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Environmental and genetic factors affecting the in vitro spontaneous mutation frequencies to aminoglycoside resistance in Escherichia coli K12 were investigated. Spontaneous mutation frequencies to kanamycin resistance were at least 100 fold higher on modified Luria agar (L2) plates, when compared to results obtained in experiments carried out with Nutrient agar (NA) plates. In contrast to rifampincin, the increased mutability to kanamycin resistance could not be attributed to a mutator phenotype expressed by DNA repair defective strains. Kanamycin mutant selection windows and mutant preventive concentrations on L2 plates were at least fourfold higher than on NA plates, further demonstrating the role of growth medium composition on the mutability to aminoglycosides. Mutability to kanamycin resistance was increased following addition of sorbitol, suggesting that osmolarity is involved on the spontaneous mutability of E. coli K12 strains to aminoglycosides. The spontaneous mutation rates to kanamycin resistance on both L2 and NA plates were strictly associated with the selective antibiotic concentrations. Moreover, mutants selected at different antibiotic concentrations expressed heterogeneous resistance levels to kanamycin and most of them expressing multiple resistance to all tested aminoglycoside antibiotics (gentamicin, neomycin, amykacin and tobramycin). These results will contribute to a better understanding of the complex nature of aminoglycoside resistance and the emergence of spontaneous resistant mutants among E. coli K12 strains.

We show a mechanism by which chaperone proteins can play a key role in maintaining the long-term evolutionary stability of mutation rates in prokaryotes with perfect genetic linkage. Since chaperones can reduce the phenotypic effects of mutations, higher mutation rate, by affecting chaperones, can increase the phenotypic effects of mutations. This in turn leads to greater mutation effect among the proteins that control mutation repair and DNA replication, resulting in large changes in mutation rate. The converse of this is that when mutation rate is low and chaperones are functioning well, then the rate of change in mutation rate will also be low, leading to low mutation rates being evolutionarily frozen. We show that the strength of this recursion is critical to determining the long-term evolutionary patterns of mutation rate among prokaryotes. If this recursion is weak, then mutation rates can grow without bound, leading to the extinction of the lineage. However, if this recursion is strong, then we can reproduce empirical patterns of prokaryotic mutation rates, where mutation rates remain stable over evolutionary time, and where most mutation rates are low, but with a significant fraction of high mutators. [ABSTRACT FROM AUTHOR]

Fungi are frequently exposed to changing environments and must adapt continuously to retain their fitness. This is especially true when changes occur rapidly and toward harsher conditions - a situation, for example, encountered by pathogenic fungi when they infect a host and during the different stages of infections. Here we provide an overview of the role of small-scale genetic exchanges for the fitness of human pathogenic fungi. These alterations can influence host-fungus interactions, and hence the outcome of an infection. We present recent experiments and observations dealing with the importance of the genetic diversity and previous selection pressures of the infecting pool. In addition, we discuss the role of specific niches and disease progression as well as antifungal treatment on fungal microevolution in vivo. We conclude that different mechanisms of small-scale genetic changes allow pathogenic fungi to increase their fitness in the host, and can thus alter the course of infections. [ABSTRACT FROM AUTHOR]

Abstract: The long-term persistance of P. aeruginosa in the cystic fibrosis (CF) lung is characterized by the selection of a variety of genotypes and phenotypes that typically descend from one infecting P. aeruginosa clone, a process known as adaptive radiation. This adaptation process of P. aeruginosa includes complex physiological changes that likely confer a selective advantage to better thrive in the diverse niches and microenvironments of the inflamed and hostile CF airways. The occurrence of P. aeruginosa variants is fixed by mutation and selection. Common loss-of-function mutations in genes such as lasR, mucA and mexT lead to a general adaptation pattern and P. aeruginosa variants with increased antimicrobial resistance, alginate overproduction, reduced acute virulence, and improved metabolic fitness. Strikingly, several virulence-associated traits and immunostimulatory components of P. aeruginosa are turned off. In contrast, other cellular factors are positively selected such as the outer membrane protein OprF, the blue copper protein azurin, the cytochrome c peroxidase c551, and the enzymes of the arginine deiminase pathway ArcA-ArcD. These metabolic components probably are required for the optimal anaerobic or microaerobic growth and viability of P. aeruginosa within CF airways. Besides these common adaptations found by the comparison of P. aeruginosa isolates from different CF patients, the overall diversity of isogenic isolates from one CF patient is extended by variable changes in the expression of regulatory-, transport-, metabolic-, and virulence-associated genes. A better understanding of the microevolution of P. aeruginosa towards niche specialists according the selection pressure in the CF lung is a prerequisite to develop new strategies for the detection of P. aeruginosa variants, the antipseudomonal treatment, the prediction of the infectious disease state, and the development of efficient vaccines. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we investigated the variability of MutS among Pseudomonas aeruginosa recovered from cystic fibrosis (CF) patients. Sequencing of the mutS gene of 15 hypermutable P. aeruginosa isolates obtained from different patients revealed high rates of nucleotide substitutions as compared to that of strain PAO1. Significantly more synonymous than non-synonymous nucleotide substitutions have been found, indicating that generally MutS is highly conserved. The functional analysis of MutS variants by complementation of a PAO1 mutS mutant revealed 5 isolates with a defective MutS due to frameshift mutations or amino acid substitutions. This work supports the hypothesis that the respiratory tract of CF patients represents an environment that favors the selection of highly adaptive mutator phenotypes. [Copyright &y& Elsevier]

Chromosome replication in Escherichia coli is accomplished by the multimeric enzyme DNA polymerase III; the relevance, in vivo, of the ∊ subunit (encoded by dnaQ) for processivity and fidelity of DNA polymerase III has been evaluated. To this aim, dnaQ has been conditionally silenced by means of in vivo expression of different antisense RNAs. Unexpectedly, the presence of the Shine–Dalgarno sequence is essential for the effectiveness of antisense constructs. Silencing of dnaQ induces a severe decrease in growth rate not paralleled by high mutation frequencies, suggesting that the ∊ subunit primarily affects the processivity of DNA polymerase III. [Copyright &y& Elsevier]

The intrinsic chlorophyll-protein CP 47 is a component of Photosystem II which functions in both light-harvesting and oxygen evolution. Using the Escherichia coli mutator strain XL-1 Red, we introduced mutations at 14 sites in the large extrinsic loop E of CP 47 and its adjacent transmembrane α-helix VI. Four mutant cell lines were recovered in which the histidyl residues 455H, 466H and 469H were altered. The cell lines H455T, H455Y, H469Y, and the double mutant F432L,H466R exhibited phenotypes that supported the identification of the histidyl residues 455H, 466H and 469H as chlorophyll ligands. Four additional mutant cell lines were recovered which contained mutations at positions 448R in the large extrinsic loop of CP 47. These mutants, R448K, R448Q, R448S, and R448W, exhibited variable phenotypes ranging from moderate alteration of photoautotrophic growth and oxygen evolution rates to a complete inhibition of these parameters. Those mutants exhibiting photoautotrophic growth and oxygen evolution capability under standard conditions were unable to grow photoautotrophically or evolve oxygen when grown at low chloride concentrations. Finally, a mutant cell line exhibiting a substitution at position 342G was recovered. The mutant G342D exhibited moderate alterations of photoautotrophic growth and oxygen evolution. In addition to these alterations, mutants were recovered in which deletions and insertions (leading to frame shifts) and stop codons were introduced. These mutants uniformly lacked the ability to either grow photoautotrophically or evolve oxygen. [ABSTRACT FROM AUTHOR]