A Suitable Streptomycin-Resistant Mutant for Constructing Unmarked In-Frame Gene Deletions Using rpsL as a Counter-Selection Marker

The streptomycin counter-selection system is a useful tool for constructing unmarked in-frame gene deletions, which is a fundamental approach to study bacteria and their pathogenicity at the molecular level. A prerequisite for this system is acquiring a streptomycin-resistant strain due to rpsL mutations, which encodes the ribosomal protein S12. However, in this study no streptomycin resistance was found to be caused by rpsL mutations in all 127 clinical strains of Klebsiella pneumoniae isolated from liver abscess patients. By screening 107 spontaneous mutants of streptomycin resistance from a clinical strain of K. pneumoniae, nucleotide substitution or insertion located within the rpsL was detected in each of these strains. Thirteen different mutants with varied S12 proteins were obtained, including nine streptomycin-dependent mutants. The virulence of all four streptomycin-resistant mutants was further evaluated. Compared with the parental strain, the K42N, K42T and K87R mutants showed a reduction in growth rate, and the K42N and K42T mutants became susceptible to normal human serum. In the mice LD₅₀ (the bacterial dose that caused 50% death) assay, the K42N and K42T mutants were ∼1,000-fold less lethal (∼2×10⁵ CFU) and the K87R mutant was ∼50-fold less lethal (∼1×10⁴ CFU) than the parental strain (∼2×10² CFU). A K42R mutant showed non-observable effects on the above assays, while this mutant exhibited a small cost (P<0.01) in an in vitro growth competition experiment. In summary, most of the K. pneumoniae strains with streptomycin resistance caused by rpsL mutations are less virulent than their parental strain in the absence of streptomycin. The K42R mutant showed similar pathogenicity to its parental strain and should be one of the best choices when using rpsL as a counter-selection marker.     

Improvement of antibiotic titers from Streptomyces bacteria by interactive continuous selection

We have applied a technique of interactive continuous selection (ICS) to the isolation of streptomycin-resistant mutants of the streptomycin-producing organism, Streptomyces griseus. A series of mutants, each with a different colonial morphology and expressing successively greater resistance to streptomycin, was isolated during the course of selection. Takeover of the mutants has been correlated with changes in on-line estimates of streptomycin concentration such that these estimates may be used as a real-time measure of the genetic state of the cell population. When grown in the medium employed for ICS, mutants expressed increased antibiotic production titers; the best mutant produced 10 to 20 times more streptomycin than the parent strain. Absolute improvements in the maximum specific growth rate and intrinsic resistance to streptomycin did not account for the observed growth advantage of all mutants. Rather, each mutant exhibited relative increases in specific growth rate at increasing concentrations of streptomycin. (c) 1996 John Wiley & Sons, Inc.     

In vitro measurement of translation accuracy of ribosomes isolated from streptomycin-resistant mutant ofStreptomyces granaticolor

Accuracy of activity of ribosome isolated from UV-light-induced streptomycin-resistant R-21 mutant ofStreptomyces granaticolor was measured in anE. coli-derived system translating poly(U) with a high rate and accuracy. Ribosomes from the R-21 mutant strain were shown to be resistant to streptomycin and about two-fold more accurate than those from the wild type. The mutant strain was found to be resistant to 1000 mg/L streptomycin (Stm) during vegetative growth while it sporulated on agar plates containing only up to 200 mg/L of Stm. The growth rate of the R-21 mutant in complex liquid medium was indistinguishable from that of the wild-type strain.     

微波诱变结合抗性突变筛选放线菌素D高产菌株

采用微波结合链霉素抗性筛选法选育放线菌素D的高产菌株。通过考察链霉素对Streptomyces rubiginosohelvolus FIM-N31菌株孢子生长情况的影响确定链霉素致死浓度,出发菌株FIM-N31的孢子经微波辐照处理后,涂布在含链霉素致死浓度（50 μg/mL）的培养基平板上培养,获得了大量的链霉素抗性基因突变株。摇瓶发酵筛选突变株,结果获得一株遗传性状稳定的放线菌素D高产菌Str186,其产放线菌素D的能力比出发菌株提高了8倍以上。     

Revertants of a streptomycin-resistant, oligosporogenous mutant ofBacillus subtilis

Summary Revertants of a streptomycin-resistant (Str^R), oligosporogenous (Spo^-) mutant ofBacillus subtilis were selected for the ability to sporulate. The revertants obtained fell into two phenotypic classes: Str^S Spo⁺ (streptomycin-sensitive, sporeforming), which arose by reversion of the streptomycin resistance mutations of the parent strain; and Str^R Spo⁺, which arose by the acquisition of additional mutations, some of which were shown to affect ribosomal proteins. Alterations of ribosomal proteins S4 and S16 in the 30S subunit and L18 in the 50S subunit were detected in Str^R Spo⁺ revertants by polyacrylamide gel electrophoresis. Streptomycin resistance of the parental strain and the Str^R revertants was demonstrated to reside in the 30S ribosomal subunit. The second site mutations of the revertants depressed the level of streptomycin resistance in vivo and in the in vitro translation of phage SP01 messenger ribonucleic acid (mRNA) relative to the resistance exhibited by the Str^R parental strain. The Str^R parent grew slowly and sporulated at approximately 1% of the wild type level. The Str^S revertants closely resembled the wild type strain with regard to growth and sporulation. The Str^R revertants grew at rates intermediate between those of the Str^R parent and wild type, and sporulated at wild type levels.     

Mutagenic effects of streptomycin in chlamydomonas

Summary Growth of a green streptomycin-resistant strain of Chlamydomonas reinhardi on a sub-lethal concentration of streptomycin on agar led to the appearance of yellow mutant cells in almost every colony. The time of appearance of the mutants varied greatly among the 9 isolates studied, each of which was selected as a single colony after repeated cloning of the parental strain. 2 isolates gave rise to colonies which responded rapidly to streptomycin (class I), 2 isolates produced yellow sub-clones as papillae only after formation of normal green colonies (class II), and 2 isolates produced stable yellow sub-clones only after a second subculture on streptomycin-agar (class III). 3 isolates were mixtures of classes II and III.The evidence that these yellow mutants arose under the mutagenic action of streptomycin is discussed in relation to the alternative possibility of their selection by the drug from a pool of pre-existing mutants. The physiological and genetic effects of streptomycin upon chlorophyll formation in Chlamydomonas are compared with reported effects of the drug upon the green flagellate, Euglena gracilis.Dedicated with appreciation and affection to Professor Dr. E. G. Pringsheim on the occasion of his 80th birthday.     

Mechanism of Resistance to Antibiotic Synergism in Enterococci

Enterococci exhibit two types of resistance to streptomycin. Moderately high-level resistance is observed in most naturally occurring strains and can be overcome by simultaneous exposure to penicillin. In addition, very high-level resistance is found in those strains against which penicillin plus streptomycin fail to produce synergism in vitro. To study the mechanism of streptomycin resistance in enterococci, ribosomes from a wild-type strain and from a highly streptomycin-resistant mutant were isolated, characterized, and studied in an in vitro amino acid incorporation system. The ribosomes from the organism with moderately high-level streptomycin resistance were sensitive to streptomycin in vitro, suggesting that this type of resistance is caused by failure of streptomycin to reach the ribosomes. Very high-level resistance (and lack of penicillin-streptomycin synergism), on the other hand, appears to be due to ribosomally mediated streptomycin resistance.     

Mendelian inheritance of streptomycin resistance in Nicotiana plumbaginifolia

In a previous study two haploid streptomycin-resistant clones of Nicotiana plumbaginifolia were isolated. The chromosome number of one of these clones has now been doubled through leaf-midvein culture and the resultant diploids were characterized genetically. Our results show that streptomycin resistance in this clone is conditioned by a recessive nuclear gene. Haploid protoplasts of this streptomycin-resistant mutant were selected for chlorate resistance. All clones obtained from the selection were deficient in nitrate reductase activity in addition to resistance to streptomycin. Genetic analysis of progeny of one of these clones revealed that the genes for streptomycin resistance and for the apoenzyme of nitrate reductase are unlinked.     

Isolation and characterization of a new globomycin-resistant dnaE mutant of Escherichia coli.

We isolated a globomycin-resistant, temperature-sensitive mutant of Escherichia coli K-12 strain AB1157. The mutation mapped in dnaE, the structural gene for the alpha-subunit of DNA polymerase III. The in vivo processing of lipid-modified prolipoprotein was more resistant to globomycin in the mutant strain 307 than in its parent. The prolipoprotein signal peptidase activity was also increased twofold in the mutant, and there was a threefold increase in the activity of isoleucyl-tRNA synthetase. The results suggest that a mutation in dnaE may affect the expression of the ileS-lsp operon in E. coli. In addition, strain 307 showed a reduced level of streptomycin resistance compared with its parental strain AB1157 (rpsL31). Strain 307 was killed by streptomycin at a concentration of 200 micrograms/ml, which did not affect the rate of bulk protein synthesis in this mutant. A second mutation which was involved in the reduced streptomycin resistance in strain 307 was identified and found to be closely linked to or within the rpsD (ramA, ribosomal ambiguity) gene. Both dnaE and rpsD were required for the reduced streptomycin resistance in strain 307.     

Induction and characterization of streptomycin-resistant mutants in Capsicum praetermissum

Streptomycin-resistant mutants were isolated from mutagenised cotyledon explants of Capsicum praetermissum Heiser & Smith. The explants were mutagenised with N-ethyl-N-nitrosourea, which resulted in a high frequency of streptomycin-resistant mutants (18.0%) and a low frequency of chlorophyll-deficient (albino) mutants (8.0%). Complete streptomycin-resistant plantlets were obtained after rooting of the regenerated green shoots on rooting medium containing 1.0 mg L-1 IAA and 500 mg L-1 streptomycin sulphate. Leaf-segment assay of these plantlets revealed that they were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, and spectinomycin. Reciprocal crosses between streptomycin-resistant and -sensitive plants showed a non-Mendelian transmission of resistance by female parents.     

THE EFFECT OF PROFLAVINE ON DIFFERENT STRAINS OF THE BLUE-GREEN ALGA ANACYSTIS NIDULANS

The growth of the blue-green alga Anacystis nidulans was inhibitedby relatively low concentrations of proflavine. The durationof the lag phase increased linearly with increasing proflavineconcentrations. The alga neither acquired increased toleranceto the drug during the course of six subcultures in proflavine-supplementedmedia nor was there any shortening of lag-phase or increasein final population density following repeated subculture inproflavine medium. The strain obtained after six subcultures in proflavine wasfound to have become more sensitive to streptomycin than theuntreated stock strain. A comparison of sensitivity to proflavine of two streptomycin-resistantstrains and of untreated stock strain of the alga revealed thatthe streptomycin-resistant strains were less sensitive to proflavinethan the untreated strain. The resistance of a strain resistant to 200 µg streptomycin/mlwas lost after it had been cultured once in streptomycin-freebasal medium that had been supplemented with 1 µg proflavineµml.The results obtained provide some indirect evidence for theexistence of episome in A. nidulans. ¹Present temporary address: Division of Biosciences, NationalResearch Council, Ottawa-2, Canada.     

Defective lysis of streptomycin-resistant escherichia coli cells infected with bacteriophage f2

A lysis defect was found to account for the failure of a streptomycin-resistant strain of Escherichia coli to form plaques when infected with the male-specific bacteriophage f2. The lysis defect was associated with the mutation to streptomycin resistance. Large amounts of apparently normal bacteriophage accumulated in these cells. Cell-free extracts from both the parental and mutant strains synthesized a potential lysis protein in considerable amounts in response to formaldehyde-treated f2 RNA but not in response to untreated RNA. As predicted from the nucleotide sequence of the analogous MS2 phage, the protein synthesized in vitro had the expected molecular weight and lacked glycine. The cistron for the lysis protein overlapped portions of the coat and replicase cistrons and was translated in the +1 reading frame. Initiation at the lysis protein cistron may be favored by translation errors that expose the normally masked initiation site, and streptomycin-resistant ribosomes, known to have more faithful translation properties, may be unable to efficiently synthesize the lysis protein.     

Single gene alteration of plasma and mitochondrial membrane function in Saccharomyces cerevisiae.

Summary Some physiological properties of a multiple-drug-resistant mutant with a permeability barrier to chloramphenicol and its isogenic parental strain were compared. The ATPase specific activity of plasma and mitochondrial membranes isolated from the mutant strain was approximately 20% lower (P(0.001, Tables 1 and 2) than that of membranes isolated from the isogenic parental strain. Additional evidence of altered mitochondrial function was: (i) the enhanced growth of the parental strain was eliminated by the [rho-] state (Table 3); (ii) the mutant strain had a greater resistance to petite induction by ethidium bromide (Table 4); (iii) the mutant strain was unable to use a nonfermentable energy source for respiratory adaptation (Table 5). It is proposed that a single gene mutation has resulted in an alteration of some physiological properties of the plasma and mitochondrial membranes.     

Interactions between viomycin resistance and streptomycin resistance on ribosomes of Mycobacterium smegmatis.

We have investigated the mechanism of the expression of resistance to high levels of viomycin and coresistance to streptomycin in a mutant strain of Mycobacterium smegmatis ATCC 14468 (AC-13) which was obtained by serial transfers of parental cells to media containing increasing concentrations of viomycin. It was shown previously that resistance to viomycin by strain AC-13 was due to an alteration in the 50 S ribosomal subunit (20). However, genetic analysis has shown that mutation in 50 S subunits alone gave only low level resistance to viomycin. When a streptomycin resistant mutation (caused by an alteration in the 30 S subunit) was introduced into the low level viomycin resistant recombinant strains, most of them were highly resistant to viomycin. Some recombinants were resistant to intermediate levels of viomycin, and the remainder were not affected by the introduction of the str^r allele. Studies with in vitro cell-free systems have shown that streptomycin resistant 30 S ribosomal subunits obtained from a high level viomycin resistant recombinant were able to modify the levels of resistance to viomycin expressed by the 50 S ribosomal subunit. These findings provide additional evidence concerning the functional relationship between 30 S and 50 S ribosomal components in ribosomes.     

A point mutation in the chloroplast rps12 gene from Nicotiana plumbaginifolia confers streptomycin resistance

In an effort to understand the mechanism of streptomycin resistance in Nicotiana plumbaginifolia, we have sequenced the chloroplast rps12 gene, a potential molecular target. We report that a streptomycin-resistant mutant isolated from protoplast cultures of N. plumbaginifolia contains an A-to-G transition at nucleotide position 149 in exon 2 of the chloroplast rps12 gene. The detected point mutation predicts a substitution of arginine for lysine in a phylogenetically conserved region.     

Transmission of paternal chloroplasts in tobacco (Nicotiana tabacum)

Medgyesy et al. (1986, Mol. Gen. Genet. 204, 195–198) have described in Nicotiana plumbaginifolia and in an interspecific cross involving N. plumbaginifolia and N. tabacum a procedure for selecting cell lines derived from seedlings carrying paternal chloroplasts by taking advantage of a plastid-encoded mutation which confers resistance to streptomycin. We have extended their demonstration of occasional transmission of chloroplasts through pollen to the case of an intraspecific cross in N. tabacum. The line used as maternal parent, ITB19(sua), displayed a cytoplasmic male sterility due to the presence of a cytoplasm originating from N. suaveolens. The line used as paternal parent, SR1, was fertile and possessed mutant chloroplasts conferring resistance to streptomycin. From cell lines derived from 204 seedlings, three were regenerated into streptomycin-resistant buds. The plants derived from these three clones were male-sterile. Their progeny, after crossing with a wild type tobacco line, XHFD8, was resistant to streptomycin. Tests of resistance of the seedlings to tentoxin and restriction analyses of the chloroplast DNA indicated that two clones still had the maternal chloroplasts and were thus probably new streptomycin-resistant mutants, whereas the third one had acquired the chloroplasts of the paternal parent, but had retained the mitochondria of the maternal parent.Abbreviations cp-DNA chloroplast DNA - mt-DNA mitochondrial DNA - Np Nicotiana plumbaginifolia - Nt Nicotiana tabacum     

Role of pyrimidine dimer excision in loss of potential streptomycin resistance mutations of ultraviolet-irradiated Escherichia coli on phosphate-buffered agar.

The frequency of ultraviolet (UV)-induced mutations to streptomycin resistance dropped rapidly when starved Escherichia coli strains WP-2 B/r and B/r T- were incubated on phosphate-buffered agar (PBA), but was reduced only slightly in a WP-2 hcr- mutant. During postirradiation, incubation viability remained approximately constant. Cells given an optimal recovery treatment with photo-reactivating light showed no further recovery if subsequently incubated on PBA. At least 70% of the mutations induced to streptomycin resistance by UV could be repaired. The loss of potential streptomycin-resistant mutants was markedly reduced in strain B/r T- when 5 mug of acriflavin or 700 mug of caffeine per ml was added to PBA. The excision of UV-induced thymine-containing dimers from E. coli tb/r T- was investigated. Dimer excision progressed more slowly when the cells were incubated on PBA containing acriflavin or caffeine. There was no congruity between the kinetics of dimer excision and the kinetics of mutant loss. Our results indicate that removal of potential streptomycin-resistant mutants is considerably faster than the excision of pyrimidine dimers.     

Induction of streptomycin-resistant plantlets in <Emphasis Type="Italic">Solanum surattense</Emphasis> through <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis

The species Solanum surattense Burm.f. has importance in ayurvedic medicine and also as vegetable. Streptomycin-resistant plantlets were induced showing chloroplast encoded mutants in S. surattense from mutagenised (ethyl methane sulphonate and gamma-rays) cotyledon explants. Chloroplast encoded – streptomycin resistant – shoots were developed from green (unbleached) sectors of the cotyledons. The streptomycin-resistant plants were similar to parental plants in morphology and ploidy level (2n=2x=24). Reciprocal crosses between streptomycin-resistant and the original streptomycin sensitive plants have shown the non-Mendelian transmission under the control of chloroplast – DNA. These antibiotic resistant plants are useful in designing biochemical selection schemes aimed at somatic hybrid/cybrid recovery in S. surattense.     

A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids

A plasmid cloning vector with ampicillin-resistance and streptomycin-sensitivity markers is suitable for the direct selection of strains carrying recombinant plasmids. The selection for plasmid transformants utilizes their ampicillin resistance whereas selection for recombinant plasmids is based on the inactivation of the rpsL gene contained on the plasmid. When streptomycin-resistant Escherichia coli strains are used as recipients in transformation, transformants carrying the parental plasmid are phenotypically sensitive to streptomycin while those carrying hybrid plasmids are resistant to streptomycin.     

Mutant ribosomes can generate dominant kirromycin resistance.

Mutations in the two genes for EF-Tu in Salmonella typhimurium and Escherichia coli, tufA and tufB, can confer resistance to the antibiotic kirromycin. Kirromycin resistance is a recessive phenotype expressed when both tuf genes are mutant. We describe a new kirromycin-resistant phenotype dominant to the effect of wild-type EF-Tu. Strains carrying a single kirromycin-resistant tuf mutation and an error-restrictive, streptomycin-resistant rpsL mutation are resistant to high levels of kirromycin, even when the other tuf gene is wild type. This phenotype is dependent on error-restrictive mutations and is not expressed with nonrestrictive streptomycin-resistant mutations. Kirromycin resistance is also expressed at a low level in the absence of any mutant EF-Tu. These novel phenotypes exist as a result of differences in the interactions of mutant and wild-type EF-Tu with the mutant ribosomes. The restrictive ribosomes have a relatively poor interaction with wild-type EF-Tu and are thus more easily saturated with mutant kirromycin-resistant EF-Tu. In addition, the mutant ribosomes are inherently kirromycin resistant and support a significantly faster EF-Tu cycle time in the presence of the antibiotic than do wild-type ribosomes. A second phenotype associated with combinations of rpsL and error-prone tuf mutations is a reduction in the level of resistance to streptomycin.