Streptomycin-resistant Mutants Research Articles

A novel thermostable TP-84 capsule depolymerase: a method for rapid polyethyleneimine processing of a bacteriophage-expressed proteins

BackgroundIn spite of the fact that recombinant enzymes are preferably biotechnologically obtained using recombinant clones, the purification of proteins from native microorganisms, including those encoded by bacteriophages, continues. The native bacteriophage protein isolation is often troubled by large volumes of the infected bacterial cell lysates needed to be processed, which is highly undesired in scaled-up industrial processing. A well-known ammonium sulphate fractionation is often a method of choice during purification of the native bacteriophage protein. However, this method is time-consuming and cumbersome, and requires large amounts of the relatively expensive reagent. Thus, other effective and inexpensive methods of reversible protein precipitation are highly desirable. We have previously characterized thermophilic TP-84 bacteriophage, defined a new genus TP84virus within Siphoviridae family, conducted the TP-84 genome annotation and proteomic analysis. The longest Open Reading Frame (ORF) identified in the genome is TP84_26. We have previously annotated this ORF as a hydrolytic enzyme depolymerizing the thick polysaccharides host’s capsule.ResultsThe TP84_26 ‘capsule depolymerase’ (depolymerase) is a large, 112 kDa protein, biosynthesized by the infected Geobacillus stearothermophilus 10 (G. stearothermophilus 10) cells. The TP84_26 protein biosynthesis was confirmed by three approaches: (i) purification of the protein of the expected size; (ii) mass spectrometry (LC–MS) analysis and (iii) detection of the enzymatic activity toward G. stearothermophilus polysaccharide capsules. Streptomycin-resistant mutant of the host was generated and microbiological aspects of both the TP-84 and G. stearothermophilus 10 were determined. A new variant of polyethyleneimine (PEI)-mediated purification method was developed, using the novel TP-84 depolymerase as a model. The enzyme was characterized. Three depolymerase forms were detected: soluble, unbound proteins in the bacteriophage/cells lysate and another integrated into the TP-84 virion.ConclusionsThe novel TP-84 depolymerase was purified and characterized. The enzyme exists in three forms. The soluble, unbound forms are probably responsible for the weakening of the capsules of the uninfected bacterial cells. The form integrated into virion particles may generate a local passage for the invading TP-84. The developed PEI purification method appears well suited for the scaled-up or industrial production of bacteriophage proteins.

Colonization efficiency of multidrug-resistant Neisseria gonorrhoeae in a female mouse model.

The rapid occurrence of gonococcal resistance to all classes of antibiotics could lead to untreatable gonorrhea. Thus, development of novel anti-Neisseria gonorrhoeae drugs is urgently needed. N. gonorrhoeae FA1090 is the most used in gonococcal infection mouse models because of its natural resistance to streptomycin. Streptomycin inhibits the urogenital commensal flora that permits gonococcal colonization. However, this strain is drug-susceptible and cannot be used to investigate the efficacy of novel agents against multidrug-resistant N. gonorrhoeae. Hence, to test the in vivo efficacy of new therapeutics against N. gonorrhoeae resistant to the frontline antibiotics, azithromycin or ceftriaxone, we constructed streptomycin-resistant mutants of N. gonorrhoeae CDC-181 (azithromycin-resistant) and WHO-X (ceftriaxone-resistant). We identified the inoculum size needed to successfully colonize mice. Both mutants, CDC-181-rpsLA128G and WHO-X-rpsLA128G, colonized the genital tract of mice for 14 days with 100% colonization observed for at least 7 days. CDC-181-rpsLA128G demonstrated better colonization of the murine genital tract compared to WHO-X-rpsLA128G. Lower inoculum of WHO-X-rpsLA128G (105 and 106 CFU) colonized mice better than higher inoculum. Overall, our results indicate that CDC-181-rpsLA128G and WHO-X-rpsLA128G can colonize the lower genital tract of mice and are suitable to be used in mouse models to investigate the efficacy of anti-gonococcal agents.

Site-specific target-modification mutations exclusively induced by the coexposure to low levels of pesticides and streptomycin caused strong streptomycin resistance in clinically relevant Escherichia coli

Environmentally relevant levels of pesticides may promote the emergence of stronger streptomycin-resistant mutants in an Escherichia coli K-12 strain in the presence of sublethal levels of streptomycin. However, it is not clear whether this synergistic effect exists in other strains within and outside the Escherichia genus. Here, we investigated the long-term evolution toward stronger antibiotic resistance under the pesticides and streptomycin coexposure in bacterial strains of three different genera, including pathogenic E. coli strains O157:H7 and O103:H2, one Pseudomonas strain, and one Staphylococcus strain. Consistently, the coexposure induced significantly stronger streptomycin-resistant mutants in the two E. coli strains. However, it did not promote any evolution toward stronger streptomycin resistance in the Pseudomonas and Staphylococcus strains. Site-specific mutations of genes, such as rpsL(Lys88Arg), which encode streptomycin target proteins were exclusively evolved in the coexposed E. coli strains and conferred 80-fold increase in streptomycin resistance. These findings imply that a higher risk of strong and inheritable streptomycin resistance of E. coli strains, including the pathogenic ones, may exist in certain environments where pesticides and antibiotics cooccur.

Physiological Responses of Ribosomal Protein S12 K43 Mutants of Corynebacterium glutamicum.

Bacterial resistance to streptomycin is often acquired as a consequence of mutations in rpsL, the gene encoding ribosomal protein S12. Corynebacterium glutamicum is a non-pathogenic Gram-positive soil bacterium that has been widely used in industry. In a previous study, we screened several streptomycin-resistant rpsL K43 mutants of C. glutamicum, and surprisingly found that two of them also confer chloramphenicol and/or kanamycin resistance. In order to understand whether or not a single mutation of rpsLK43 could confer resistance to multiple antibiotics, in this study we attempted to construct saturation mutagenesis of rpsL K43 by rational genetic manipulation. Despite many efforts had been made, only nine mutants were successfully constructed. They were indeed resistant to streptomycin, but not to other antibiotics. This suggested that other mutations should be acquired, contributing to multiple antibiotics in the screened strains. The growth and enhanced green fluorescent protein (eGFP) expression of these nine mutants were then investigated. The results showed that they grew differently in CGXII minimal medium, but not in BHI medium. When cultured in the absence of streptomycin, the expression of eGFP was positively proportional to the growth, approximately, while in the presence of streptomycin, the expression of eGFP was proportional to the ability of streptomycin resistance.

Unmarked Gene Editing in Clavibacter michiganensis Using CRISPR/Cas9 and 5-Fluorocytosine Counterselection.

Plant-pathogenic bacteria in the genus Clavibacter are important quarantine species that cause considerable economic loss worldwide. The development of effective gene editing techniques and additional selectable markers is essential to expedite gene functional analysis in this important Gram-positive genus. The current study details a highly efficient unmarked CRISPR/Cas9-mediated gene editing system in Clavibacter michiganensis, which couples the expression of cas9 and single-guide RNA with homology-directed repair templates and the negative selectable marker codA::upp within a single plasmid. Initial experiments indicated that CRISPR/Cas9-mediated transformation could be utilized for both site-directed mutagenesis, in which an A to G point mutation was introduced at the 128th nucleotide of the C. michiganensis rpsL gene to generate a streptomycin-resistant mutant, and complete gene knockout, in which the deletion of the C. michiganensis celA or katA genes resulted in transformants that lacked cellulase and catalase activity, respectively. In subsequent experiments, the introduction of the codA::upp cassette into the transformation vector facilitated the counterselection of unmarked transformants by incubation in the absence of the selective antibiotic, followed by plating on M9 agar containing 5-fluorocytosine at 100 μg/ml, in which an unmarked katA mutant lacking the transformation vector was recovered. Compared with conventional homologous recombination, the unmarked CRISPR/Cas9-mediated system was more useful and convenient because it allowed the template plasmid to be reused repeatedly to facilitate the editing of multiple genes, which constitutes a major advancement that could revolutionize research into C. michiganensis and other Clavibacter spp.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Enhancement of angucycline production by combined UV mutagenesis and ribosome engineering and fermentation optimization in Streptomyces dengpaensis XZHG99T

Strain improvement of Streptomyces dengpaensis XZHG99T was performed by combined UV mutagenesis and ribosome engineering, as well as fermentation optimization for enhanced angucycline production (rabelomycin and saquayamycin B1). First, four streptomycin-resistant mutants were obtained after screening of UV mutagenesis and ribosome engineering. Then a rpsL mutant (HTT7) with higher productivity of rabelomycin and saquayamycin B1 was selected according to genetic screening and HPLC/LC-MS analyses, whose maximum titers of rabelomycin and saquayamycin B1 were 3.6 ± 0.02 mg/L and 7.5 ± 0.04 mg/L, respectively, about fourfold higher than those produced by XZHG99T. Next, fermentation optimization of HTT7 was successively carried out by single-factor experiments in shake flasks. The titers of rabelomycin and saquayamycin B1 were increased to 11.2 ± 0.04 mg/L and 20.5 ± 0.02 mg/L after optimization of shake flask fermentation conditions, respectively, which was increased about sixfold compared with those produced by XZHG99T. Finally, the titers of rabelomycin and saquayamycin B1 reached 15.7 ± 0.05 mg/L and 39.9 ± 0.05 mg/L after the scaled-up fermentation, which was 7.8-fold and 11.4-fold higher than those produced by XZHG99T, respectively. These data demonstrate that the combined empirical strain-breeding approaches are still an effective and convenient pathway to improve strain production ability.

Increasing production of the antibiotic-abyssomicin C in Micromonospora maris

Abyssomicin C is a polyketide antibiotic produced by Micromonospora maris AB-18-032. Previous work on abyssomicin C indicated that it inhibits growth of infectious pathogens such as Methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). It does this by suppressing para-aminobenzoic acid (pABA) synthesis, which is required for folic acid biosynthesis in bacteria. This makes abyssomicin C an appealing antibiotic drug, as it is specific only to bacteria. However, its yield in chemical synthesis (4 %) and biosynthesis (60.0 mg/L) are low. Ribosome engineering, through the selection of streptomycin-resistant and rifampin-resistant mutants, of M. maris may result in strains with a higher titer of production. After screening by bioassay and sequencing, the mutant genes in six Ochi mutants were identified. Of these mutants, four of them are able to produce a higher titer of abyssomicin C. The other two strains were detected to produce the other secondary metabolite.

Novel Fredericamycin Variant Overproduced by a Streptomycin-resistant Streptomyces albus subsp. chlorinus Strain.

Streptomycetes are an important source of natural products potentially applicable in the pharmaceutical industry. Many of these drugs are secondary metabolites whose biosynthetic genes are very often poorly expressed under laboratory cultivation conditions. In many cases, antibiotic-resistant mutants exhibit increased production of natural drugs, which facilitates the identification and isolation of new substances. In this study, we report the induction of a type II polyketide synthase gene cluster in the marine strain Streptomyces albus subsp. chlorinus through the selection of streptomycin-resistant mutants, resulting in overproduction of the novel compound fredericamycin C2 (1). Fredericamycin C2 (1) is structurally related to the potent antitumor drug lead fredericamycin A.

Spatial structure increases the benefits of antibiotic production inStreptomyces*

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.

Comparison of the Wild-Type Obligate Methylotrophic Bacterium Methylophilus quaylei and its Isogenic Streptomycin-Resistant Mutant via Metal Nanoparticle Generation

Metal nanoparticles synthesized by green methods with the use of microorganisms are currently one of the most closely studied types of nanomaterials. It has accurately been shown that the characteristics of metal nanoparticles generated in the presence of different bacteria vary. For the two isogenic strains of obligate methylotrophic bacteria of the wild type (M. quaylei MTT) and its streptomycin-resistant mutant (M. quaylei SMR), the pleiotropic character of streptomycin resistance mutation in the SMR cells has been revealed. It has been shown that both cultures can generate silver nanoparticles. There is a dramatic difference in the formation of palladium nanoparticles, which are formed only in the presence of cells of the streptomycin-resistant mutant M. quaylei SMR. This study shows that closely related isogenic strains of obligate methylotrophic bacteria can be distinguished by the spectra of biogenic nanoparticles of two noble metals. While palladium nanoparticles are only generated by the cells of the streptomycin-resistant mutant M. quaylei SMR, biogenic silver nanoparticles can be generated from both cultures. Thus, the assessment of the ability of microorganisms to form biogenic nanoparticles of different metals allows the revelation of subtle metabolic differences of even close cultures.

Differential protein expression of a streptomycin-resistant Streptomyces albulus mutant in high yield production of ε-poly-l-lysine: a proteomics study.

ε-Poly-l-lysine (ε-PL), produced by Streptomyces albulus, is an excellent antimicrobial agent which has been extensively used in the field of food and medicine. In our previous study, we have improved ε-PL production by S. albulus M-Z18 through iterative introduction of streptomycin resistance. To decipher the overproduction mechanism of high-yielding mutant S. albulus SS-62, we conducted a comparative proteomics analysis between S. albulus SS-62 and its parent strain S. albulus M-Z18. Approximately 11.5% of the predicted S. albulus proteome was detected and 401 known or putative regulatory proteins showed statistically differential expression levels. Expression levels of proteins involved in ε-PL precursor metabolism and energy metabolism, and proteins in the pathways related to transcriptional regulation and translation were up-regulated. It was indicated that mutant SS-62 could not only strengthen the ε-PL precursor metabolism and energy metabolism but also tune the pathways related to transcriptional regulation and translation, suggesting a better intracellular metabolic environment for the synthesis of ε-PL in mutant SS-62. To confirm these bioinformatics analyses, qRT-PCR was employed to investigate the transcriptional levels of pls, frr and hrdD and their transcription levels were found to have increased more than 4-fold. Further, overexpression of pls and frr resulted in an increase in ε-PL titer and the yield of ε-PL per unit cell. This report not only represents the first comprehensive study on comparative proteomics in S. albulus, but it would also guide strain engineering to further improve ε-PL production.

Structure of the LPS O-chain from Fusobacterium nucleatum strain ATCC 23726 containing a novel 5,7-diamino-3,5,7,9-tetradeoxy-l-gluco-non-2-ulosonic acid presumably having the d-glycero-l-gluco configuration

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and still births. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report the following structure of the lipopolysaccharide O-chain of a spontaneous streptomycin resistant (SmR) mutant of F. nucleatum strain ATCC 23726:-4-β-Non5Am7Ac-4-β-d-GlcNAcyl3NFoAN-3-β-d-FucNAc4N-where GlcNAcyl3NFoAN indicates 2,3-diamino-2,3-dideoxyglucuronic acid amide with Fo at N-3 being formyl and Acyl at N-2 being propanoyl (∼70%) or butanoyl (∼30%); Non5Am7Ac indicates 7-acetamido-5-acetimidoylamino-3,5,7,9-tetradeoxy-l-gluco-non-2-ulosonic acid presumably having the d-glycero-l-gluco configuration. To our knowledge, no l-gluco isomer of higher sugars of this class as well as no N-propanoyl or N-butanoyl group have so far been found in bacterial polysaccharides.

Molecular Mechanisms of Intrinsic Streptomycin Resistance in Mycobacterium abscessus.

Streptomycin, the first drug used for the treatment of tuberculosis, shows limited activity against the highly resistant pathogen Mycobacterium abscessus We recently identified two aminoglycoside-acetylating genes [aac(2') and eis2] which, however, do not affect susceptibility to streptomycin. This suggests the existence of a discrete mechanism of streptomycin resistance. M. abscessus BLASTP analysis identified MAB_2385 as a close homologue of the 3″-O-phosphotransferase [APH(3″)] from the opportunistic pathogen Mycobacterium fortuitum as a putative streptomycin resistance determinant. Heterologous expression of MAB_2385 in Mycobacterium smegmatis increased the streptomycin MIC, while the gene deletion mutant M. abscessus ΔMAB_2385 showed increased streptomycin susceptibility. The MICs of other aminoglycosides were not altered in M. abscessus ΔMAB_2385. This demonstrates that MAB_2385 encodes a specific and prime innate streptomycin resistance determinant in M. abscessus We further explored the feasibility of applying rpsL-based streptomycin counterselection to generate gene deletion mutants in M. abscessus Spontaneous streptomycin-resistant mutants of M. abscessus ΔMAB_2385 were selected, and we demonstrated that the wild-type rpsL is dominant over the mutated rpsLK43R in merodiploid strains. In a proof of concept study, we exploited this phenotype for construction of a targeted deletion mutant, thereby establishing an rpsL-based counterselection method in M. abscessus.

Влияние условий культивирования Bacillus subtilis ИМВ B-7023 и его стрептомицинустойчивого мутанта на свойства поверхности этих бактерий

Влияние условий культивирования Bacillus subtilis ИМВ B-7023 и его стрептомицинустойчивого мутанта на свойства поверхности этих бактерий

Enhanced ε-poly-L-lysine production by inducing double antibiotic-resistant mutations in Streptomyces albulus.

ε-Poly-L-lysine (ε-PL), as a food additive, has been widely used in many countries. However, its production still needs to be improved. We successfully enhanced ε-PL production of Streptomyces albulus FEEL-1 by introducing mutations related to antibiotics, such as streptomycin, gentamicin, and rifampin. Single- and double-resistant mutants (S-88 and SG-31) were finally screened with the improved ε-PL productions of 2.81 and 3.83g/L, 1.75- to 2.39-fold compared with that of initial strain FEEL-1. Then, the performances of mutants S-88 and SG-31 were compared with the parent strain FEEL-1 in a 5-L bioreactor under the optimal condition for ε-PL production. After 174-h fed-batch fermentation, the ε-PL production and productivity of hyper-strain SG-31 reached the maximum of 59.50g/L and 8.21g/L/day, respectively, which was 138 and 105% higher than that of FEEL-1. Analysis of streptomycin-resistant mutants demonstrated that a point mutation occurred in rpsL gene (encoding the ribosomal protein S12). These single and double mutants displayed remarkable increases of the activities and transcriptional levels of key enzymes in ε-PL biosynthesis pathway, which may be responsible for the enhanced mycelia viability, respiratory activity, and ε-PL productions of SG-31. These results showed that the new breeding method, called ribosome engineering, could be a novel and effective breeding strategy for the evolution of ε-PL-producing strains.

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 (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR 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.

UV - Induced Mutagenesis in Lactic Acid Bacteria

Induced mutagenesis is widely used for selection of microorganisms producing biologically active substances and further improving of their activities. However, it is rarely used toward lactic acid bacteria (LAB) due to their genetic specificity. Determination of LABs sensitivity to UV light and evaluate the effectiveness of UV-induced mutagenesis by positive selection of antibiotic resistant mutants. Bacterial cells grown in LAPTg and NB broth up to late log phase were harvested by centrifugation and resuspended in phosphate buffer (pH 6.8) and irradiated by UV (15 w paired lamps) at distance of 30 cm and plated on LAPTg and NB agars with and without antibiotics for survivors count and positive selection of Rif and Str colonies. It was revealed that lactic acid bacteria were 1 to 2 log more resistant to UV in compression with E. coli. At dose 40 sec the difference in survival between L. lactis and E. coli was not so significant. The yield of rifampicin and streptomycin resistant mutants of LABs vary depending of UV exposure. The maximum yield about 2-3 log of both types of resistant mutants in lactobacilli and lactococcus were observed at dose 20 sec (0.1% survival). LAB possesses intrinsic resistance to UV irradiation 1-2 log higher than E. coli. The maximum yield of UV induced Rif and Str mutants in all LABs occurs at survival about 0.1%. Thus UV rays as an effective mutagen can be used in selection of dairy starters with improved technological and probiotics characteristic.

Improved ethanol tolerance and ethanol production from glycerol in a streptomycin-resistant Klebsiella variicola mutant obtained by ribosome engineering

To improve the ethanol tolerance of the Klebsiella variicola strain TB-83, we obtained the streptomycin-resistant, ethanol-tolerant mutant strain TB-83D by a ribosome engineering approach. Strain TB-83D was able to grow in the presence of 7% (v/v) ethanol and it showed higher ethanol production than strain TB-83. Examination of various culture conditions revealed that yeast extract was essential for ethanol production and bacterial growth. In addition, ethanol production was elevated to 32g/L by the addition of yeast extract; however, ethanol production was inhibited by formate accumulation. With regard to cost reduction, the use of corn steep liquor (CSL) markedly decreased the formate concentration, and 34g/L ethanol was produced by combining yeast extract with CSL. Our study is the first to improve ethanol tolerance and productivity by a ribosome engineering approach, and we found that strain TB-83D is effective for ethanol production from glycerol.

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.

Phenotypic interactions among mutations in a Thermus thermophilus 16S rRNA gene detected with genetic selections and experimental evolution.

During protein synthesis, the ribosome undergoes conformational transitions between functional states, requiring communication between distant structural elements of the ribosome. Despite advances in ribosome structural biology, identifying the protein and rRNA residues governing these transitions remains a significant challenge. Such residues can potentially be identified genetically, given the predicted deleterious effects of mutations stabilizing the ribosome in discrete conformations and the expected ameliorating effects of second-site compensatory mutations. In this study, we employed genetic selections and experimental evolution to identify interacting mutations in the ribosome of the thermophilic bacterium Thermus thermophilus. By direct genetic selections, we identified mutations in 16S rRNA conferring a streptomycin dependence phenotype and from these derived second-site suppressor mutations relieving dependence. Using experimental evolution of streptomycin-independent pseudorevertants, we identified additional compensating mutations. Similar mutations could be evolved from slow-growing streptomycin-resistant mutants. While some mutations arose close to the site of the original mutation in the three-dimensional structure of the 30S ribosomal subunit and probably act directly by compensating for local structural distortions, the locations of others are consistent with long-range communication between specific structural elements within the ribosome.