A new class of mutations altering the response of the ribosome to streptomycin

Summary Mutants were isolated from high-level streptomycin dependent strains of Escherichia coli B, which do not spontaneously revert to antibiotic independence. In these mutants the requirement for streptomycin was much reduced, but not abolished. The relieving of the antibiotic dependence was caused by qui (for quasi-independent) mutations. These were analogous to the ramA (rpsD) mutations which relieve the streptomycin requirement of other classes of streptomycin dependent mutants, but strains harboring qui mutations exhibited novel streptomycin phenotypes in conjunction with all rpsL (strA) alleles. RamA mutations increase ribosomal misreading; qui mutations either did not significantly alter misreading, or else reduced it.This work was done in partial fulfilment of the requirements for the Ph. D. degree in the Division of Medical Sciences of Harvard University     

Variations in xanthan production by antibiotic-resistant mutants of Xanthomonas campestris

Mutants resistant to different antibiotics (streptomycin, tetracycline, ampicillin and penicillin) were obtained from several strains of Xanthomonas campestris and evaluated for xanthan production. Most of the mutants showed alterations in their polysaccharide production, either increasing, decreasing or totally losing their polymer-production capacity. The existence of two types of antibiotic-resistance mechanisms for the assayed drugs is suggested: one that affects xanthan production and another that does not. Differences in outer-membrane protein patterns of mutants that were simultaneously altered in antibiotic resistance and xanthan production were found, in comparison with their parental strains. These findings suggest the existence of a genetic relationship between antibiotic-resistance mechanisms and xanthan production. Some of the mutants obtained showed significant increases in broth viscosity and xanthan concentration. These results suggest that resistance to streptomycin and ampicillin can be used to obtain improved strains in plate screening assays. Received: 8 January 1997 / Received revision: 13 June 1997 / Accepted: 4 July 1997     

Streptomycin-phosphorylating Enzyme Produced by Streptomyces griseus

Streptomycin-phosphorylating enzyme was reported previously to be produced in mycelium of Streptomyces griseus HUT 6037 at late stage of growth. In the present investigation, this enzyme was purified 200 times as high in specific activity as cell-free extract by means of salting out, chromatography on DEAE-Sephadex A-25 and gel filtration with Sephadex G-100. This enzyme was most stable at pH 8.0 and required 10^?2mMg²⁺ in the reaction mixture for the highest activity. It lost the activity by heat treatment at 40°C for 15 min in absence of the substrate.

Mutant cultures were prepared on productivity of or tolerance to streptomycin, and their capacity to produce streptomycin-phosphorylating enzyme was examined. The cultures which had low to no capacity to produce streptomycin produced a small amount to none of the enzyme, suggesting that production of the streptomycin-phosphorylating enzyme had some correlation with streptomycin productivity of the culture. But no definite correlation was observed between productivity of the enzyme and the capacity to tolerate streptomycin.     

Bacterial ribosomes with two ambiguity mutations: Effects on translational fidelity, on the response to aminoglycosides and on the rate of protein synthesis

Summary A set of mutants affected in translational fidelity was constructed by transduction within an otherwise isogenic Escherichia coli B argF40 argR11 background. Alterations in ribosomal proteins S4, S5, S12 and L6 either as single mutations or in various combinations were compared for their effects on aminoglycoside phenotypes, on in vivo and in vitro mis-reading and on the rate of peptide bond formation. Results may be summarized as follows: (i) Strains carrying two ambiguity mutations on the ribosome without any restrictive mutation are viable. When together, they only weakly increase the level of mistranslation as judged by several in vivo and in vitro test systems. (ii) The combination of two ram mutations causes a very strong cooperative increase of streptomycin sensitivity, irrespective of whether the strains have a wild-type S12 or mutationally altered S12 proteins (of the drug-resistant or-dependent types) on their ribosomes; (iii) The S4 and S5 ram mutations do not alter the response of the ribosome to aminoglycosides of the 2-desoxystreptamine group which are structurally unrelated to streptomycin. This is interpreted in terms of an effect of these ram mutations on the streptomycin binding site but not on the site(s) of binding of the other aminoglycosides. (iv) The rate of polypeptide bond formation which was determined from the kinetics of -galactosidase induction is not significantly changed in strains bearing the ram and the strA (streptomycin-resistant) alleles. In contrast, the L6 and the strA (streptomycin-dependent) alleles strongly reduce the rate of polypeptide elongation which mechanistically might be connected with restriction of ambiguity (Ninio, 1974) in these cases.     

Discrete differences between strains of different Rhizobium spp. for competitive nodule occupancy on beans

Rhizobium etli strain TAL182 and R. leguminosarum bv phaseoli strain 8002, both of which produce melanin pigment, were tested for their nodulation competitiveness on beans by paired inoculation with two strains which do not produce melanin: R. tropici strain CIAT899 and Rhizobium sp. strain TAL1145. An assay was developed to distinguish nodules formed by the melanin-producing and non-producing strains. Strain TAL182 had discrete competitive superiority over CIAT899 and TAL1145 for nodulation of beans. Nodulation competitiveness was not correlated with the ability to produce melanin pigment or the host range of the Rhizobium strains tested.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gillmore 402, Honolulu, HI 96822, USA     

Spatial structure increases the benefits of antibiotic production in Streptomyces*

Bacteria in the soil compete for limited resources. One of the ways they might do this is by producing antibiotics, but the metabolic costs of antibiotics and their low concentrations have caused uncertainty about the ecological role of these products for the bacteria that produce them. Here, we examine the benefits of streptomycin production by the filamentous bacterium Streptomyces griseus. We first provide evidence that streptomycin production enables S. griseus to kill and invade the susceptible species, S. coelicolor, but not a streptomycin-resistant mutant of this species. Next, we show that the benefits of streptomycin production are density dependent, because production scales positively with cell number, and frequency dependent, with a threshold of invasion of S. griseus at around 1%. Finally, using serial transfer experiments where spatial structure is either maintained or destroyed, we show that spatial structure reduces the threshold frequency of invasion by more than 100-fold, indicating that antibiotic production can permit invasion from extreme rarity. Our results show that streptomycin is both an offensive and defensive weapon that facilitates invasion into occupied habitats and also protects against invasion by competitors. They also indicate that the benefits of antibiotic production rely on ecological interactions occurring at small local scales.     

Antibacterial and Antifungal Spectra of Antibiotics Produced by Different Strains of Erwinia herbicola (= Pantoea agglomerans)

Nine antibiotic producer strains of Erwinia herbicola (=Pantoea agglomerans), belonging to different groups, strongly inhibited growth of 21 streptomycin sensitive strains and 6 streptomycin resistant strains of E. amylovora. The antibacterial spectra of antibiotics produced by the tested strains of E. herbicola were mainly limited to E. amylovora and related tested species. The tested strains of E. amylovora that are resistant to streptomycin did not show cross-resistance to the different types of antibiotics produced by the tested strains of E. herbicola. The antibiotics produced by the different tested strains of E. herbicola did not exert any activity on tested fungi with the exception that strains Eh 153 and Eh 351 slightly inhibited the growth of Verticillium dahliae.     

Entamoeba histolytica: Collagenolytic Activity and Virulence1

Several axenic strains of pathogenic and nonpathogenic Entamoeba histolytica were tested for their capacity to digest native radioactive type I collagen gels and to produce liver abscesses when injected into the liver of newborn hamsters. The results demonstrate that the pathogenic strains of amebas (HM1:IMSS, HM3:IMSS, HM38:IMSS, and HK9) have a collagenolytic activity that closely correlates with their in vivo capacity to produce liver lesions. The nonpathogenic isolate (Laredo) did not show collagenolytic activity and failed to produce lesions in the liver of newborn hamsters. The results also demonstrate that type I collagen obtained from rodents and cats is degraded less by amebic collagenase than is bovine collagen, which is similar to human collagen. These findings suggest that species susceptibility to invasive infection may depend, among other factors, on the characteristics of the extracellular components of host tissues.     

Rhizobitoxine: A phytotoxin of unknown function which is commonly produced by bradyrhizobia

Summary Fifty-six percent of 93 strains ofBradyrhizobium japonicum andBradyrhizobium sp. (various hosts) from diverse geographical areas were found to produce a chlorosis-inducing toxin. Toxin production was common among bradyrhizobia originating from the USA, Africa, Central America, and South America. Toxin produced by West African strains was compared with rhizobitoxine by cation exchange chromatography, paper chromatography, and soybean (Glycine max (L.) Merr.) bioassay. The comparison suggested that the chlorosis-inducing toxin produced by West African bradyrhizobia is rhizobitoxine. Purified toxin from a West AfricanBradyrhizobium sp. (Vigna) strain inhibited the growth ofBacillus subtilis on minimal medium. The growth inhibition was reduced by addition of yeast-extract or casamino acids but not by any of 21 individual amino acids, including methionine. The same toxin did not inhibit the growth of 14 Bradyrhizobium strains, including eight strains that did not produce toxin. Mixed inoculum experiments revealed that a toxin-producing West African strain could not assist toxin non-producingB. japonicum strains in nodulating non-nodulating (rj₁ rj₁) soybeans.     

Phenotypic suppression by streptomycin of amber mutants in the ribonucleic Acid bacteriophage coat protein cistron

After mutagenesis with nitrosoguanidine or ultraviolet light, 298 streptomycin high-resistant and 98 streptomycin high-dependent mutants were isolated from HfrC Su⁻. They were tested for their ability to phenotypically suppress five different amber ribonucleic acid (RNA) bacteriophage mutants in the presence of streptomycin. The phage mutants are all in the coat protein, which is 129 amino acids long; the uracil-adenine-guanine codons were at the following positions: sus3 and amB2, 6; amB11, 50; amB21, 54; sus11, 70. Only sus3 and amB2 could be phenotypically suppressed by streptomycin; this was clearly demonstrated in nine mutant strains, seven str-HR and two str-HD. The suppression was always dependent upon added streptomycin and was dose-dependent in all cases. None of the mutants showed measurable suppression in absence of the drug. Among revertants to streptomycin independence from streptomycin-dependent strains that could show phenotypic suppression, most of those that were still resistant to streptomycin (10 μg or more) retained the capacity to show phenotypic suppression; whereas among those revertants sensitive to 10 μg of streptomycin or less, none retained the capacity. Eight different amber polar mutants (strong and weak) in gene 34 of phage T4 were also tested for pleiotypic suppression by streptomycin in all the streptomycin-resistant and -dependent strains isolated. No suppression was found in any of the 396 strains tested.     

Distribution of Streptomycin Resistant Strains of Erwinia amylovora in Egypt during 1988

A survey of the occurrence of strains of Erwinia amylovora resistant to streptomycin in certain Egyptian pear orchards was earned out during April and May 1988. Twenty-two isolates out of 604 isolates collected from 11 orchards showed resistance to streptomycin. All the streptomycin resistant (Str^r) strains isolated in the present work were resistant to high levels of streptomycin with minimal inhibitory concentrations ranging from 1000 to 3000 μg/ml. The occurrence of Str^r strains in Egypt is still limited and the population of resistant strains was at relatively low level. However, such occurrence of E. amylovora with resistance to streptomycin is a potentially serious situation.     

Mutants of the moss Physcomitrella patens which produce leaves of altered morphology

Abstract

After screening large numbers of survivors of mutagenic treatment with MNNG, 165 strains of the moss Physcomitrella patens altered in stem or leaf development were isolated. Of these, seven strains which produce leaves of greatly reduced size and cell number were analysed genetically, using protoplast fusion to obtain hybrids for dominance and complementation tests, since the strains are sterile. Three of the mutant alleles are recessive to the wild-type allele, but this is not true of the other four. Four of the mutant alleles can be assigned to one complementation group, and two to a second group.     

Dissociation of peptidyl-tRNA from ribosomes is perturbed by streptomycin and by strA mutations

Summary Peptidyl-tRNA may dissociate preferentially from ribosomes during protein synthesis when it is inappropriate to, does not correctly complement, the messenger RNA. To test this idea, growing cultures of Escherichia coli were treated with streptomycin to increase the frequency of errors during protein synthesis. Since the treated cells had a temperature-sensitive peptidyl-tRNA hydrolase and could not destroy dissociated peptidyl-tRNA, it was possible to measure the rate of its accumulation after raising the temperature to non-permissive conditions. Both low and high doses of streptomycin enhanced the rate of dissociation and accumulation of peptidyl-tRNA. The rank order of rates of dissociation/accumulation of various isoaccepting tRNA families was not significantly altered by the drug treatment. We concluded that streptomycin stimulated a normal pathway for dissociation of peptidyl-tRNA. Two streptomycin-resistent strains of E. coli had higher rates of dissociation of peptidyl-tRNA than did their sensitive parent strain. When treated with high doses of the drug, the resistant strains showed slightly reduced rates of dissociation of peptidyl-tRNA. These results were interpreted in terms of a two state, two site model for protein synthesis: streptomycin enhances the binding of aminoacyl-tRNA to a tight state of the ribosome A site; the strA mutation enhances translocation to a loose state of the ribosome P site.     

Alcohol metabolism in Drosophila melanogaster: Uselessness of the most active aldehyde oxidase produced by the Aldox locus

Alcohol dehydrogenase is necessary for ethanol detoxification and metabolic utilization. It has been generally assumed that aldehyde oxidase (AO) produced by the Aldox locus (3–56.7) is necessary for a further transformation of acetaldehyde into acetate. We find that various mutant strains (ma-l or Aldox ⁿ) which do not produce an active enzyme show about the same tolerance to alcohol as do wild strains. This physiological paradox is probably to be explained by the discovery of another locus (not localized) which produced a small amount of AO in all tested strains. The adaptive significance of the genetically polymorphic Aldox locus is probably to be looked for in physiological pathways other than ethanol metabolism.     

Transfer of Streptomycin Biosynthesis Gene Clusters within Streptomycetes Isolated from Soil

Streptomyces strains isolated from soil were found to possess various numbers of genes from the streptomycin biosynthesis cluster. The strains missing genes from the cluster also lacked the ability to produce streptomycin. Two of the isolates which contain only part of the cluster are apparently recipients of a gene transfer event. The implications for the role of gene transfer in antibiotic evolution are discussed.     

Functional properties of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from dairy products

Twenty-four acid- and bile-tolerant lactobacilli isolates from dairy products were identified and further in vitro characterized for the presence of functional traits potentially useful for probiotic applications, which included desirable and undesirable traits, such as biofilm formation, ability to inhibit intestinal pathogens, antibiotic susceptibility, and enzyme activity. The majority of examined strains were susceptible to certain antimicrobial agents (streptomycin, gentamicin, clindamycin, erythromycin, tetracycline, quinupristin–dalfopristin), except for three strains of Lactobacillus rhamnosus with minimal inhibitory concentration levels for streptomycin higher than the microbiological breakpoints (≥32 μg/mL), which are considered as resistant. Undesirable traits such as α-chymotrypsin or N-acetyl-β-glucosaminidase activities were not detected, but low β-glucuronidase, and moderate and high β-glucosidase activities were recorded in nine strains, which were eliminated from further examination together with three isolates showing unsuitable antibiotic resistance. Of the remaining 12 isolates, 4 (Lactobacillus fermentum 202, Lactobacillus gallinarum 7001, L. rhamnosus 183, and Lactobacillus plantarum L2-1) manifested an outstanding potential to inhibit selected intestinal pathogens in an agar spot test, including Escherichia coli and Salmonella spp., and simultaneously demonstrated strong biofilm-forming capacity. In conclusion, the results of our in vitro experiments showed that the above four strains had a potential probiotic value and met the criteria to be identified as a possible probiotic microorganism, with the necessity of verification through well-designed in vivo experimental, clinical, and technological studies before the strains can be used as probiotics or as starter probiotic cultures.     

Coevolution of antibiotic production and counter‐resistance in soil bacteria

We present evidence for the coexistence and coevolution of antibiotic resistance and biosynthesis genes in soil bacteria. The distribution of the streptomycin (strA) and viomycin (vph) resistance genes was examined in Streptomyces isolates. strA and vph were found either within a biosynthetic gene cluster or independently. Streptomyces griseus strains possessing the streptomycin cluster formed part of a clonal complex. All S. griseus strains possessing solely strA belonged to two clades; both were closely related to the streptomycin producers. Other more distantly related S. griseus strains did not contain strA. S. griseus strains with only vph also formed two clades, but they were more distantly related to the producers and to one another. The expression of the strA gene was constitutive in a resistance‐only strain whereas streptomycin producers showed peak strA expression in late log phase that correlates with the switch on of streptomycin biosynthesis. While there is evidence that antibiotics have diverse roles in nature, our data clearly support the coevolution of resistance in the presence of antibiotic biosynthetic capability within closely related soil dwelling bacteria. This reinforces the view that, for some antibiotics at least, the primary role is one of antibiosis during competition in soil for resources.     

recB recJ mutants ofSalmonella typhimurium are deficient in transductional recombination, DNA repair and plasmid maintenance

recB recJ mutants ofSalmonella typhimurium are deficient in transduction of chromosomal markers and ColE1-derived plasmids, and also in the maintenance of ColE1 and F plasmids. Plasmid instability is less severe inrecD recJ strains; ColE1 plasmid DNA preparations from these strains show an increased yield of high molecular weight (HMW) linear multimers and a concomitant reduction in plasmid monomers compared to the wild type. Plasmids remain unstable inrecA recD recJ mutants; since these do not produce HMW linear concatemers, we propose that a decrease in monomer production leads to plasmid instability.recB recJ strains also display decreased viability, a component of which may be related to their deficiency in DNA repair. In contrast to their severe defects in recombination, DNA repair and plasmid maintenance,recB recJ mutants ofS. typhimurium behave similarly to the wild type in the segregation of chromosome duplications. The latter observation suggests that neither RecBCD nor RecJ functions are required for chromosomal recombination events that do not involve the use of free ends as recombination substrates.     

Physiological and biochemical contributions to the taxonomy of the genus Prototheca

16 strains of the genus Prototheca do not produce extracellular amylolytic enzymes. The base composition of their DNA shows rather continuous values from 62% to 78% GC (guanine + cytosine). Their assignment to four species and their possible relationship with Chlorella protothecoides are discussed.Abbreviations Used GC guanine + cytosine - SSC saline sodium citrate     

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.