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PubMed2002

Post-termination complex disassembly by ribosome recycling factor, a functional tRNA mimic.

Ribosome recycling factor (RRF) together with elongation factor G (EF-G) disassembles the post- termination ribosomal complex. Inhibitors of translocation, thiostrepton, viomycin and aminoglycosides, inhibited the release of tRNA and mRNA from the post-termination complex. In contrast, fusidic acid and a GTP analog that fix EF-G to the ribosome, allowing one round of tRNA translocation, inhibited mRNA but not tRNA release from the complex. The release of tRNA is a prerequisite for mRNA release but partially takes place with EF-G alone. The data are consistent with the notion that RRF binds to the A-site and is translocated to the P-site, releasing deacylated tRNA from the P- and E-sites. The final step, the release of mRNA, is accompanied by the release of RRF and EF-G from the ribosome. With the model post-termination complex, 70S ribosomes were released from the post-termination complex by the RRF reaction and were then dissociated into subunits by IF3.

Escherichia coli↗
PubMed1987

The aacA-aphD gentamicin and kanamycin resistance determinant of Tn4001 from Staphylococcus aureus: expression and nucleotide sequence analysis.

The aacA-aphD aminoglycoside resistance determinant of the Staphylococcus aureus transposon Tn4001, which specifies resistance to gentamicin, tobramycin and kanamycin, has been cloned and shown to express these resistances in Escherichia coli. The determinant encoded a single protein with an apparent size of 59 kDa which specified both aminoglycoside acetyltransferase [AAC(6')] and aminoglycoside phosphotransferase [APH(2")] activities. Nucleotide sequence analysis of the determinant showed it to be capable of encoding a 479-amino-acid protein of 56.9 kDa. analysis of Tn1725 insertion mutants of the determinant indicated that resistance to tobramycin and kanamycin is due to the AAC activity specified by, approximately, the first 170 amino acids of the predicted protein sequence and is consistent with the gentamicin resistance, specified by the APH activity, being encoded within the C-terminal region of the protein. Comparison of the C-terminal end of the predicted amino acid sequence with the reported sequences of 13 APHs and a viomycin phosphotransferase revealed a region which is highly conserved among these phosphotransferases.

Amino Acid Sequence↗
PubMed1991

Plasmid effects on secondary metabolite production by a streptomycete synthesizing an anthelmintic macrolide.

Transformation of the thermotolerant streptomycete, soil isolate S541, with plasmid cloning vectors of varying size, copy number, and parent replicon (derived from pIJ101, SCP2* and SLP1.2) depressed the biosynthesis of nemadectins (polyketide-derived secondary metabolites possessing anthelmintic activity). However, production of the chemically distinct 21-hydroxyl-oligomycin A, also produced by S541, was either unaffected or increased in plasmid-containing strains. A causal relationship between plasmid carriage and the changes in secondary metabolite yield was confirmed since cured strains were restored to normal production levels and their subsequent retransformation by plasmid DNA was followed by the same effects on nemadectin and oligomycin biosynthesis as before. All the plasmids tested were highly unstable in S541 and it was generally necessary to include an appropriate selective antibiotic (usually thiostrepton) in the growth medium. Thiostrepton was not responsible for the depressive effect, since this was also observed in plasmid-containing strains (i) when grown in antibiotic-free media and (ii) when alternative selective antibiotics such as neomycin were used. In addition, the plasmid-free strain produced both nemadectins and 21-hydroxyl-oligomycin A in the presence of sub-inhibitory levels of thiostrepton. The thiostrepton resistance gene, which was present on many of the plasmids tested, did not mediate the effect since plasmids carrying other selectable markers (pIJ58, neomycin, and pIJ355, viomycin) also depressed nemadectin but not 21-hydroxyl-oligomycin A production. No obvious recombination or integration events between S541 chromosomal DNA and any of the plasmids tested were revealed by DNA-DNA Southern hybridization.

Anti-Bacterial Agents↗
PubMed1997

Gene disruption and replacement in the rapamycin-producing Streptomyces hygroscopicus strain ATCC 29253.

A system for gene disruption and replacement based on a streptomycete temperate phage vector was developed to introduce DNA in the rapamycin-producing Streptomyces hygroscopicus strain ATCC 29253. This will be useful in attempts to produce, through genetic manipulation, novel forms of the therapeutically important immunosuppressive drug rapamycin. Recombinant phages were constructed from the phi C31 phage derivative KC515 (C+ attp) carrying a thiostrepton or viomycin resistance gene along with segments of the S. hygroscopicus chromosome. Each of the cloned segments also contained the aphll neomycin/kanamycin resistance gene to enable gene replacement by loss of the phage-derived DNA. Specific deletion of the entire polyketide synthase (PKS) believed to govern rapamycin biosynthesis resulted in the loss of rapamycin production. In contrast, disruption or deletion of a region predicted to encode four PKS open reading frames, or another region predicted to encode another PKS plus a cytochrome P450 hydroxylase and ferredoxin, had no effect on the production of rapamycin or nigericin, a polyether antibiotic also produced by S. hygroscopicus. Therefore, S. hygroscopicus may have the capacity to produce polyketides additional to rapamycin and nigericin.

Bacteriophages↗
PubMed1980

Studies on the cross-resistance of Mycobacterium tuberculosis, strain H37Rv, to aminoglycoside- and peptide-antibiotics.

Various phenotypes of the resistance to aminoglycoside- and peptide-antibodies of Mycobacterium tuberculosis strain H37Rv were produced by single- and/or two-step selection of the parent strain. Mutants obtained by single-step selection with antibiotics were classified into ten phenotypes; one of single resistance, two of triple resistance, three of quadruple resistance, and four of sextuple resistance. There were two kinds of sextuple resistance (high resistance to enviomycin, viomycin, capreomycin, kanamycin, lividomycin). One was isolated from the parent strain by single-step selection and could be eliminated by mutation to isoniazid resistance, the other was obtained by two-step selections and was not eliminated by mutation to isoniazid resistance. Interaction between mutation to streptomycin resistance and mutation to quadruple resistance (4R phenotype) was observed. Streptomycin resistance interfered with the formation of the 4R phenotype and produced a different phenotype, KR instead of the 4R phenotype. The existence of mutation of the 4R phenotype did not usually interfere with mutation to streptomycin resistance, but a small portion of the mutants with the 4R phenotype were altered in their phenotype from 4R to KR after addition of the mutation to streptomycin resistance. This effect of the mutation to streptomycin resistance was not observed in mutants which already had a mutation to klR phenotype (mutation to low concentrations of kanamycin only).

Aminoglycosides↗
PubMed2005

Characterization of the tRNA and ribosome-dependent pppGpp-synthesis by recombinant stringent factor from Escherichia coli.

Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl transferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; (b) a low ribosome-dependent activity that was inhibited by the A-site specific antibiotics thiostrepton, micrococcin, tetracycline and viomycin; (c) a tRNA- and ribosome-dependent activity amounting to 4500 pmol pppGpp per pmol stringent factor per minute. Footprinting analysis showed that stringent factor interacted with ribosomes that contained tRNAs bound in classical states. Maximal activity was seen when the ribosomal A-site was presaturated with unacylated tRNA. Less tRNA was required to reach maximal activity when stringent factor and unacylated tRNA were added simultaneously to ribosomes, suggesting that stringent factor formed a complex with tRNA in solution that had higher affinity for the ribosomal A-site. However, tRNA-saturation curves, performed at two different ribosome/stringent factor ratios and filter-binding assays, did not support this hypothesis.

Base Sequence↗
PubMed1979

In vitro activity of 39 antimicrobial agents against Treponema hyodysenteriae.

The in vitro activities of 39 antimicrobial agents against 23 isolates of Treponema hyodysenteriae, the majority of which were field isolates, were determined by the agar dilution technique. Quinoxalines, pleuromutilin, nitroimidazoles, and nitrofuran were the most active. Their activities ranged from </=0.10 to 1.56 mug/ml. Lincomycin, penicillins, chloramphenicol, tetracyclines, cephalosporins, three peptides (virginiamycin, thiopeptin, and bacitracin), and one aminoglycoside (gentamicin) exhibited intermediate levels of activity ranging from 0.39 to 50 mug/ml. Four peptides (enduracidin, viomycin, bicyclomycin, and colistin), three aminoglycosides (kanamycin, streptomycin, and neomycin), polyene, and other agents, including novobiocin, vancomycin, rifampin, nalidixic acid, and p-arsanilic acid, displayed limited activities ranging from 12.5 to >/=100 mug/ml. Macrolides showed varying degrees of activity depending upon isolates.

Anaerobiosis↗
PubMed1984

In vitro antimicrobial susceptibility of a Mycobacterium sp. isolated from patients with Crohn's disease.

The in vitro susceptibility of three strains of an unclassified Mycobacterium sp., isolated from three patients with Crohn's disease, to 23 antimicrobial agents was determined by a modified broth dilution method with 7H9 broth containing oleic acid-albumin-dextrose-catalase, Tween 80, and mycobactin J. All three strains were susceptible to streptomycin, viomycin, rifampin, clofazimine, cefazolin, amikacin, and kanamycin and resistant to p-aminosalicylic acid, cycloserine, 2-thiophenecarboxylic acid hydrazide, trimethoprim, diaminodiphenylsulfone, sulfamethoxazole, sulfadimethoxine, polymyxin B, metronidazole, neomycin, and carbenicillin. Variable results between strains were encountered with ethambutol, ethionamide, capreomycin, amoxicillin, and cephalothin.

Adolescent↗
PubMed1992

Stability of antimycobacterial drugs in susceptibility testing.

Aqueous solutions of 0.02% isoniazid, 0.2% streptomycin, 0.2% para-aminosalicylate, and 0.5% ethambutol and ethylene glycol solutions of 0.5% ethionamide stored at 3 to 7 degrees C remained stable for 1 year, as did aqueous solutions of 0.05% ethionamide hydrochloride, 0.05% kanamycin, 0.05% viomycin, and 0.1% capreomycin stored at -20 degrees C. The ethambutol and capreomycin solutions were tested by microbiologic methods; the other solutions were tested by both spectrophotometric and microbiologic methods. Prepared susceptibility testing media made with cycloserine, rifampin, and the above solutions incorporated into Middlebrook 7H10 medium showed acceptable stability when stored at 3 to 7 degrees C for 1 month. During incubation of the test medium at 37 degrees C, approximately half of the activity of isoniazid, ethionamide, ethambutol, cycloserine, and rifampin was lost after periods ranging from 2 to 4 days for ethambutol to 2 weeks for rifampin.

Antitubercular Agents↗
PubMed1981

Plasmid deoxyribonucleic acid and translucent-to-opaque variation in Mycobacterium intracellulare 103.

After treatment with mitomycin D and other antibacterial agents, a translucent, smooth-colony-forming mycobacterium, isolated from sputum and designated as Mycobacterium intracellulare strain 103, gave rise to variants forming opaque colonies. These opaque variants were more sensitive streptomycin, kanamycin, viomycin, and rifampin than were the wild-type translucent variants. Plasmid deoxyribonucleic acids taken from translucent strain cells and from cells of certain opaque variants were analyzed by agarose gel electrophoresis. Two plasmids of molecular weights of approximately 2 x 10(6) and 50 x 10(6), respectively, were found in the wild-type translucent cells; one of them, the 2 x 10(6)-molecular-weight plasmid, was always missing from deoxyribonucleic acids of the opaque variant cells. The results suggested that translucent colonial appearance and antibiotic resistance of the strain are plasmid-determined functions.

Anti-Bacterial Agents↗
PubMed1982

Biochemical characterization of resistance determinants cloned from antibiotic-producing streptomycetes.

Determinants of antibiotic resistance have been cloned from four antibiotic-producing streptomycetes into Streptomyces lividans. Biochemical analyses of resistant clones revealed the presence of enzymes that had previously been characterized as likely resistance determinants in the producing organisms. These included: 23S rRNA methylases from S. azureus and S. erythreus, which confer resistance to thiostrepton and erythromycin, respectively; viomycin phosphotransferase from S. vinaceus; and aminoglycoside phosphotransferase and acetyltransferase from the neomycin producer S. fradiae. In general, the levels of antibiotic resistance of the clones were similar to those of the producing organisms. Although the two aminoglycoside-modifying enzymes from S. fradiae could independently confer only low-level resistance to neomycin, the presence of both enzymes in the same strain resulted in a level of resistance comparable with that of the producing organism.

Acetyltransferases↗
PubMed1987

Tn4556, a 6.8-kilobase-pair transposable element of Streptomyces fradiae.

A 6.8-kilobase-pair (kbp) transposable element (Tn4556) was found in a neomycin-producing strain of Streptomyces fradiae. This element was first observed in two 30.3-kbp plasmids (pUC1123 and pUC1124) which arose when a thiostrepton resistance gene (1 kbp) was ligated with the BclI-2 fragment (22.5 kbp) that contains the origin of replication of phage SF1. The Tn4556 segment was deleted when these plasmids were transduced into another S. fradiae host with phage SF1. These deletion plasmids (pUC1210 and pUC1211) had copy numbers of less than 1 per chromosome and were unstable. In contrast, pUC1123 and pUC1124, with copy numbers of 12 to 15 per chromosome, respectively, were relatively stable. When pUC1210 and pUC1211 were reintroduced into S. fradiae by protoplast transformation, the Tn4556 element transposed again to the plasmids at numerous new locations in either of two orientations. A copy of Tn4556 was found in the S. fradiae chromosome by hybridization studies. It appears that Tn4556 originated from the chromosome, transposed into unstable pUC1210 and pUC1211, and made stable plasmids. A temperature-sensitive hybrid plasmid carrying a viomycin resistance derivative of Tn4556 (pMT660::Tn4556::vph) was constructed. When Streptomyces lividans UC8390 containing the hybrid plasmid was grown at 39 degrees C, Tn4556::vph (Tn4560) transposed to random positions in the host chromosome.

Cloning, Molecular↗
PubMed1992

Construction and application of plasmid- and transposon-based promoter-probe vectors for Streptomyces spp. that employ a Vibrio harveyi luciferase reporter cassette.

Several versatile promoter-probe vectors have been constructed for Streptomyces strains which utilize the production of blue-green light as a measure of transcription activity. Three plasmid vectors (two high and one low copy number) and two transposons are described. The multicopy plasmids pRS1106 and pRS1108 contain a transcription terminator and multiple-cloning polylinker upstream of promoterless luciferase (lux) and neomycin resistance reporter genes. Plasmid pHI90 is similar in structure to the pRS vectors except that its single copy number is an advantage for regulation studies or situations in which overexpression is otherwise toxic to the cell. The two transposons carry a promoterless lux cassette cloned such that transposition into a target DNA and fusion to the target's transcription unit occur simultaneously. Tn5351 was created by inserting the luciferase genes near the right end of the viomycin resistance transposon Tn4563. Tn5353 carries the luciferase genes near the right end of a neomycin resistance transposon derived from Tn4556. The size of Tn5353 was minimized by deleting nonessential transposon sequences, making this element small enough to be cloned into phi C31 bacteriophages for efficient transposon delivery to target cells of Streptomyces strains. The two Tnlux transposons have been used to generate Streptomyces coelicolor morphological mutants and to monitor transcription from chromosomal promoters during development.

Chromosomes, Bacterial↗
PubMed1977

Symbiotic effectiveness of antibiotic-resistant mutants of fast- and slow-growing strains of Rhizobium nodulating Lotus species.

Mutants resistant ot 16 individual antibiotics were isolated from two fast-growing and two slow-growing strains of Lotus rhizobia and their symbiotic effectiveness on Lotus pedunculatus evaluated. Resistance to streptomycin, spectinomycin, chloramphenicol, and tetracycline (inhibitors of protein synthesis) was associated with little or no loss of effectiveness with all four strains but resistance to nalidixic acid and rifampicin (inhibitors of nucleic acid synthesis), and to D-cycloserine, novobiocin, and penicillin (inhibitors of cell wall-cell membrane synthesis) was associated with significant loss of effectiveness in 20-100% of the mutants. Resistance to viomycin, neomycin, kanamycin, and vibramycin was associated with loss of effectiveness with mutants of the two fast-growing strains but not with mutants of the two slow-growing strains. When tested on four alternate host legumes individual mutants of a slow-growing strain showed significantly different levels of effectiveness. The results suggest that both the inherent characteristics of the bacterium and of the host plant will influence the symbiotic effectiveness of antibiotic-resistant mutants of Rhizobium.

Anti-Bacterial Agents↗
PubMed1979

Primary drug resistance in children. Drug susceptibility of strains of Mycobacterium tuberculosis isolated from children during the years 1973 through 1977 at the Kings County Hospital Center of Brooklyn.

A continuing study of the frequency of primary drug resistance among children treated at the Kings County Hospital Center of Brooklyn during the years 1973 through 1977 showed a high incidence of primary drug resistance to isoniazid (8.8 per cent) and to streptomycin (12.3 per cent). In contrast, there were no strains resistant to cycloserine, viomycin, ethambutol, or rifampin, and only one of 57 strains (1.8 per cent) was resistant to ethionamide, and one (1.8 per cent) was resistant to para-aminosalicylic acid. Comparison with previous studies begun in 1961 showed no significant increase in resistance to isoniazid during 3 prior periods of study and no increase in resistance to streptomycin during the last 2 periods of study. It must be emphasized that these findings relate only to the children of a local community, and do not reflect the prevalence of primary drug resistance elsewhere in this country or among different age groups.

Adolescent↗
PubMed1989

Analyses of specific and total antibody responses of rabbits to four kinds of immunogens.

As a basic study to investigate suitable conditions to immunize rabbits with drug-immunogens, two highly sensitive and accurate enzyme immunoassays (EIAs) for specific antibody to viomycin (VM) and blasticidin S (BLS) were developed using the corresponding standard antibody, the solid-phase antigens, and enzyme-labeled goat anti-rabbit immunoglobulin G (IgG) antibody as immunological reagents. The accuracy of the assay results with these newly developed EIAs was demonstrated. The new EIAs as well as two previously developed EIAs, EIA for antibody specific to neocarzinostatin (NCS) and a sandwich EIA for rabbit IgG, were applied for analyses of the changes in contents of total and specific antibodies in rabbit antisera samples collected during immunizations with four antigens. Total IgG levels increased from 7.0-9.9 mg/ml to 30-50 mg/ml in all rabbits immunized under the same immunizing schedule, despite the use of four kinds of antigens. The highest level of specific antibodies, anti-BLS, anti-VM and anti-NCS, was 0.5 mg/ml in each case.

Animals↗
PubMed1989

Comparative study of complete and incomplete Freund's adjuvants for immunization of a drug immunogen using enzyme immunoassay methods.

Highly sensitive and accurate enzyme immunoassays (EIAs), a sandwich EIA for mouse immunoglobulin G (IgG) and an enzyme linked immunosorbent assay for mouse antibody specific to viomycin (VM), were developed. Accuracy and specificity of the assay results were confirmed before their application. The changes of total IgG and antibody specific to VM in mice, immunized with a VM-immunogen with or without two types of Freund's adjuvants under various conditions, were assessed by means of the newly developed EIA methods. Both methods were very useful tools to follow the immunization processes of mice, and complete and incomplete Freund's adjuvant were found to have similar adjuvant activities for production of antibody specific to VM, judging from the amounts of anti-VM antibody formed. It seems to be important that too many booster injections should be avoided in the immunization of mice with a hapten immunogen.

Animals↗
PubMed1990

Studies on the optimal immunization schedule of experimental animals. V. The effects of the route of injection, the content of Mycobacteria in Freund's adjuvant and the emulsifying antigen.

The effects of several conditions for the immunization of mice was studied using an aliquot of a viomycin (VM) protein conjugate as the common primary or booster antigen. Responses of the mice were assessed by measuring mouse serum levels of total immunoglobulin G (IgG) and anti-VM antibody responses using the newly improved two assay methods. The choice of route was found to be a very important factor in immunization and intraperitoneal injection was the most optimal among the four routes studied. The effect of the concentration of Mycobacteria in Freund's complete adjuvant (FCA) was also studied, and it was found that a diluted FCA was more effective than a commercial FCA. The effect of the controlled release of the antigen was studied and three important phenomena were observed: The mice immunized by the mini-osmotic pump-aided controlled release of the antigen responded with similar small amounts of both total IgG and anti-VM antibody regardless of the presence or absence of FCA in the antigen; emulsifying the antigen with FCA was a very important condition for the effective elicitation of the specific antibody; a mixture of antigen and FCA without emulsifying produced little specific antibody and a large amount of total IgG. The more effectively immunized mice responded with a larger decrease in body weight soon after the primary injection.

Animals↗