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.     

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.     

Streptomycin affinity depends on 13 amino acids forming a loop in homology modelled ribosomal S12 protein (rpsL gene) of Lysinibacillus sphaericus DSLS5 associated with marine sponge (Tedania anhelans)

Streptomycin, an antibiotic used against microbial infections, inhibits the protein synthesis by binding to ribosomal protein S12, encoded by rpsL12 gene, and associated mutations cause streptomycin resistance. A streptomycin resistant, Lysinibacillus sphaericus DSLS5 (MIC >300 µg/mL for streptomycin), was isolated from a marine sponge (Tedania anhelans). The characterisation of rpsL12 gene showed a region having similarity to long terminal repeat sequences of murine lukemia virus which added 13 amino acids for loop formation in RpsL12; in addition, a K56R mutation which corresponds to K43R mutation present in streptomycin-resistant Escherichia coli is also present. The RpsL12 protein was modelled and compared with that of Lysinibacillus boronitolerans, Escherichia coli and Mycobacterium tuberculosis. The modelled proteins docked with streptomycin indicate compound had less affinity. The effect of loop on streptomycin resistance was analysed by constructing three different models of RpsL12 by, (i) removing both loop and mutation, (ii) removing the loop alone while retaining the mutation and (iii) without mutation having loop. The results showed that the presence of loop causes streptomycin resistance (decreases the affinity), and it further enhanced in the presence of mutation at 56th codon. Further study will help in understanding the evolution of streptomycin resistance in organisms.     

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.     

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.     

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.     

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     

Modulation of allele leakiness and adaptive mutability inEscherichia coli

It is shown that partial phenotypic suppression of two ochre mutations (argE3 andlacZU118) and an amber mutation (inargE) by sublethal concentrations of streptomycin in anrpsL ⁺ (streptomycin-sensitive) derivative of theEscherichia coli strain AB1157 greatly enhances their adaptive mutability under selection. Streptomycin also increases adaptive mutability brought about by theppm mutation described earlier. Inactivation ofrecA affects neither phenotypic suppression by streptomycin nor replication-associated mutagenesis but abolishes adaptive mutagenesis. These results indicate a causal relationship between allele leakiness and adaptive mutability.     

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.     

Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates from China as determined by denaturing HPLC analysis and DNA sequencing

China is regarded by the World Health Organization as a major hot-spot region for Mycobacterium tuberculosis infection. Streptomycin has been deployed in China for over 50 years and is still widely used for tuberculosis treatment. We have developed a denaturing HPLC (DHPLC) method for detecting various gene mutations conferring drug resistance in M. tuberculosis. The present study focused on rpsL and rrs mutation analysis. Two hundred and fifteen M. tuberculosis clinical isolates (115 proved to be streptomycin-resistant and 100 susceptible by a routine proportional method) from China were tested to determine the streptomycin minimal inhibitory concentration (MIC), and subjected to DHPLC and concurrent DNA sequencing to determine rpsL and rrs mutations. The results showed that 85.2% (98/115) of streptomycin-resistant isolates harbored rpsL or rrs mutation, while rpsL mutation (76.5%, 88/115) dominated. MIC of 98 mutated isolates revealed no close correlation between mutation types and levels of streptomycin resistance. No mutation was found in any of the susceptible isolates. The DHPLC results were completely consistent with those of sequencing. The DHPLC method devised in this study can be regarded as a useful and powerful tool for detection of streptomycin resistance. This is the first report to describe DHPLC analysis of mutations in the rpsL and rrs genes of M. tuberculosis in a large number of clinical isolates.     

Detection of Streptomycin Resistance in Mycobacterium tuberculosis Clinical Isolates Using Four Molecular Methods in China

To evaluate the relationship between mutations in rpsL or rrs genes and streptomycin (SM) resistance, we compared four molecular methods for their clinical value in the detection of SM resistance. Genotypic analysis of SM resistance in 167 M. tuberculosis clinical strains isolated from Chinese patients was performed by direct DNA sequencing, SSCP, RFLP, and reverse dot-blot hybridization (RDBH) assays. Of the 98 SM-resistant isolates, 78 (79.6%) had missense mutations in codon 43 or 88 of rpsL resulting in a Lys to Arg substitution, 6 (6.1%) had mutations of the rrs gene at positions 513 A to C or T or 516 C to T, and 14 (14.3%) had the wild-type sequence. None of the 69 SM-susceptible isolates examined had alterations in rpsL or rrs. The results of the SSCP, RFLP, and RDBH analyses for these mutations and wild-type sequences were completely consistent with DNA sequencing data. Five distinct single-nucleotide substitutions in codon 43 or 88 of rpsL gene or in position 513 or 516 of rrs gene were correctly identified in 84 of 98 (85.7%) phenotypically SM-resistant isolates by RDBH assay. Molecular analyses of the rpsL and rrs genes are useful for rapid prediction of SM resistance in most clinical strains of M. tuberculosis. Reverse dot-blot hybridization assay is a rapid, simple, and reliable method for the detection of drug resistance.     

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.     

Comparative genomic analysis of Erwinia amylovora reveals novel insights in phylogenetic arrangement,plasmid diversity,and streptomycin resistance

Erwinia amylovora is a destructive pathogen of Rosaceous plants and an economic concern worldwide. Herein, we report 93 new E. amylovora genomes from North America, Europe, the Mediterranean, and New Zealand. This new genomic information demonstrates the existence of three primary clades of Amygdaloideae (apple and pear) infecting E. amylovora and suggests all three independently originate from North America. The comprehensive sequencing also identified and confirmed the presence of 7 novel plasmids ranging in size from 2.9 to 34.7 kbp. While the function of the novel plasmids is unknown, the plasmids pEAR27, pEAR28, and pEAR35 encoded for type IV secretion systems. The strA-strB gene pair and the K43R point mutation at codon 43 of the rpsL gene have been previously documented to confer streptomycin resistance. Of the sequenced isolates, rpsL-based streptomycin resistance was more common and was found with the highest frequency in the Western North American clade.     

Site-directed mutagenesis of UbiA to promote menaquinone biosynthesis in Elizabethkingia meningoseptica

To increase the menaquinone (MK) content of an Elizabethkingia meningoseptica, site-directed mutagenesis was generated to suppress 4-hydroxybenzoate octaprenyl transferase (UbiA) activity and subsequently blocked the ubiquinone (UQ) biosynthesis pathway. Fourteen conserved residues except L174 and G211 were mutated to analyze the effect of site-directed mutagenesis. The expression of UbiA in twelve mutants was decreased in both mRNA and protein levels, which resulted in the decrease of UQ concentration. Based on MenA expression level, 12 mutants were divided into two groups. Second group such as N72A, D76A, K81A, L139A, and D198A enhanced the expression of MenA, which increased MK production by 127.1%, 87.9%, 96.2%, 109.7% and 130.0% in wt-EmUbiA, respectively. In general, blocking UQ synthesis pathway for by site-directed mutagenesis of the active site of UbiA in E. meningoseptica was a promising strategy to increase MK production in E. meningoseptica.     

The expression of plasmid-determined resistance to ampicillin in colicin K-tolerant mutant of Escherichia coli

A large fraction of colicin K-tolerant mutants of Escherichia coli with R28K plasmid expressed the lowered resistance to ampicillin (Lra phenotype). Transduction experiments have shown that in HC-108 mutant the tolPAB and lra mutations are cotransducible. The tolPAB mutation is responsible specifically for the Lra phenotype due to changes in the cell envelope which result in the elimination of some form of barrier whose effect is to restrict the access of substrate to the antibiotic inactivating β-lactamase. The tolPA mutation has no effect on the stability of R28K. Neither does it affect the susceptibility of the cell to chloramphenicol, kanamycin, and streptomycin nor the resistance to these antibiotics determined by R1drd19.     

Leigh syndrome associated with mitochondrial complex I deficiency due to novel mutations In NDUFV1 and NDUFS2

Leigh syndrome (LS) is a progressive neurodegenerative disease caused by either mitochondrial or nuclear DNA mutations resulting in dysfunctional mitochondrial energy metabolism. Mutations in genes encoding for subunits of the respiratory chain or assembly factors of respiratory chain complexes are often documented in LS cases. Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) enzyme deficiencies account for a significant proportion of mitochondrial disorders, including LS. In an attempt to expand the repertoire of known mutations accounting for LS, we describe the clinical, radiological, biochemical and molecular data of six patients with LS found to have novel mutations in two complex I subunits (NDUFV1 and NDUFS2). Two siblings were homozygous for the previously undescribed R386C mutation in NDUFV1, one patient was a compound heterozygote for the R386C mutation in NDUFV1 and a frameshift mutation in the same gene, one patient was a compound heterozygote for the R88G and R199P mutations in NDUFV1, and two siblings were compound heterozygotes for an undescribed E104A mutation in NDUFS2. After the novel mutations were identified, we employed prediction models using protein conservation analysis (SIFT, PolyPhen and UCSC genome browser) to determine pathogenicity. The R386C, R88G, R199P, and E104A mutations were found to be likely pathogenic, and thus presumably account for the LS phenotype. This case series broadens our understanding of the etiology of LS by identifying new molecular defects that can result in complex I deficiency and may assist in targeted diagnostics and/or prenatal diagnosis of LS in the future.