Cellular Lipid and Fatty Acid Compositions of Burkholderia pseudomallei Strains Isolated from Human and Environment in Viet Nam

Viet nam is known as an endemic area of melioidosis but its etiologic agent originated in Viet nam was not extensively studied. For the first time, we analyzed the cellular lipid and fatty acid compositions of 15 Vietnamese isolates of Burkholderia pseudomallei, 10 from humans and 5 from the environment. Cellular lipid compositions were analyzed by two-dimensional thin-layer chromatography on silica gel G plates. Cellular fatty acid methyl esters were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The major lipids in all the isolates were phosphatidylglycerol (PG), two forms of phosphatidylethanolamine (PE-1 and PE-2), and two forms of ornithine-containing lipid (OL-1 and OL-2). PE-1 contained non-hydroxy fatty acids at both sn-1 and ?2 positions, while PE-2 possessed 2-hydroxy fatty acids and non-hydroxy fatty acids in a ratio of 1: 1. Since snake venom phospholipase A₂ digestion of PE-2 liberated 2-hydroxy fatty acids, it was confirmed that these acids are at the sn-2 position of glycerol moiety. In both OL-1 and OL-2, amide-linked fatty acid was 3-hydroxy palmitic acid (3-OH-C16: 0), while ester-linked fatty acids were non-hydroxy acids in OL-1 and 2-hydroxy acids in OL-2. The total cellular fatty acid compositions of the test strains were characterized by the presence of 2-hydroxy palmitic (2-OH-C16: 0), 2-hydroxy hexadecenoic (2-OH-C16: 1), 2-hydroxy octadecenoic (2-OH-C18: 1), 2-hydroxy methylene octadecanoic (2-OH-C19CPA), 3-hydroxy myristic (3-OH-C14: 0) and 3-hydroxy palmitic (3-OH-C16: 0) acids. There were significant differences in the concentration of hexadecenoic (C16: 1), methylene hexadecanoic (C17CPA), octadecenoic (C18: 1) and methylene octadecanoic (C19CPA) acids among the Vietnamese isolates of B. pseudomallei. However, no significant difference was observed in cellular lipid and fatty acid components between strains of human and environmental origins.     

Enzyme-linked immunosorbent assay (ELISA) using a glycolipid antigen for the serodiagnosis of melioidosis

Abstract The serodiagnosis of melioidosis is commonly performed with tests using protein or polysaccharide as antigen. However, due to the low sensitivity, specificity and difficulty in the preparation of the antigens, more simple, precise and reproducible diagnostic tests were required. A purified glycolipid antigen (GL) which is a specific lipid component of Burkholderia pseudomallei has been used in an ELISA. With this antigen, specific immunoglobulin G (IgG) was detected in 49 out of 50 melioidosis sera. IgG was also detected in 2 out of 185 (Japanese) and 16 out of 181 (Vietnamese) control sera. Thus, the sensitivity was 98.0%, and specificity was 98.9% and 91.1% in the Japanese and Vietnamese sera, respectively. When the ELISA and indirect haemagglutination (IHA) tests were combined, a sensitivity of 100% and specificity of 97.8% were achieved. The advantages of the glycolipid antigen are ease of preparation, stability, high sensitivity and specificity.     

Effects of Calcium and Potassium Ions on Phototaxis in Cryptomonas

Effects on positive phototaxis and the cell motility of 7 cationsin 5mM MOPS (morpholinopropane sulfonic acid) buffer (pH 7.0)containing 0.16 mM NaCl, 0.68 mM KCl, 0.5 mM CaCl₂ and 0.16mM MgCl₂ were studied in the unicellular flagellate Cryptomonaswith a photoelectrical measuring apparatus and photomicrography.When calcium ion was removed from the medium by adding 1 mMEGTA (ethylene glycol-bis-(ß-amino-ethylether)-N,N'-tetraaceticacid), the phototactic response was totally inhibited, but theswimming rate was not much affected. The effect of EGTA waspartially reversed by the addition of 1 mM CaCl₂. When 15mMKCl or RbCl was added to the medium, phototaxis was greatlyinhibited, but there was no significant influence on the swimmingrate. Similar but less inhibitory effects were induced in thepresence of NaCl, LiCl and CsCl. KCl-induced inhibition waspartially removed by the addition of 15 mM CaCl₂ or MgCl₂. (Received June 25, 1982; Accepted September 27, 1982)     

Unidirectional replication in Escherichia coli of three small plasmids derived from R factor R12

Replicating molecules of three small plasmids, pSM1, pSM2, and pSM3, were isolated from a CsCl density gradient containing ethidium bromide. These plasmids are all derived from R12, a mutant of NR1 (same as R100). By means of pulse-labeling experiments, the replicating forms were located at buoyant densities intermediate between those of the closed circular and open circular DNA bands. These molecules were analyzed by electron microscopy following digestion with restriction endonucleases. Digestion of pSM2 with EcoR1 and with HindIII revealed the presence of a single origin of replication located 1.72 kilobases (kb) from the EcoR1 cutting site (2.04 kb from the HindIII cutting site). These experiments also demonstrated that replication occurs in a unidirectional mode from the origin. Analysis of EcoR1-cleaved replicating molecules of pSM1 and pSM3, which carry common sequences completely or partly homologous to pSM2, provides further evidence for the unidirectional replication of these plasmids from a common origin. The site of the origin of replication was fixed at 85.5 on the kilobase map of R100. This origin, which is located in the RTF region, probably corresponds to one of the replication origins of R100.     

Characterization of a plant SINE, p-SINE1, in rice genomes.

We have previously found that a short interspersed element (SINE), named p-SINE1, is present in the Waxy gene of Oryza sativa in two copies. Here, we cloned five members of p-SINE1 located at other loci in O. sativa and determined their nucleotide sequences. These sequences had a T-rich pyrimidine tract at their defined 3' end and were flanked by direct repeats of a sequence of mostly 14-15 bp long like p-SINE1s in the Waxy gene. The consensus sequence derived from total seven members of p-SINE1 was 123 bp in length and had an internal promoter region for RNA polymerase III. The 5'-half region of the sequence was partially homologous to the tRNA-related block of rabbit C family, one of SINEs in the animal system. Two of the seven p-SINE1 members were not present in the corresponding loci in African rice, Oryza glaberrima, and may thus be available for classification of some rice strains. Comparison of the nucleotide sequences of the Waxy gene between O. sativa and O. glaberrima showed that base substitutions have frequently occurred in a p-SINE1 member (p-SINE1-r1) and a transposable element Tnr1 also present in the Waxy gene, suggesting that these elements, which appear as repetitive sequences in the rice chromosome, tend to acquire base substitutions at a higher frequency than do unique sequences.     

The stable maintenance system pem of plasmid R100: degradation of PemI protein may allow PemK protein to inhibit cell growth.

We constructed plasmids carrying heat-inducible pemI and pemK genes, which were fused with the collagen-lacZ sequence in frame. The PemK-collagen-LacZ (PemK*) protein produced from the fusion gene upon heat induction inhibited the growth of cells and killed most of the cells in the absence of the PemI protein but did not do so in the presence of the PemI protein. This supports our previous assumption that the PemK protein inhibits cell division, leading to cell death, whereas the PemI protein suppresses the function of the PemK protein. We also constructed the plasmid carrying the heat-inducible pem operon which consists of the intact pemI gene and the pemK gene fused with collagen-lacZ. The simultaneously induced PemI and PemK* proteins did not inhibit the growth of cells. However, the temperature shift to 30 degrees C after induction of both proteins at 42 degrees C caused inhibition of cell growth and death of most cells. This suggests that the PemI protein is somehow inactivated upon the arrest of de novo synthesis of the PemI and PemK* proteins, allowing the PemK* protein to function. We observed that the PemI-collagen-LacZ (PemI*) protein was degraded faster than the PemK* protein, perhaps by the action of a protease(s). In fact, the lon mutation, which caused no apparent degradation of the PemI* protein, did not allow the PemK* protein to function, supporting the suggestion described above. Instability of the PemI protein would explain why the cells which have lost the pem+ plasmid are preferentially killed.     

The amino acid sequence of a 20 kDa bifunctional subtilisin/alpha-amylase inhibitor from bran [correction of brain] of rice (Oryza sativa L.) seeds.

A 20 kDa bifunctional inhibitor of the microbial proteinase, subtilisin, and the alpha-amylase from the larvae of the red flour beetle (Tribolium castaneum) was purified from bran of rice seeds by saline extraction, precipitation with ammonium sulphate, ion-exchange chromatography on DEAE-Cellulose and Toyopearl CM-650, and preparative HPLC on Vydac C18. The complete primary structure was determined by automatic degradation of the intact, reduced and S-alkylated protein, and by manual DABITC/PITC micro-sequencing of peptides obtained from the protein following separate enzymic digestions with trypsin, pepsin, chymotrypsin, elastase and the protease from S. aureus V8. The protein sequence, which contained 176 residues, showed strong homology with similar bifunctional inhibitors previously isolated from wheat and barley which are related to the Kunitz family of proteinase inhibitors from legume seeds.     

A simple method for shortening a plasmid genome using a system of plasmid cointegration mediated by a Tn3 mutant

This paper describes a simple method for the isolation of small plasmids of various sizes from pSMI, a derivative of the resistance plasmid R 100. The method is based on the observation that a repressor-negative mutant of the ampicillin-resistance (amp^r) transposon Tn3, Tn3 No. 5, mediates cointegration of a plasmid carrying Tn 3 No. 5 (pMB8::Tn 3 No. 5) into virtually any site on pSMI. The resulting cointegrate plasmids contain the pSMI sequence which is joined with the amp^r gene of the Tn 3 mutant. This cointegration is so frequent that large cointegrate plasmids can be readily detected in the total plasmid DNA prepared from cells carrying pSMI and pMB8::Tn3 No. 5. We were able to isolate small plasmids of various sizes by digesting the total plasmid DNAs with restriction endonucleases which cut both pSM 1 and Tn3 No. 5 sequences present in the cointegrates and subsequently ligating the restriction fragment containing both the amp^r gene and the region necessary for replication of pSMI. Analysis of these plasmids, named pBV plasmids, with restriction endonucleases and by nucleotide sequencing allowed us to determine regions necessary or unnecessary for replication, thus defining a minimal replication region of pSMI. The present method is generally useful for the isolation of small derivatives from any large plasmid for the study of genes and sites adjacent to or within the minimal replication region of the plasmid.