Jeffrey S. Kavanaugh

Rot is a key regulator of Staphylococcus aureus biofilm formation.

Staphylococcus aureus is a significant cause of chronic biofilm infections on medical implants. We investigated the biofilm regulatory cascade and discovered that the repressor of toxins (Rot) is part of this pathway. A USA300 community‐associated methicillin‐resistant S. aureus strain deficient in Rot was unable to form a biofilm using multiple different assays, and we found rot mutants in other strain lineages were also biofilm deficient. By performing a global analysis of transcripts and protein production controlled by Rot, we observed that all the secreted protease genes were up‐regulated in a rot mutant, and we hypothesized that this regulation could be responsible for the biofilm phenotype. To investigate this question, we determined that Rot bound to the protease promoters, and we observed that activity levels of these enzymes, in particular the cysteine proteases, were increased in a rot mutant. By inactivating these proteases, biofilm capacity was restored to the mutant, demonstrating they are responsible for the biofilm negative phenotype. Finally, we tested the rot mutant in a mouse catheter model of biofilm infection and observed a significant reduction in biofilm burden. Thus S. aureus uses the transcription factor Rot to repress secreted protease levels in order to build a biofilm.

High-resolution X-ray study of deoxy recombinant human hemoglobins synthesized from beta-globins having mutated amino termini.

The crystal structures of three mutant hemoglobins reconstituted from recombinant beta chains and authentic human alpha chains have been determined in the deoxy state at 1.8-A resolution. The primary structures of the mutant hemoglobins differ at the beta-chain amino terminus. One mutant, beta Met, is characterized by the addition of a methionine at the amino terminus. The other two hemoglobins are characterized by substitution of Val 1 beta with either a methionine, beta V1M, or an alanine, beta V1A. All the mutation-induced structural perturbations are small intrasubunit changes that are localized to the immediate vicinity of the beta-chain amino terminus. In the beta Met and beta V1A mutants, the mobility of the beta-chain amino terminus increases and the electron density of an associated inorganic anion is decreased. In contrast, the beta-chain amino terminus of the beta V1M mutant becomes less mobile, and the inorganic anion binds with increased affinity. These structural differences can be correlated with functional data for the mutant hemoglobins [Doyle, M. L., Lew, G., DeYoung, A., Kwiatkowski, L., Noble, R. W., & Ackers, G. K. (1992) Biochemistry preceding paper is this issue] as well as with the properties of ruminant hemoglobins and a mechanism [Perutz, M., & Imai, K. (1980) J. Mol. Biol. 136, 183-191] that relates the intrasubunit interactions of the beta-chain amino terminus to changes in oxygen affinity. Since the structures of the mutant deoxyhemoglobins show only subtle differences from the structure of deoxyhemoglobin A, it is concluded that any of the three hemoglobins could probably function as a surrogate for hemoglobin A.(ABSTRACT TRUNCATED AT 250 WORDS)

Site‐directed mutations of human hemoglobin at residue 35β: A residue at the intersection of the α1β1, α1β2, and α1α2 interfaces

Because Tyr35β is located at the convergence of the α1β1, α1β2, and α1α2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation‐induced changes in functional and structural properties are found for the recombinant hemoglobins βY35F and βY35A. Oxygen equilibrium‐binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35β results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen‐binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in βY35F and increases in βY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in βY35F or βY35A. X‐ray crystal structures of deoxy βY35F and βY35A are highly isomorphous with the structure of wild‐type deoxyhemoglobin. The βY35F mutation repositions the carboxyl group of Asp126α1 so that it may form a more favorable interaction with the guanidinium group of Arg141α2. The βY35A mutation results in increased mobility of the Arg141α side chain, implying that the interactions between Asp126α1 and Arg141α2 are weakened. Therefore, the changes in the functional properties of these 35β mutants appear to correlate with subtle structural differences at the C terminus of the α‐subunit.