Biomedical subjects
Publications and source records attributed to Y Ike.
Streptococcus faecalis strains harboring the conjugative plasmid pAD1 excrete a small peptide, iAD1, which inhibits the sex pheromone cAD1. Studies making use of the host strain Streptococcus faecium 9790, which normally does not excrete peptide pheromones, suggest that iAD1 may be determined directly by pAD1.
Hemolysin production, clumping (pheromone) response, transferability of the hemolytic trait, and drug resistance were examined in 97 clinical isolates of Enterococcus (Streptococcus) faecalis. The isolates were derived from various sources (i.e., urine, pus, vagina, sputum, bile, and blood), and approximately 60% were found to be hemolytic. About 85% of the hemolytic strains exhibited a clumping response, compared with about 49% of the nonhemolytic strains. Over 50% of the hemolytic strains carried transferable hemolysin determinants, and in no case were drug resistance genes linked. The hemolytic strains exhibited multiple drug resistance more frequently than did the nonhemolytic strains. In contrast to the high frequency of hemolysin producers among parenteral isolates, strains derived from fecal specimens of healthy individuals exhibited a low (17%) incidence of hemolysin production.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
The conjugative plasmid pAD1 (56.7 kilobases) in Streptococcus faecalis confers hemolysin-bacteriocin (Hly-Bcn) expression and a mating response to the sex pheromone cAD1 excreted by recipient cells. We examined the contribution of hemolysin to pathogenicity in intraperitoneally infected mice by using Tn916 and Tn917 insertion mutants altered in hemolysin expression. Strains exhibiting the normal hemolysin phenotype were significantly more virulent than the nonhemolytic insertion mutants. A mutant plasmid with an increased copy number which gave rise to a larger-than-normal zone of hemolysis on blood agar rendered host strains more virulent than the wild-type streptococci in mice.
The conjugative plasmid pAD1 (56.7 kilobases) in Streptococcus faecalis has been shown to confer a mating response to the sex pheromone cAD1 excreted by recipient strains. The response is characterized by the synthesis of a proteinaceous adhesin which coats the surface of the pAD1 -containing donor cell and facilitates the formation of mating aggregates. Donors exposed to cAD1 -containing filtrates of recipients undergo self-aggregation (clumping), an event believed to be associated with an interaction between the adhesin and a binding substance always present on the surface of both recipients and donors. To analyze the molecular processes involved in the mating response, mutants were generated by the erythromycin resistance transposon Tn917 . Transpositions to pAD1 in S. faecalis DS16 gave rise to a number of derivatives that exhibited "constitutive clumping" and the ability to transfer at high frequencies in short (10-min) matings. These mutants fell into two subclasses, which exhibited colony morphologies that were "dry" or "normal". The Tn917 insertions were mapped by restriction enzyme analysis to two separate clusters, designated traA and traB. The dry colony subclass corresponded to traA and represented a span of 1.5 kilobases, whereas the normal subclass corresponded to traB and spanned 1.3 kilobases. The two clusters were separated by 1.7 kilobases in which insertions of Tn917 did not affect the ability to respond normally to cAD1 . Neither type of constitutive clumper produced cAD1 . Another series of insertions exhibited reduced donor potential. In two cases, the reduction in transfer was three to four orders of magnitude; these mapped in traA . In two other cases, the reduction was one to two orders of magnitude. These mapped outside of traA and traB, and one was associated with an increase in plasmid copy number.
A solid phase method for the simultaneous synthesis of mixed oligonucleotides using a phosphotriester approach has been developed. For this synthesis, a mixture of mono or dimeric coupling units is used, and a slight difference in the reactivity of those units is found. However, this difference does not hamper the simultaneous, mixed oligonucleotide synthesis, and the sequence analysis of a product demonstrates the existence of all desired sequences in the final mixture.
Protein S, a development-specific protein of Myxococcus xanthus is produced only during fruiting body formation. More than 15% of total protein synthesis during this period is accounted for by the production of protein S. The genes for protein S were identified and cloned with the use of mixed probes consisting of eight synthetic oligodeoxyribonucleotides (tetradecamers) which correspond to a carboxyl-terminal portion of protein S. The two genes are oriented in the same direction and are separated approximately 1.2 kilobases. The DNA sequences of the carboxyl-terminal portions of the two genes reveal that both can code for the identical eleven amino acid sequence which corresponds to the carboxyl-terminal end of protein S. However, there are a few base substitutions upstream of these regions. This duplication of genes in M. xanthus may facilitate the extremely rapid synthesis of protein S during fruiting body formation.
Recipient strains of Streptococcus faecalis excrete multiple, peptide sex pheromones that induce mating responses in donors harboring certain conjugative plasmids. Acquisition of plasmid DNA leads to a "shutting off" of pheromone excretion, and such cells become responsive to exogenous pheromone. Data are presented showing that donors excrete low levels of a modified, inactive form of the pheromone. This substance, when mixed in excess with active pheromone, inhibits pheromone activity (probably by competition for a receptor site on the donor). Modified forms of both cPD1 and cAD1 were revealed, and each appeared to have a mass about 350-400 daltons larger than the active pheromone. In both cases, pheromone activity could be regenerated by treatment with phosphodiesterase II.
The recent development of the amino acid microsequence technique allows us to obtain partial sequence information using an extremely small amount of protein. Two sets of mixed oligonucleotide probes were chemically synthesized using the amino acid sequence information for the heavy chain of human HLA-DR antigen obtained by the microsequence technique. These two hybridization probes were used to screen cDNA clones constructed from cytoplasmic poly(A)+ mRNA from a human B lymphoblastoid homozygous cell line (LG-2). Of the 10,000 clones screened, two clones hybridized with the probes. DNA sequence analysis showed that the longer one of the two cDNA clones was 1183 nucleotides long, including the entire coding region, the signal peptide region, and the complete 3'-noncoding region. The deduced amino acid sequence of the HLA-DR alpha chain is identical to that of other cell lines with a different HLA-DR typing. However, several nucleotide differences are found in the 3'-untranslated region compared with that of other DR haplotypes.
A simple solid phase method for the synthesis of oligodeoxyribonucleotides has been developed using the phosphotriester approach. Mononucleotide coupling units are sequentially added to the polystyrene copolymer with 1% divinylbenzene and two kinds of oligonucleotides, d(CACGACCCCTCCACGT) and d(AACTGGTATTACTGGGCG), are synthesized in a relatively high yield. One cycle of the mononucleotide addition is about 70 minutes, and this method is particularly suitable for the automation of the synthesis upon availability of an automatic synthesizer.
Based on the published amino-acid sequence of H-2Kb, we synthesized a mixture of eight 16-base long oligodeoxyribonucleotides representing all possible coding sequences for residues 51-56 (Trp-Met-Glu-Gln-Glu-Gly). The hexadecanucleotide mixture was used as a probe to screen recombinant DNA clones constructed from cytoplasmic PolyA+ RNA isolated from the murine thymoma cell line EL4(b haplotype). Of the 30 000 independent clones screened, one clone was found to hybridize with the probe. DNA sequence analysis showed that the cDNA clone was derived from a portion of an H-2Kb -related mRNA. The clone encodes a protein sequence identical with a region of H-2Kb in 42 consecutive residues (50 through 91). The sequence than diverges from the H-2Kb sequence and, after a single Glu codon, a termination codon is encountered. It is possible that this mRNA codes for a small 92 amino-acid protein with a sequence identical (except for a carboxy-terminal Glu residue) with the amino terminus of H-2Kb. It is further speculated that this mRNA is coded for by the H-2Kb gene and differs from the H-2Kb mRNA in the pattern of posttranscriptional splicing.
We isolated 1,120 drug-resistant Escherichia coli strains from pigs, slaughterers, breeders of pigs, and urban residents and examined their drug resistance. With reference to resistance to tetracycline (Tc) chloramphenicol (Cm), streptomycin (Sm), and sulfanilamide (Su), Tcr, Smr, and Sur strains were most frequently isolated from pigs (57.9%), followed by slaughterers (50.2%) and breeders (42.4%). By contrast, the isolation frequencies of Tcr, Smr, and Sur strains from urban residents were low (12.0%). The isolation frequencies of R plasmids from resistant strains were more than 50% in the case of pigs, slaughterers, and breeders, whereas they were only 13.8% in urban residents. Kanamycin-resistant strains were frequently isolated from pigs (40.3%), slaughterers (22.6%), and breeders of pigs (25.4%), but seldom (only 3.4%) from urban residents. With ampicillin resistance, the frequencies of strains from pigs, slaughterers, breeders, and urban residents were 30.0, 21.5, 5.6, and 13.8%, respectively. R plasmids specifying these latter resistances were frequently seen from pigs (24.0%), slaughterers (23.6%), and breeders (21.1%), but they were not isolated from urban residents. The results suggest that the porcine gut can function as a reservoir of antibiotic resistance plasmids which can then be directly transferred to humans, with the highest frequency of such plasmids appearing in people in the most immediate contact with these animals.
Eight gentamicin-resistant Escherichia coli were identified from among 630 E. coli strans isolated from parenteral infections. All eight strains were multiply resistant and harbored R plasmids mediating gentamicin resistance. The R plasmids specified the formation of two types of aminoglycoside-inactivating enzymes AAD (2") and APH (3').
Escherichia coli harboring mutant plasmids defective in maintenance stability (from the conjugative plasmid Rms201) showed a wide distribution of ampicillin resistance levels, as well as increased frequency of plasmid loss from the cell. The amounts of covalently closed circular deoxyribonucleic acid of mutant plasmid Rms268 and parental plasmid Rms201 per chromosome were 5.3 and 6.1%, respectively. The beta-lactamase activities of strains W3630(Rms268) and W3630(Rms201) were 0.56 and 0.44 U/mg of protein, respectively. Frequency of plasmid loss from W3630(Rms268) was about 0.8 to 1.2% per cell generation, 100 times more than that of the wild-type strain. Ampicillin resistance levels of the colonies harboring the mutant plasmid showed a wide distribution, from low (100 micrograms/ml) to high (1,600 micrograms/ml). A miniplasmid (pMS268) with a mass of 7 X 10(6) daltons and encoding ampicillin resistance was isolated from Rms268. Frequency of pMS268 loss from W3630(pMS268) was about 0.8 to 1.9% per cell generation. W3630(pMS268) also showed a wide range of distribution in the levels of ampicillin resistance. These results indicated that the copies of Rms268 in E. coli did not segregate evenly between daughter cells at cell division and that the gene involved was located on the miniplasmid.
Copy number mutants Rms201ts15 and Rms201ts16 were isolated at 30 degrees C from a temperature-sensitive replication mutant (Rms201ts14) of the conjugative plasmid Rms201. The numbers of plasmids per chromosome of ML1410(Rms201ts14), ML1410(Rms201ts15), and ML1410(Rms201ts16) grown at 30 degrees C were 2.2, 7.4, and 20, respectively. The synthesis of covalently closed circular plasmid deoxyribonucleic acid stopped in Rms201ts14, Rms201ts15, and Rms201ts16 immediately after a "shift-up" in temperature (42 degrees C). At 42 degrees C, antibiotic-sensitive derivatives appeared after a certain lag time: the lag times of ML1410(Rms201ts14), ML1410(Rms201ts15), and ML1410(Rms201ts16) were 2.5, 5, and 6.8 generations, respectively. After these times, plasmid-positive cells in the populations decreased at a rate of about 50% per generation in all of the mutants. From these results we conclude that plasmid segregation (partition) of Rms201 occurs by regular segregation (partition).
A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37 degrees C. When Escherichia coli ML1410(Rms201ts14)(+) was grown at temperatures between 40 and 42 degrees C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)(+) in L-broth was shifted to 42 from 30 degrees C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42 degrees C. At 30 degrees C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42 degrees C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42 degrees C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42 degrees C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 x 10(6) and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 x 10(6). These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.
Explore the source record for details and available documents.