Streptomycin-resistant Mutants Research Articles

Symbiotic effectiveness of streptomycin-resistant Rhizobium spp. mutants of chickpea (Cicer arietinum L.)

Antibiotic-resistant Rhizobium spp. strains have been used in ecological studies of legumerhizobia symbiosis. It has been suggested that in the course of acquiring resistance against high doses of antibiotics, rhizobia might lose their symbiotic effectiveness. Evidence both for and against this argument has been presented (Kremer and Peterson 1982; Materon and Hagedron 1983). This communication reports our experience with streptomycin-resistant (Str+) mutants of chickpea Rhizobium spp. strains. Parent strains were used as controls.

Myomycin: mode of action and mechanism of resistance.

Myomycin is an unusual pseudodisaccharide antibiotic with a beta-lysyl oligopeptide ester side chain that has structural similarities with kasugamycin, streptomycin and streptothricin. We show that the mode of action of myomycin in vivo and in vitro closely resembles that of streptomycin; in addition, spontaneous myomycin-resistant mutants of Escherichia coli are essentially indistinguishable from streptomycin-resistant mutants at the rRNA and r-protein level. Myomycin is not a substrate for the known streptomycin-modifying enzymes and could be useful in the characterization of natural streptomycin-resistant isolates and in counterselecting against the presence of streptomycin-modifying enzymes. The relationship between structure and inhibition of protein synthesis has been examined for a series of derivatives of myomycin.

Response of soybean [ Glycine max (L.) merrill.] to Bradyrhizobium japonicum inoculation in three soils in ghana

Three antigenically distinguishable Bradyrhizobium japonicum streptomycin-resistant mutant strains (SRJ-S, S14-S and SX-S) were evaluated in three soil series in Ghana (Tikobo, pH 4.3; Amo, pH 5.4; Akuse, pH 6.1), for nodulating ability and competitiveness for nodule occupancy on soybean var. Williams. Few nodules developed on plants grown in the uninoculated soils. The most probable number counts on sand-grown soybean indicated 23, 280 and 330 native B. japonicum cells g −in the Tikobo, Akuse and Amo soils, respectively. Inoculation with single or mixed strain combinations of SRJ-S. S14-S and SX-S, at 10 5 cells g −1 soil greatly improved nodulation (>10-fold), N accretion (50–470%) and shoot dry weight (40–90%). When independently inoculated into soil, these strains formed between 75–100% of the soybean nodules, with no significant nodulation differences between any of the single strain inoculation treatments in each soil. However, strain identification from nodules formed by mixed strain inoculation treatments (in population ratios of 1:1 or 1:1:1) revealed marked variations in competitiveness between the three strains for nodule occupancy. Nodules formed by the most competitive strain, SRJ-S (obtained from Kenya), were also the most effective in N 3 fixation (as measured by the total N in plants). Of the locally isolated strains, S14-S was more competitive than SX-S. The nodulation, N content and dry matter yield results showed that, in general, mixed strain inoculation resulted in similar effects as those by the best individual in a particular strain-combination.

Competitiveness of introduced and indigenous cowpea Bradyrhizobium strains for nodule formation on cowpeas [ Vigna unguiculata (L.) walp.] in three soils

Streptomycin-resistant mutant strains of cowpea Bradyrhizobium. designated CP3-S, CBD-S and CKF-S were inoculated into three soils (Tikobo, Amo and Akuse) containing indigenous bradyrhizobia and assessed for relative nodule occupancy on cowpea plants [ Vigna unguiculata (L.) Walp.]. The nodule-forming strains were identified by the ability to grow on streptomycin-containing yeast extract mannitol medium and by serological differences. Each strain competed successfully against the native soil bradyrhizobia. Except for CKF-S in a few cases, the addition of about 10 5 cells of each strain g − soil, or various strain combinations, always increased nodule numbers, N yield and shoot dry weight of plants in all three soils, compared with the uninoculated soil. The magnitude of these inoculation responses was influenced by the soil type, with the highest effect occurring in the Amo soil. The mixed strain inoculation frequently gave plant responses intermediate to those obtained using the individual strains constituting the particular mixture. At 10 2 cells of CP3-S or CBD-S g −1 soil, the inoculation responses disappeared. suggesting that the observed increases in harvest measurements were due to the large cell populations used. The strains differed in competitiveness for nodule formation, with strain CP3-S being the most competitive. Inoculated alone, or in competition with the other strains and native soil bradyrhizobia, CP3-S formed between 60 and 94% of the nodules sampled. Strain CP3-S formed more nodules than strain CBD-S even when the CBD-S population was ten times greater. However, when the relative CBD-S :CP3-S population was increased to 1000:1, CBD-S occupied more nodules than CP3-S. These results indicate the possibility of increasing nodulation, N3 fixation and yield of field grown cowpeas through inoculation with selected strains.

Utility ofBacillus sphaericusin controllingCulex tritaeniorhynchusbreeding

Freeze-dried powder preparation of a streptomycin-resistant (Str) mutant of Bacillus sphaericus B.42 was applied against Culex tritaeniorhynchus larvae breeding in paper factory effluent. Whether applied as single, double or triple sprays on days 0, 1 and 2, respectively, a complete control of larvae was observed only until day 7 post application. Though the applied strain of B. sphaericus persisted (in the sprayed pits) at least until 91 days, as evident from larval resurgence, the sprayed B. sphaericus did not recycle in paper factory effluent. Whenever the heat-resistant spore counts dropped below approx. 600/ml, a build-up in larval population was observed. The Str mutant strain of B. sphaericus used in the present study seems to be an effective biocide capable of persisting and controlling C. tritaeniorhynchus larvae breeding in such a highly polluted water habitat as paper factory effluent.

Bacteriostatic action of streptomycin on ribosomally resistant mutants (rpsL) of Salmonella typhimurium

Incubation of streptomycin-resistant (rpsL) mutants of Salmonella typhimurium in alkaline nutrient medium containing streptomycin brought about an inhibition of cell growth that was readily reversed by removing the antibiotic or neutralizing the medium. Growth inhibition was maximal at pH 8.2 and a streptomycin concentration of 800 micrograms/ml. A similar amount of dihydrostreptomycin had a negligible effect, and 10-times-higher concentrations of this antibiotic were required to reproduce the streptomycin action. Addition of streptomycin (400 micrograms/ml) to rpsL cells in alkaline (pH 8.2) nutrient medium caused inhibition of protein and DNA synthesis and also, but to a lower degree, of RNA synthesis. This effect on macromolecular synthesis was not due to ATP deprivation, since ATP content rose after addition of the antibiotic. At pH 8.2, the rate of entrance of streptomycin increased fourfold with respect to the rate at pH 7.0, leading to a large accumulation of streptomycin into rpsL cells. Uptake of the antibiotic was halted by addition of KCN or chloramphenicol. Equal uptake was obtained with 800 micrograms of dihydrostreptomycin or 400 micrograms of streptomycin per ml, yet the former did not affect cell growth at that concentration. It is concluded that high pH stimulates streptomycin and dihydrostreptomycin uptake by rpsL strains but only streptomycin accumulation causes growth inhibition in cells lacking the high-affinity ribosomal site.

A point mutation in the chloroplast 16 S rRNA gene of a streptomycin resistant Nicotiana tabacum

We sequenced a 82 bp fragment in the chloroplast 16 S rRNA gene of the streptomycin resistant mutant SR1 of Nicotiana tabacum. The sequence contains a transition from cytosine to adenine in position 860 of the 16 S rRNA. We suggest that this mutation is sufficient for the streptomycin resistance phenotype in the SR1 mutant.

Somatic Hybridization between Nicotiana tabacum and Nicotiana plumbaginifolia by Electrofusion of Mesophyll Protoplasts

Summary Leaf mesophyll protoplasts of a streptomycin resistant nitrate reductase deficient mutant of Nicotiana tabacum were fused with leaf mesophyll protoplasts of a wild type variety of N. plumbaginifolia in a simple electrofusion apparatus. Somatic hybrid colonies, and plants were readily recovered after fusion by selection of colonies in medium with nitrate as sole nitrogen source and also containing streptomycin. The value of this simple electrofusion apparatus (Watts and King, 1984) lies in its simplicity of construction and use and also in its ability to deal with literally millions of protoplasts in any one experiment.

Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp.

Genetic transformation of auxotrophs of the extreme thermophile Thermus thermophilus HB27 to prototrophy was obtained at high frequencies of 10(-2) to 10(-1) when proliferating cell populations were exposed to chromosomal DNA from a nutritionally independent wild-type strain. The transformation frequency was proportional to the DNA concentration from 10 pg/ml to 100 ng/ml. T. thermophilus HB27 cells did not require chemical treatment to induce competence, although optimal transformation was obtained by the addition of a divalent cation (Ca2+ or Mg2+). Competence was maintained throughout the growth phase, with the highest transformation frequencies at pH 6 to 9 and at 70 degrees C. T. thermophilus HB27 and four other typical Thermus strains, T. thermophilus HB8, T. flavus AT62, T. caldophilus GK24, and T. aquaticus YT1, were also transformed to streptomycin resistance by DNA from their own spontaneous streptomycin-resistant mutants. A cryptic plasmid, pTT8, from T. thermophilus HB8 was introduced into T. thermophilus HB27 Pro- at a frequency of 10(-2).

Somatic hybridization of Nicotiana tabacum and Petunia hybrida

Leaf mesophyll protoplasts of a nitrate reductase deficient streptomycin resistant mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type Petunia hybrida. Somatic hybrid cell colonies were selected for streptomycin resistance and nitrate reductase proficiency. Six independent cell lines, capable of growth in selection medium, were analysed by electrophoresis of callus peroxidases and leucine aminopeptidases and also by hybridization with rDNA and a chloroplast encoded gene as molecular probes. The results show that all six lines represented nuclear somatic hybrids, possessing the chloroplast of N. tabacum, at an early stage of development. However, after 6–12 months in culture, genomic incompatibility was observed resulting in the loss of most of the tobacco nuclear genome in the majority of the cell lines. One of the latter cell lines regenerated plants which possessed the chloroplast of N. tabacum in a predominantly P. hybrida nuclear background.

Enteroadhesion fimbriae and enterotoxin of Escherichia coli: genetic transfer to a streptomycin-resistant mutant of the galE oral-route live-vaccine Salmonella typhi Ty21a.

An enterotoxigenic Escherichia coli plasmid encoding colonization factor antigen (CFA) I fimbriae and heat-stable toxin was transferred into a streptomycin-resistant mutant of the Salmonella typhi galE strain Ty21a (a live attenuated oral typhoid vaccine). The virulence plasmid-carrying transconjugants produced CFA I fimbriae and heat-stable toxin. The marked production of CFA I fimbriae was observed even in a vaccine medium for Ty21a. The data lead to a new type of potential live oral vaccine, S. typhi Ty21a producing enteroadhesion fimbriae.

Mutagenicity of ozone in different repair-deficient strains of Escherichia coli.

The mutagenic activity of ozone was investigated by the isolation of streptomycin-resistant mutants (Smr) in different strains of Escherichia coli. RecA, lexA, polA and parental strains were ozonated and streptomycin-resistant mutants were scored after a short or long phenotypic delay. Our results suggest that ozone is an active mutagen for forward mutation and that this oxidizing agent could be able to induce mutations via two mechanisms: directly and indirectly by the rec-lex error-prone repair system.

Effect of incubation in natural and autoclaved soil upon potency and viability of Bacillus thuringiensis

Spores and parasporal crystals of a Bacillus thuringiensis var. aizawai H-serotype 7, strain HD137, streptomycin-resistant mutant, were added to normal and autoclaved aliquots of pH 5 soil incubated at 25°C and −0.10 MPa water availability. Viable B. thuringiensis in soil samples were estimated by dilution-plating on a streptomycin-based medium, and combined spore and crystal insecticidal activity was bioassayed with larvae of Galleria mellonella. Populations of B. thuringiensis in both soil treatments suffered exponential rates of mortality, which were represented by segmented linear regression. Mortality was far greater in natural than autoclaved soil. Potency also fell in both soil treatments. This loss of potency was greater in natural soil, although the rates of potency loss in either soil treatment correlated poorly with the respective mortality rates of the B. thuringiensis populations, as potency losses were not exponential functions. The results suggest that the presence of indigenous microorganisms in natural soil accelerated the rate of mortality and loss of potency of B. thuringiensis.

Persistence of Bacillus thuringiensis parasporal crystal insecticidal activity in soil

Spores and parasporal crystals of a Bacillus thuringiensis var. aizawai (H-serotype 7), strain HD137, streptomycin-resistant mutant were added to acidic ( pH 5.0) natural and autoclaved soil and incubated at −0.10 MPa, 25°C. Populations of B. thuringiensis in both soil treatments showed exponential rates of mortality which were represented by linear regression, the loss of viability being greater in natural than autoclaved soil. In natural soil, parasporal crystal insecticidal activity was lost at a complex, nonexponential rate. The initial, rapid decrease of activity gradually slowed, and the level of activity stabilized at 10% of the original inoculum level after 250 days incubation, until the cessation of sampling at >2 years. In autoclaved soil no significant ( P > 0.2) loss of parasporal crystal insecticidal activity was detected over the same period, which suggested that soil microorganisms were responsible for the loss of crystal insecticidal activity in the natural, nonsterilized soil. The rate of loss of crystal activity in natural soil correlated well with assay data reported in the literature using Galleria mellonella, which measures the combined activity of spore and crystal. In autoclaved soil correlation was poor, probably due to variability in the bioassay data.

Somatic hybridisation using a double mutant of nicotiana tabacum

Leaf mesophyll protoplasts of a nitrate reductase deficient, streptomycin resistant double mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type N. rustica. Hybrid colonies were selected for nitrate reductase proficiency and streptomycin resistance. Green, actively proliferating colonies were recovered in the selection medium. Eleven of twenty green colonies, transferred to regeneration medium, produced plants. Regenerated plants were analysed for their vegetative and floral characteristics, Fraction 1 protein polypeptide composition and leaf esterases. All the eleven regenerants were nuclear somatic hybrids possessing the chloroplast of the N. tabacum parent. By developing a double mutant with a negative (auxotrophic) and a positive (resistance) selection marker in a species of interest, this species can be hybridised by somatic cell fusion with wild type species lacking selectable markers.

Isolation and Characterization of Fast Acid-Producing Antibiotic-Resistant Mutants of Lactic Streptococci

Single-step streptomycin-resistant mutants of nine strains of lactic streptococci used in commercial Cheddar cheese manufacture were isolated. In each case a 24-h broth culture was concentrated by centrifugation and pour plated in medium containing 600μg/ml of streptomycin. Mutant colonies were selected and subcultured in broth and then streaked onto antibiotic-containing fast-slow differential agar, which allowed isolation of fast acid-producing organisms. A fast rifampin-resistant mutant also was obtained by this technique. Characterization involved comparing parent and mutant strains for growth, acid production, proteolytic abilities, optimum temperatures, and phage sensitivities. The use of this technique to obtain isolates resistant to other antibiotics and their possible use in commercial cheese manufacture to overcome antibiotics in the milk supply is discussed.

Examination of Strain Interaction in a Multiple-Strain Lactic Starter Culture by Streptomycin-Resistant Mutants of Lactic Streptococci

Streptomycin-resistant mutants of lactic streptococci were used to determine strain dominance and suppression in a multiple-strain Cheddar cheese starter culture composed of two Streptococcus lactis and four Streptococcus cremoris strains. This was accomplished by growing the six strains together in six separate batches of nonfat milk and substituting a different streptomycin-resistant mutant in each experiment. Samples were taken periodically and plated onto media with and without added streptomycin. In this manner, the resistant strain in each batch could be identified and counted during growth in mixed culture. Two-strain combinations were studied similarly. Both Streptococcus lactis strains significantly inhibited two of the four Streptococcus cremoris strains, and one instance of Streptococcus cremoris-Streptococcus cremoris inhibition was found. Speculation regarded a dominance pattern in this starter.

Rhizobium strain evaluation of Glycine max cv. Davis, Vigna mungo cv. Regur and V. unguiculata cv. Caloona for three soils in glasshouse and field experiments

The need for inoculation of soybean (Glycine max cv. Davis), gram (Vigna mungo cv. Regur) and cowpea (V. unguiculata cv. Caloona) with six appropriate strains of Rhizobium in two grain legume, clay soil growing areas (Narayen and Emerald, central Queensland) and a Rhizobium free (to sparse) soil (Beerwah, in south-eastern Queensland) was investigated in glasshouse and field experiments. Inoculated plants generally had vegetative and grain yields which were between those of the nitrogen and nil nitrogen uninoculated controls, although for the freely nodulating Vigna species the nil nitrogen control plants were sometimes as large as those in the inoculated treatments. The strains of Rhizobium CB1015 and CB1243 performed well on cowpea and gram as did most strains used as inoculum on soybean. Strain USDA94 was often poor on soybean. In the two heavy clay soils tested, plants inoculated with CB756 and the streptomycin-resistant mutant CB756str developed nitrogen deficiency symptoms in their leaves, suggesting that the strains were not suited to those soil types. There was not a close relationship between the glasshouse and field means due to large residual errors in the field.

Plasmid-coded ampicillin resistance in Haemophilus ducreyi.

Seven of the 96 ampicillin-resistant isolates of Haemophilus ducreyi reported in the preceding article (Bilgeri et al., Antimicrob. Agents Chemother. 22:686-688, 1982) were investigated and found to harbor plasmids of 3.95, 5.2, 5.8, and 6.4 megadaltons. All except the 5.8-megadalton plasmid have been shown to code for beta-lactamase. The 6.4- and 5.2-megadalton plasmids of three isolates were conjugally transferable to a streptomycin-resistant mutant of H. ducreyi at high frequencies, perhaps due to the presence in these strains of a high-molecular-weight plasmid.

Isolation and genetic characterization of streptomycin and chloramphenicol resistant mutants in the mossPhyscomitrella patens

Mutant strains of the mossPhyscomitrella patens that were resistant to the antibiotics streptomycin and chloramphenicol were isolated following UV irradiation. Mutants resistant to streptomycin at 1.7×10−4M showed both dominant and recessive Mendelian and inheritance patterns. Mutants resistant to streptomycin at 1.7 ¢ 10−4M and to chloramphenicol at 3.1×10−4M were, with one exception, cytoplasmically inherited.