Controlling the hypermutation: Exploitation of the MutS protein in Pseudomonas aeruginosa

Pseudomonas aeruginosa infections commonly develop in individuals with cystic fibrosis (CF), and its adaptation in such an unfavourable condition is always found to be related to hypermutation. In fact, most of the hypermutation is due to the defects in mutS gene which involves in the mismatch repair mechanism, causing the acceleration of mutation rate and adaptive evolution. In order to rheostatically express the MutS protein and achieve “hypomutation” (in which the rate of mutation is lower than that of wild type strain), an exogenous mutS gene with rhamnose-inducible promoter was cloned into MPAO1 mutS::Tn mutant strain. Present findings demonstrate that this system is tightly-controlled and stable, with less rifampicin-resistant mutant frequency and more fluorescence intensity from a GFP-tagged MutS expressing cells were observed when the concentration of the inducer increases. Interestingly, the results from Western blot analysis show that less MutS protein is required to suppress hypermutation in the wild type strain, as compared to our construct that behaves similar to the wild type but obviously needs more MutS expression to achieve such state. This indicates that the exogenous MutS might be lacking of other important protein to work efficiently in mismatch recognition. Therefore, based on our cDNA analysis, we found that fdxA gene next to the mutS gene is in the same operon, which could suggest that they might be functionally related in the DNA repair machinery.

Inactivation of Genes Encoding MutL and MutS Proteins Influences Adhesion and Biofilm Formation by Neisseria gonorrhoeae

Neisseria gonorrhoeae is an etiological agent of gonorrhea, which remains a global health problem. This bacterium possesses MutL and MutS DNA repair proteins encoded by mutL and mutS genes, whose inactivation causes a mutator phenotype. We have demonstrated the differential gene expression in N. gonorrhoeae mutL and mutS mutants using DNA microarrays. A subset of differentially expressed genes encodes proteins that can influence adhesion and biofilm formation. Compared to the wild-type strain, N. gonorrhoeae mutL and mutS mutants formed denser biofilms with increased biofilm-associated biomass on the abiotic surface. The N. gonorrhoeae mutS::km, but not the mutL mutant, was also more adherent and invasive to human epithelial cells. Further, during infection of epithelial cells with N. gonorrhoeae mutS::km, the expression of some bacterial genes encoding proteins that can influence gonococcal adhesion was changed compared with their expression in cells infected with the wild-type gonococcus, as well as of human genes’ encoding receptors utilized by N. gonorrhoeae (CD46, CEACAM 1, HSPG 2). Thus, deficiency in the mutS gene resulting in increased mutation frequency in singular organisms can be beneficial in populations because these mutants can be a source of features linked to microbial fitness.

Genetic diversity of Ralstonia solanacearum, A phytopathogenic bacterium infecting horticultural plants in Java, Indonesia

Hemelda NM, Safitri R, Suhandono S. 2019. Genetic diversity of Ralstonia solanacearum, A phytopathogenic bacterium infecting horticultural plants in Java, Indonesia. Biodiversitas 20: 364-372. Ralstonia solanacearum is a phytopathogenic bacterium causing bacterial wilt disease which has been reported to infect many important plants in Indonesia. However, less study has been done on its genetic diversity in Indonesia. This study aimed to investigate the genetic diversity of R. solanacearum virulent strains isolated from Java Island. BOX-PCR fingerprint was carried out to investigate the genetic pattern of R. solanacearum isolates, while phylogenetic analysis of mutS gene was performed to determine the genetic diversity of R. solanacearum isolates. A total of 21 isolates was obtained from four crop species: twenty isolates identified as phylotype I while one isolate identified as phylotype II. Based on BOX-PCR, most of the isolates clustered according to their original provinces, indicating site-dependent distribution pattern. However, BOX-PCR also detected site-contaminations indicated by the similar genetic patterns found in two provinces. Phylogenetic analysis of mutS gene discovered that most of the phylotype I isolate showed 100% similarity to each other, while phylotype II isolate belonged to phylotype IIB and showed 100% similarity to IPO1609 strain. Pathogenicity and biovar test were confirmed that the phylotype IIB isolate belonged to the R3bv2 strain which was adapted in low temperature. This study provided the first description about genetic diversity patterns of R. solanacearum strains in Java Island and revealed new challenges related to how to prevent contamination of R. solanacearum from one province to another, as well as the phylotype IIB strain infection in a highland area in Indonesia.

Genetic Diversity and Distribution of Korean Isolates of Ralstonia solanacearum

Genetic diversity among 478 isolates of Ralstonia solanacearum collected from various plants in Korea between 1997 and 2005 was determined based on biovar, pathogenicity, amplified fragment length polymorphism (AFLP), 16S rRNA, endoglucanase, hrpB, and mutS gene sequence analyses. Of the isolates, 440 belonged to biovars 1, 3, or 4, and 38 belonged to biovar 2. Biovar N2 isolates were not found. The biovar 1 and 2 isolates were found mainly in southern Korea, whereas the biovar 3 and 4 isolates were widely distributed throughout all nine provinces. AFLP analysis divided the 109 representative Korean isolates into six clusters that were distinct from most of the foreign isolates. Grouping of 8 representative isolates based on their 16S rRNA gene sequences indicated that biovars 1, 3, and 4 belonged to division 1, while biovar 2 belonged to subdivision 2b. Sequence analysis of the endoglucanase, hrpB, and mutS genes from the same isolates indicated that the biovar 1, 3, and 4 isolates belonged to phylotype I, while the biovar 2 isolate belonged to phylotype IV. This study is the first comprehensive analysis of genetic diversity among Korean isolates of R. solanacearum.

The Yeast HSM3 Gene Acts in One of the Mismatch Repair Pathways

Mutants with enhanced spontaneous mutability (hsm) to canavanine resistance were induced by N-methyl-N-nitrosourea in Saccharomyces cerevisiae. One bearing the hsm3-1 mutation was used for this study. This mutation does not increase sensitivity to the lethal action of different mutagens. The hsm3-1 mutation produces a mutator phenotype, enhancing the rates of spontaneous mutation to canavanine resistance and reversions of lys1-1 and his1-7. This mutation increases the rate of intragenic mitotic recombination at the ADE2 gene. The ability of the hsm3 mutant to correct DNA heteroduplex is reduced in comparison with the wild-type strain. All these phenotypes are similar to ones caused by pms1, mlhl, and msh2 mutations. In contrast to these mutations, hsm3-1 increases the frequency of ade mutations induced by 6-HAP and UV light. Epistasis analysis of double mutants shows that the PMS1 and HSM3 genes control different mismatch repair systems. The HSM3 gene maps to the right arm of chromosome II, 25 cM distal to the HIS7 gene. Strains that bear a deleted open reading frame YBR272c have the genetic properties of the hsm3 mutant. The HSM3 product shows weak similarity to predicted products of the yeast MSH genes (homologs of the Escherichia coli mutS gene). The HSM3 gene may be a member of the yeast MutS homolog family, but its function in DNA metabolism differs from the functions of other yeast MutS homologs.

MUTATOR GENE STUDIES IN ESCHERICHIA COLI: THE mutS GENE

ABSTRACT We report here on a study of a mutator gene (mutS) that causes transition mutations in Escherichia coli. We have used the trpA system to show that A:T→G:C and G:C→A:T transitions occur. Not all A:T pairs are equally susceptible to mutS action however, since the A:T pair at the trpA223 site reverts at a frequency similar to, if not identical with, the frequency in a mut + background. Presumably this is a consequence of neighboring bases, because other A:T pairs are reverted by mutS in the same gene; and an A:T pair in the lac operon is reverted at two widely separated points on the chromosome, and in two orientations relative to the trp sense strand. In addition, we have shown that the mutS1 allele is recessive to wild type, and trans active.