We have characterized three copy number mutants of the plasmid pSC101. These mutations caused single amino acid substitutions at the 46th, 83rd and 115th codons in the rep gene and an increase in the copy number by 4- to 8-fold. Although the in vivo and in vitro repressor activities of these mutated Rep proteins were quite different from each other, the intracellular concentrations of the proteins were maintained at higher levels than the wild-type protein. It has been reported that excess amounts of Rep inhibit pSC101 replication (Ingmer and Cohen, 1993). This inhibitory activity of Rep was markedly decreased in all three mutants. When both the wild-type and one of the mutated rep genes were retained in the same plasmids, the copy number of these plasmids was decreased compared with plasmids retaining a single mutated rep gene. These results support the theory that the inhibitory activity of Rep for its own replication plays an important role in copy number regulation.
There have been many reports of primary Vibrio vulnificus septicemia in Korea since 1987. This study was undertaken to determine the cellular fatty acid (CFA) compositions of 95 clinical strains of V. vulnificus isolated in Korea during 1985-1995. We compared these results with the CFA profile of V. vulnificus in the Microbial Identification System (MIS) (CLIN library version 3.9; Microbial ID Inc., Newark, DE, U.S.A.), and the grouping of V. vulnificus by CFA analysis was also performed. The relationship between groups and serotypes of V. vulnificus was described. Although most of the CFAs in V. vulnificus strains were similar to the CFA profile of V. vulnificus in the MIS, some distinctive differences were observed between the results of this study and the MIS CFA profile of V. vulnificus. First, the means of 2 major CFAs, 16:0 and 16:1w7c, were 22.16 and 18.26% in this study but 23.52 and 25.44% in the MIS, respectively. Second, all isolates had 11:Oiso3OH, which was not present in the MIS. Of 95 strains, 10 strains (10.5%) showed ‘NO MATCH’ results by the MIS identification. Eighty five strains (89.5%) were identified as V. vulnificus by the MIS (the first choice identification), but they disclosed low SI values of <0.6 (not ‘EXCELLENT MATCH’) except 2 strains (2.1%). This showed that V. vulnificus strains isolated in Korea had different characteristics in CFA composition in comparison with the MIS V. vulnificus library. Nine groups comprising all the strains were obtained by cluster analysis and were further characterized by principal- component analysis. There was heterogeneity between the groups by CFA and serotypes of V. vulnificus. The same serovars were distributed into diverse groups.
Lactate dehydrogenase (LDH) was purified from three strains of Streptococcus bovis, and the gene Idh was cloned and sequenced. The Idh of S. bovis from a goat (TH1) was different from the Idhs of two other strains from cattle (TH2, JB1) in that Asp220 was substituted for Glu. Northern blot analysis revealed that the LDH-mRNA of S. bovis was approximately 1.0kbp, which was transcribed in amonocistronic fashion. When cells were grown at pH 6.9 in a batch culture, the level of Idh transcript decreased as the growth phase changed; from exponential growth to the cessation of growth. The level of Idh transcript was higher in cells grown at pH 4.5 than at pH 6.9. This observation was consistent with the amounts of LDH in cells and the percentages of lactate produced. These results support the hypothesis that S. bovis regulates LDH synthesis at the transcriptional level probably in response to intracellular pH.
Two different newly designed primer sets were used in PCR to amplify the fragments of chitin synthase (CHS) encoding genes (CHS) from the zygomycete fungus Phycomyces blakesleeanus, in which the cell wall is mainly made of chitin and chitosan, a deacetylated derivative from chitin. DNA sequencing and alignment analysis of the deduced amino acid sequences showed the possible existence of ten different genes. Six different DNA fragments, designated PbCHS1, PbCHS2, PbCHS3, PbCHS4, PbCHS5 and PbCHS6, homologous to chitin synthase genes were identified in 250-bp products. From 350-bp products, four different fragments, PbCHS7, PbCHS8, PbCHS9 and PbCHS10, were obtained. Clustal analysis suggested that while this fungus may not have class III- CHS, class I-, II- and IV-CHS could be present in multiple forms. Only PbCHS1, PbCHS2, PbCHS7 and PbCHS8 genes were actively expressed in the young germlings cultured in the liquid medium. Transcriptional products from PbCHS8 and PbCHS10, 8.2-kb long poly(A)+RNA, might suggest the existence of an unidentified type of CHS or the possibility of a multifunctional gene including CHS function.
Chitinase from a high producing strain (TP-1) of Bacillus licheniformis was used with B. thuringiensis subsp. aizawai (B.t.a.) in a combined larvicidal assay against the pest, Spodoptera exigua. With 10mU of this chitinase, the LD50 of B.t.a. was reduced by 7.6, 13.8 and 15 times on days 3, 5 and 7, respectively when compared to use of B.t.a. alone. In addition, a combination of chitinase (10mU) and B.t.a. at a sub-lethal dose retarded growth and development of S. exigua. In preparation for transformation of B.t.a., the TP-1 chitinase gene was cloned in E. coli DH5α and sequenced to reveal a single open reading frame of 1, 815bp. This open reading frame encoded for a protein of 604 amino acids and a characteristic signal peptide sequence of 35 amino acids. The gene was subsequently introduced into B.t.a. where it was expressed constitutively. The transformed strain showed slightly improved activity against S. exigua when compared to the non-transformed strain. This was probably due to the low chitinase activity (15mU/ml) of the transformant, which might be improved by further gene manipulation to overexpress enzyme production.
Five strains of Gram-negative, oxidase-negative, facultatively anaerobic, fermentative, motile, rodshaped bacterium with the general characteristics of the family Enterobacteriaceae were isolated from the gut of multiple specimens of the pea aphid. All the strains caused aphid mortality when ingested by insects via a synthetic diet. The results of biochemical tests showed that these strains are most related to Erwinia herbicola and Pantoea agglomerans. According to DNA-DNA hybridization, the five strains showed more than 96% relatedness to each other, indicating that these organisms are members of a single species. These strains were most closely related to Erwinia herbicola (22% DNA relatedness). Phenotypic differentiation of these strains from Erwinia herbicola, which was also detected from aphid gut, was based on negative reactions in tests of yellow pigment production, gelatin liquefaction, acid production from inulin, starch and dulcitol, and positive acid production from melibiose, inositol, cellobiose and glycerol. On the basis of these data, the name Erwinia aphidicola is proposed for the new organism. The type strain is strain X 001 (=IAM 14479).
In an attempt to define the phylogenetical relationship among 17 phenotypically related species of genera Enterobacter, Pantoea, Serratia, Klebsiella and Erwinia, we determined almost all of their groE operon sequences using the polymerase chain reaction direct sequencing method. The number of nucleotide substitutions per site was 0.12±0.030. The value was 3.6-fold higher than that of 16S rDNA. As a result, we were successful in constructing molecular phylogenetic trees which had a finer resolution than that based on the 16S rDNA sequences. The phylogenetic trees based on the nucleotide sequences and deduced amino acid sequences of groE operons indicated that the members of genera Enterobacter, Pantoea and Klebsiella were closely related to each other, while Serratia and Erwinia species except Erwinia carotovora, made distinct clades. The close relationship between Enterobacter aerogenes and Klebsiella pneumoniae, that had been suggested by biochemical tests and DNA hybridization, was also supported by our molecular phylogenetic trees.
Pea aphids were reared aseptically in the laboratory and fed on strains of Erwinia aphidicola, Erwiniaherbicola, Klebsiella pneumoniae, Escherichia coli and bacterium W, an aphid enterobacterium, by mixing with a synthetic diet to determine whether these bacteria have pathogenicity to the insect. It turned out that Er. Aphidicola and Er. Herbicola grew in aphid gut, while K. pneumoniae, Es. Coli and bacterium W were effectively eliminated. In the case of Er. aphidicola, ingestion of a single dose of 103CFU/ml was enough to infect aphid gut and to prevent post-final ecdysis growth of the insect. As a result, the body weight of the infected aphids was significantly reduced (p<0.01). A concentration of 105CFU/ml of Er. aphidicola caused aphid mortality. In contrast, Er. herbicola demanded 105CFU/ml to colonize in aphid gut, and seemed to have no effect on insect health.