Isogenic strain construction and gene mapping in Candida albicans.

Phagocyte-derived reactive oxygen and nitrogen species are of crucial importance for host resistance to microbial pathogens. Decades of research have provided a detailed understanding of the regulation, generation and actions of these molecular mediators, as well as their roles in resisting infection. However, differences of opinion remain with regard to their host specificity, cell biology, sources and interactions with one another or with myeloperoxidase and granule proteases. More than a century after Metchnikoff first described phagocytosis, and more than four decades after the discovery of the burst of oxygen consumption that is associated with microbial killing, the seemingly elementary question of how phagocytes inhibit, kill and degrade microorganisms remains controversial. This review updates the reader on these concepts and the topical questions in the field.

Antimicrobial reactive oxygen and nitrogen species: concepts and controversies

https://www.cell.com/cell/pdf/0092-8674(95)90105-1.pdf

The Bloom's syndrome gene product is homologous to RecQ helicases

In the past decade, the frequency of diagnosed fungal infections has risen sharply due to several factors, including the increase in the number of immunosuppressed patients resulting from the AIDS epidemic and treatments during and after organ and bone marrow transplants. Linked with the increase in fungal infections is a recent increase in the frequency with which these infections are recalcitrant to standard antifungal therapy. This review summarizes the factors that contribute to antifungal drug resistance on three levels: (i) clinical factors that result in the inability to successfully treat refractory disease; (ii) cellular factors associated with a resistant fungal strain; and (iii) molecular factors that are ultimately responsible for the resistance phenotype in the cell. Many of the clinical factors that contribute to resistance are associated with the immune status of the patient, with the pharmacology of the drugs, or with the degree or type of fungal infection present. At a cellular level, antifungal drug resistance can be the result of replacement of a susceptible strain with a more resistant strain or species or the alteration of an endogenous strain (by mutation or gene expression) to a resistant phenotype. The molecular mechanisms of resistance that have been identified to date in Candida albicans include overexpression of two types of efflux pumps, overexpression or mutation of the target enzyme, and alteration of other enzymes in the same biosynthetic pathway as the target enzyme. Since the study of antifungal drug resistance is relatively new, other factors that may also contribute to resistance are discussed.

Clinical, cellular, and molecular factors that contribute to antifungal drug resistance.

Biochemistry of homologous recombination in Escherichia coli.

The Escherichia coli recQ gene, a member of the RecF recombination gene family, was set in an overexpression plasmid, and its product was purified to near-homogeneity. The purified RecQ protein exhibited a DNA-dependent ATPase and a helicase activity. Without DNA, no ATPase activity was detected. The capacity as ATPase cofactor varied with the type of DNA in the following order: circular single strand greater than linear single strand much greater than circular or linear duplex. As a helicase, RecQ protein displaced an annealed 71-base or 143-base single-stranded fragment from circular or linear phage M13 DNA, and the direction of unwinding seemed to be 3'----5' with respect to the DNA single strand to which the enzyme supposedly bound. Furthermore, the protein could unwind 143-base-pair blunt-ended duplex DNA at a higher enzyme concentration. It is concluded that RecQ protein is a previously unreported helicase, which might possibly serve to generate single-stranded tails for a strand transfer reaction in the process of recombination.

https://www.pnas.org/content/pnas/87/14/5363.full.pdf

Escherichia coli RecQ protein is a DNA helicase.

An Escherichia coli K12 mutant resistant to thymineless death (TLD) was isolated, and its genetic analysis led us to identify a new mutation (recQ1) located between corA and metE on the standard linkage map. The mutation was found to result in increased sensitivity to ultraviolet light and deficiency in conjugational recombination when placed in the recBC sbcB background, indicating that it blocked the RecF pathway of recobbination. It seemed likely that this mutation is also capable of causing partial resistance to TLD, but we reserve the possibility of a separate mutation closely linked to recQ1 giving rise to this phenotype. The original mutant was shown to carry an additional mutation probably in the vicinity of the uhp locus, which was also required for the full TLD resistance of the mutant to be expressed.

Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway.

The recQ gene of Escherichia coli K12 was subcloned from plasmid pKO1 (Oeda et al. 1981) by monitoring the capacity of the resulting recombinant plasmids partially to reverse the increased ultraviolet (UV) sensitivity of a recF143 recQ1 double mutant. We were able to trace this complementation activity to a 3.4 kilobase (kb) SalI-PvuII fragment. Furthermore, analysis of the Tn3 insertion mutations that abolished the complementation revealed the exclusive localisation of such insertions in the same 3.4 kb segment. This segment was situated about 4 kb clockwise from corA on the chromosome, a result consistent with the transductional data previously reported. In addition, a comparison of our restriction endonuclease cleavage map with the published data has placed recQ between pldA and pldB. When relocated to the recQ site on the chromosome, the recQ::Tn3 mutations conferred partial resistance to thymineless death (TLD) or, in the case of a recBC sbcB background, recombination deficiency and increased UV sensitivity. This has provided the firm evidence that both the TLD resistance and the deficiency in the RecF recombination pathway result from loss of the functional recQ gene. We also identified the recQ gene product as a 74 kilodalton polypeptide by using the maxicell technique.

The recQ gene of Escherichia coli K12: molecular cloning and isolation of insertion mutants.

RecQ DNA Helicase of Escherichia coli: Characterization of the Helix-unwinding Activity with Emphasis on the Effect of Single-stranded DNA-binding Protein

Streptococcus pyogenes strains can be divided into two classes, one capable and the other incapable of producing H2O2 (M Saito, S Ohga, M Endoh, H Nakayama, Y Mizunoe, T Hara, and S Yoshida, Microbiology 147:2469-2477, 2001) In the present study, this dichotomy was shown to parallel the presence or absence of H2O2-producing lactate oxidase activity in permeabilized cells Both lactate oxidase activity and H2O2 production under aerobic conditions were detectable only after glucose in the medium was exhausted Thus, the glucose-repressible lactate oxidase is likely responsible for H2O2 production in S pyogenes Of the other two potential H2O2-producing enzymes of this bacterium, NADH and alpha-glycerophosphate oxidase, only the former exhibited low but significant activity in either class of strains This activity was independent of the growth phase, suggesting that the protein may serve in vivo as a subunit of the H2O2-scavenging enzyme NAD(P)H-linked alkylhydroperoxide reductase The activity of lactate oxidase was associated with the membrane while that of NADH oxidase was in the soluble fraction, findings consistent with their respective physiological roles, ie, the production and scavenging of H2O2 Analyses of fermentation end products revealed that the concentration of lactate initially increased with time and decreased on glucose exhaustion, while that of acetate increased during the culture These results suggest that the lactate oxidase activity of H2O2-producing cells oxidizes lactate to pyruvate, which is in turn converted to acetate This latter process proceeds presumably via acetyl coenzyme A and acetyl phosphate with formation of extra ATP

Hiroaki Nakayama

Papers

Escherichia coli RecQ protein is a DNA helicase.

Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway.

The recQ gene of Escherichia coli K12: molecular cloning and isolation of insertion mutants.

RecQ DNA Helicase of Escherichia coli: Characterization of the Helix-unwinding Activity with Emphasis on the Effect of Single-stranded DNA-binding Protein

Hydrogen Peroxide Production in Streptococcus pyogenes: Involvement of Lactate Oxidase and Coupling with Aerobic Utilization of Lactate