Structure of Mycoplasma salivarium Examined by the Freeze Substitution Method

The internal structures of Mycoplasma examined by the thin section technique mainly resemble the structure of bacteria (4-8, 11, 12). The cytoplasm is filled with many ribosomes and the nucleoid is seen as a less dense area made up of bundles of fibers. No intracellular organella such as the mitochondria, the endoplasmic reticulum or Golgi apparatus are found. The external shape, size and intracellular density of each cell vary greatly in individual cells. These morphological results have been obtained on the thin-sectioned profiles of chemically fixed cells. Recently the fixation technique for thin sectioning has been greatly improved. As a mild and rapid fixation method, the rapid freezing and substitution method is now being used in studies on the fine structure of bacterial cells. The structures of the cell walls (1, 9), nucleoid (3, 10), and capsules (2) obtained by this technique showed quite different features and seemed to represent more natural states of the bacterial structures than those obtained by conventional methods. In this communication we applied this technique to the examination of the structure of Mycoplasma and described some new structural profiles of this organism. Mycoplasma salivarium strain Hup 127 was obtained from Dr. L. Hayflick, Childrens Hospital Medical Center, Bruce Lyon Memorial Research Laboratory, Oakland, Calf., U.S.A. and cultured anaerobically on mycoplasma agar in a Gas-Pak system (BBL Microbiology Systems, Cockeysville, Md., U.S.A.) at 37 C for 4 days. The mycoplasma agar consisted of 3.4% PPLO agar (Difco Laboratories, Detroit, Mich., U.S.A.) supplemented with 0.25% (w/v) freshly prepared yeast extract, 20% (w/v) heat inactivated horse serum, 1,000 units penicillin and 0.025% thallium acetate. The method of rapid freezing and substitution fixation was the same as described in a previous paper (1). Briefly, the colonies developed on an agar plate were cut out together with surrounding agar medium in the form of 5-7 mm squares. The agar square was then applied to the plunger of a rapid freezing device (Type RF-1, Eiko Co., Ltd., Tokyo) and frozen by pressing it on the surface of a metal block standing in liquid nitrogen. The substitution fixation was performed in 4% OsO4 in acetone in a mixture of dry-ice and acetone for 20 hr. Thin sections were cut with a diamond knife equipped on Porter-Blum ultramicrotome MT-2B and stained with uranyl acetate and lead citrate.

Antigenic Determinants on Fimbriae of Serratia marcescens US5 Analyzed Using Monoclonal Antibodies

The antigenic sites on small thin fimbriae of Serratia marcescens strain US5 were investigated using immunoelectron microscopy and monoclonal antibodies (MAbs). Negative staining of the fimbriae after treatment with MAbs showed a regularly spaced arrangement of the antibody molecules. When the subunit peptide was subjected to immunoblotting using the MAbs, a single band with a molecular weight of approximately 19kD was evident. This binding of the MAbs to the subunit peptide was completely abrogated after treatment with 2‐mercaptoethanol, thereby suggesting the important role of disulfide linkage in the maintenance of the conformation of the antigenic site reacted with MAbs. Amino acid analysis of the subunit peptide revealed two cysteine residues, and cysteine residues were absent in the N‐terminal portion.