Ribosomal Protein S12 and Aminoglycoside Antibiotics Modulate A-site mRNA Cleavage and Transfer-Messenger RNA Activity in Escherichia coli

Translational pausing in Escherichia coli can lead to mRNA cleavage within the ribosomal A-site. A-site mRNA cleavage is thought to facilitate transfer-messenger RNA (tmRNA)·SmpB- mediated recycling of stalled ribosome complexes. Here, we demonstrate that the aminoglycosides paromomycin and streptomycin inhibit A-site cleavage of stop codons during inefficient translation termination. Aminoglycosides also induced stop codon read-through, suggesting that these antibiotics alleviate ribosome pausing during termination. Streptomycin did not inhibit A-site cleavage in rpsL mutants, which express streptomycin-resistant variants of ribosomal protein S12. However, rpsL strains exhibited reduced A-site mRNA cleavage compared with rpsL⁺ cells. Additionally, tmRNA·SmpB-mediated SsrA peptide tagging was significantly reduced in several rpsL strains but could be fully restored in a subset of mutants when treated with streptomycin. The streptomycin-dependent rpsL(P90K) mutant also showed significantly lower levels of A-site cleavage and tmRNA·SmpB activity. Mutations in rpsD (encoding ribosomal protein S4), which suppressed streptomycin dependence, were able to partially restore A-site cleavage to rpsL(P90K) cells but failed to increase tmRNA·SmpB activity. Taken together, these results show that perturbations to A-site structure and function modulate A-site mRNA cleavage and tmRNA·SmpB activity. We propose that tmRNA·SmpB binds to streptomycin-resistant rpsL ribosomes less efficiently, leading to a partial loss of ribosome rescue function in these mutants.     

Improvement of α-Amylase Production by Modulation of Ribosomal Component Protein S12 in Bacillus subtilis 168

The capacity of ribosomal modification to improve antibiotic production by Streptomyces spp. has already been demonstrated. Here we show that introduction of mutations that produce streptomycin resistance (str) also enhances α-amylase (and protease) production by a strain of Bacillus subtilis as estimated by measuring the enzyme activity. The str mutations are point mutations within rpsL, the gene encoding the ribosomal protein S12. In vivo as well as in vitro poly(U)-directed cell-free translation systems showed that among the various rpsL mutations K56R (which corresponds to position 42 in E. coli) was particularly effective at enhancing α-amylase production. Cells harboring the K56R mutant ribosome exhibited enhanced translational activity during the stationary phase of cell growth. In addition, the K56R mutant ribosome exhibited increased 70S complex stability in the presence of low Mg²⁺ concentrations. We therefore conclude that the observed increase in protein synthesis activity by the K56R mutant ribosome reflects increased stability of the 70S complex and is responsible for the increase in α-amylase production seen in the affected strain.     

Targeted Curing of All Lysogenic Bacteriophage from Streptococcus pyogenes Using a Novel Counter-selection Technique

Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (Kan^R) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (Sm^S), into a targeted prophage on the chromosome of a streptomycin resistant (Sm^R) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the Kan^S/Sm^R phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and further elucidate how the presence of prophage may affect overall streptococcal survival, pathogenicity, and evolution.     

Development of Antibiotic-Overproducing Strains by Site-Directed Mutagenesis of the rpsL Gene in Streptomyces lividans

Certain rpsL (which encodes the ribosomal protein S12) mutations that confer resistance to streptomycin markedly activate the production of antibiotics in Streptomyces spp. These rpsL mutations are known to be located in the two conserved regions within the S12 protein. To understand the roles of these two regions in the activation of silent genes, we used site-directed mutagenesis to generate eight novel mutations in addition to an already known (K88E) mutation that is capable of activating antibiotic production in Streptomyces lividans. Of these mutants, two (L90K and R94G) activated antibiotic production much more than the K88E mutant. Neither the L90K nor the R94G mutation conferred an increase in the level of resistance to streptomycin and paromomycin. Our results demonstrate the efficacy of the site-directed mutagenesis technique for strain improvement.     

Use of a Dominant rpsL Allele Conferring Streptomycin Dependence for Positive and Negative Selection in Thermus thermophilus

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.     

Innovative Approach for Improvement of an Antibiotic-Overproducing Industrial Strain of Streptomyces albus

Working with a Streptomyces albus strain that had previously been bred to produce industrial amounts (10 mg/ml) of salinomycin, we demonstrated the efficacy of introducing drug resistance-producing mutations for further strain improvement. Mutants with enhanced salinomycin production were detected at a high incidence (7 to 12%) among spontaneous isolates resistant to streptomycin (Str^r), gentamicin, or rifampin (Rif^r). Finally, we successfully demonstrated improvement of the salinomycin productivity of the industrial strain by 2.3-fold by introducing a triple mutation. The Str^r mutant was shown to have a point mutation within the rpsL gene (encoding ribosomal protein S12). Likewise, the Rif^r mutant possessed a mutation in the rpoB gene (encoding the RNA polymerase β subunit). Increased productivity of salinomycin in the Str^r mutant (containing the K88R mutation in the S12 protein) may be a result of an aberrant protein synthesis mechanism. This aberration may manifest itself as enhanced translation activity in stationary-phase cells, as we have observed with the poly(U)-directed cell-free translation system. The K88R mutant ribosome was characterized by increased 70S complex stability in low Mg²⁺ concentrations. We conclude that this aberrant protein synthesis ability in the Str^r mutant, which is a result of increased stability of the 70S complex, is responsible for the remarkable salinomycin production enhancement obtained.     

Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and “Sarcina lutea” did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and “S. lutea” were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.     

A novel mechanism of streptomycin resistance in Yersinia pestis: Mutation in the rpsL gene

Streptomycin is considered to be one of the effective antibiotics for the treatment of plague. In order to investigate the streptomycin resistance of Y. pestis in China, we evaluated streptomycin susceptibility of 536 Y. pestis strains in China in vitro using the minimal inhibitory concentration (MIC) and screened streptomycin resistance-associated genes (strA and strB) by PCR method. A clinical Y. pestis isolate (S19960127) exhibited high-level resistance to streptomycin (the MIC was 4,096 mg/L). The strain (biovar antiqua) was isolated from a pneumonic plague outbreak in 1996 in Tibet Autonomous Region, China, belonging to the Marmota himalayana Qinghai–Tibet Plateau plague focus. In contrast to previously reported streptomycin resistance mediated by conjugative plasmids, the genome sequencing and allelic replacement experiments demonstrated that an rpsL gene (ribosomal protein S12) mutation with substitution of amino-acid 43 (K43R) was responsible for the high-level resistance to streptomycin in strain S19960127, which is consistent with the mutation reported in some streptomycin-resistant Mycobacterium tuberculosis strains. Streptomycin is used as the first-line treatment against plague in many countries. The emergence of streptomycin resistance in Y. pestis represents a critical public health problem. So streptomycin susceptibility monitoring of Y. pestis isolates should not only include plasmid-mediated resistance but also include the ribosomal protein S12 gene (rpsL) mutation, especially when treatment failure is suspected due to antibiotic resistance.     

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.     

Rational optimization of tolC as a powerful dual selectable marker for genome engineering

Selection has been invaluable for genetic manipulation, although counter-selection has historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport in E. coli, has been implemented as a selectable/counter-selectable marker, but counter-selection escape frequency using colicin E1 precludes using tolC for inefficient genetic manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting counter-selection escape to identify loss-of-function mutations, which offered mechanistic insight and guided strain engineering to reduce counter-selection escape frequency by ∼40-fold. We fundamentally improved the tolC counter-selection by supplementing a second agent, vancomycin, which reduces counter-selection escape by 425-fold, compared colicin E1 alone. Combining these improvements in a mismatch repair proficient strain reduced counter-selection escape frequency by 1.3E6-fold in total, making tolC counter-selection as effective as most selectable markers, and adding a valuable tool to the genome editing toolbox. These improvements permitted us to perform stable and continuous rounds of selection/counter-selection using tolC, enabling replacement of 10 alleles without requiring genotypic screening for the first time. Finally, we combined these advances to create an optimized E. coli strain for genome engineering that is ∼10-fold more efficient at achieving allelic diversity than previous best practices.     

How do combinations of rpsL- and miaA- generate streptomycin dependence?

Summary Petrullo et al. (1983) have studied the consequences of combining a mutation (rpsL ^–) that normally generates streptomycin resistant (Sm^r) ribosomes with a mutation (miaA ^–) that leads to loss of a tRNA hypermodification. They found surprisingly that such doubly mutant bacteria become streptomycin dependent (Sm^d). Here, we show in vitro that ribosomes purified from an Sm^r mutant behave very like Sm^d ribosomes when they are combined with tRNA from an miaA ^– mutant. Our analysis suggests that proofreading becomes excessively intense when the mutant components are combined, and that this reduces the efficiency of translation to the very low levels characteristic of Sm^d ribosomes. We show that Sm increases the efficiency of translation in vitro by suppressing the proofreading flows. We suggest that this will explain the growth stimulatory effect of Sm on the rpsL ^–, miaA ^–double mutants.     

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.     

Action of mutagenic chemicals on Anacystis nidulans

Summary A mutant strain of the blue-green alga Anacystis nidulans showing a 1000-fold increased resistance to streptomycin over the parental strain was isolated following treatment with diethyl sulphate. The mutant strain was found to show profuse filamentation, partial requirement of streptomycin for pigment production, and greater resistance to ultraviolet radiation. It also showed greater sensitivity to dimethyl sulphate than the parental strain. It seems that the streptomycin-resistant strain is a composite one comprising at least four kinds of mutations, namely streptomycin-resistance, filamentation, u.v.-resistance and partial dependence on streptomycin for pigment accumulation. Further. the streptomycin-resistant mutant seems nuclear rather than extra-nuclear in nature.Treatment with diethyl sulphate also conferred a slight increase in resistance to penicillin in this alga.     

The novel mutation K87E in ribosomal protein S12 enhances protein synthesis activity during the late growth phase in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

Resistance to streptomycin in bacterial cells often results from a mutation in the rpsL gene that encodes the ribosomal protein S12. We found that a particular rpsL mutation (K87E), newly identified in Escherichia coli, causes aberrant protein synthesis activity late in the growth phase. While protein synthesis decreased with age in cells in the wild-type strain, it was sustained at a high level in the mutant, as determined using living cells. This was confirmed using an in vitro protein synthesis system with poly(U) and natural mRNAs (GFP mRNA and CAT mRNA). Other classical rpsL mutations (K42N and K42T) tested did not show such an effect, indicating that this novel characteristic is typical of ribosomes bearing the K87E mutant form of S12, although the K87E mutation conferred the streptomycin resistance and error-restrictive phenotypes also seen with the K42N and K42T mutations. The K87E (but not K42N or K42T) mutant ribosomes exhibited increased stability of the 70S complex in the presence of low concentrations of magnesium. We propose that the aberrant activation of protein synthesis at the late growth phase is caused by the increased stability of the ribosome.Communicated by W. Goebel     

Improvement of A21978C production in Streptomyces roseosporus by reporter-guided rpsL mutation selection

Aims:  Daptomycin, one of the A21978C factors produced by Streptomyces roseosporus, is an acidic cyclic lipopeptide antibiotic with potent activity against a variety of Gram‐positive pathogens. To increase the titre of this extensively used and clinically important antibiotic, we applied a reported‐guided rpsL mutation selection system to generate strains producing high levels of A21978C. Methods and Results:  In the reporter design, dptE was chosen as the overexpressing target, and neo‐encoding neomycin phosphotransferase as the reporter. Using this reporter‐guided selection system, 20% of the selected, streptomycin‐resistant mutants produced greater amounts of A21978C than the starting strain. The selection system increased the screening efficiency about 10‐fold with a frequency of 1·7% A21978C overproducing strains among str^r mutants. A21978C production was increased approximately 2·2‐fold in the rpsL K43N mutant. Conclusions:  The combination of ribosome engineering and reporter‐guided mutant selection generated an A21978C overproducing strain that produced about twice as much A21978C as the parental strain. Significance and Impact of the Study:  The strategies presented here, which integrated the advantages of both ribosome engineering and reporter‐guided mutation selection, could be applied to other bacteria to improve their yield of secondary metabolites.     

Streptomycin resistance in Rhizobium japonicum

Mutants resistant to varying concentrations of streptomycin were recovered from two streptomycin-sensitive, effective nitrogen-fixing strains of Rhizobium japonicum. To determine if there were an upper limit of resistible antibiotic concentration, 3 mutants which were resistant to 10000 μg/ml were challenged by higher concentrations of streptomycin. Only one grew well at 25000 μg/ml, and none grew at 50000 μg/ml. All mutants maintained a smooth colonial morphology, and none exhibited streptomycin-dependence. Streptomycin-resistant mutants of both strains were examined for properties of infectivity and effectiveness. All mutants tested retained the symbiotic properties of the parental strains. The retention of these parental properties by the streptomycin-resistant mutants of R. japonicum is different from the properties described for phenotypically similar mutants in certain other rhizobial species.     

Streptomycin Induced Stress Response in Salmonella enterica Serovar Typhimurium Shows Distinct Colony Scatter Signature

We investigated the streptomycin-induced stress response in Salmonella enterica serovars with a laser optical sensor, BARDOT (bacterial rapid detection using optical scattering technology). Initially, the top 20 S. enterica serovars were screened for their response to streptomycin at 100 μg/mL. All, but four S. enterica serovars were resistant to streptomycin. The MIC of streptomycin-sensitive serovars (Enteritidis, Muenchen, Mississippi, and Schwarzengrund) varied from 12.5 to 50 μg/mL, while streptomycin-resistant serovar (Typhimurium) from 125–250 μg/mL. Two streptomycin-sensitive serovars (Enteritidis and Mississippi) were grown on brain heart infusion (BHI) agar plates containing sub-inhibitory concentration of streptomycin (1.25–5 μg/mL) and a streptomycin-resistant serovar (Typhimurium) was grown on BHI containing 25–50 μg/mL of streptomycin and the colonies (1.2 ± 0.1 mm diameter) were scanned using BARDOT. Data show substantial qualitative and quantitative differences in the colony scatter patterns of Salmonella grown in the presence of streptomycin than the colonies grown in absence of antibiotic. Mass-spectrometry identified overexpression of chaperonin GroEL, which possibly contributed to the observed differences in the colony scatter patterns. Quantitative RT-PCR and immunoassay confirmed streptomycin-induced GroEL expression while, aminoglycoside adenylyltransferase (aadA), aminoglycoside efflux pump (aep), multidrug resistance subunit acrA, and ribosomal protein S12 (rpsL), involved in streptomycin resistance, were unaltered. The study highlights suitability of the BARDOT as a non-invasive, label-free tool for investigating stress response in Salmonella in conjunction with the molecular and immunoassay methods.