Evolutionary biologists have postulated that several fitness advantages may be conferred by the maintenance of duplicate genes, including environmental adaptation resulting from differential regulation. We examined the expression and physiological contributions of two redundant operons in the adaptable bacterium Pseudomonas aeruginosa PA14. These operons, phzA1-G1 (phz1) and phzA2-G2 (phz2), encode nearly identical sets of proteins that catalyze the synthesis of phenazine-1-carboxylic acid, the precursor for several phenazine derivatives. Phenazines perform diverse roles in P. aeruginosa physiology and act as virulence factors during opportunistic infections of plant and animal hosts. Although reports have indicated that phz1 is regulated by the Pseudomonas quinolone signal, factors controlling phz2 expression have not been identified, and the relative contributions of these redundant operons to phenazine biosynthesis have not been evaluated. We found that in liquid cultures, phz1 was expressed at higher levels than phz2, although phz2 showed a greater contribution to phenazine production. In colony biofilms, phz2 was expressed at high levels, whereas phz1 expression was not detectable, and phz2 was responsible for virtually all phenazine production. Analysis of mutants defective in quinolone signal synthesis revealed a critical role for 4-hydroxy-2-heptylquinoline in phz2 induction. Finally, deletion of phz2, but not of phz1, decreased lung colonization in a murine model of infection. These results suggest that differential regulation of the redundant phz operons allows P. aeruginosa to adapt to diverse environments. G ene duplications give rise to genetic redundancy, an unstable condition that would not be expected to persist over time (1). This property is exhibited by diverse genomes (2-4), however, and evolutionary theorists have proposed several mechanisms whereby duplicate genes might provide selective advantages (5); for example, redundancy may be favored when spatial or temporal differences in expression enable tissue-specific variation or survival under varying environmental conditions (6, 7). Products of duplicated genes are involved in crucial cellular processes, including signal transduction, development, and metabolism (8).The genome of the bacterium Pseudomonas aeruginosa, an opportunistic pathogen that thrives in both soil and host environments, contains two redundant seven-gene operons termed phzA1-G1 (phz1) and phzA2-G2 (phz2). These operons are nearly identical (∼98% similarity at the DNA level), and each encodes the biosynthetic enzymes for phenazine-1-carboxylic acid (PCA) (9). Downstream modifications derivatize this precursor, generating other phenazines (SI Appendix, Fig. S1A). Pseudomonad phenazines are toxic to many other organisms and cell types due to their inherent redox activity (10, 11). Studies conducted in various plant and animal models of infection have implicated phenazines in colonization and pathogenicity (12, 13). In addition, recent studies have elucidated beneficial roles...

Integration of EM, protein–protein interaction, and phenotypic data reveals novel insights into the structure and function of the nuclear pore complex’s ∼600-kD heptameric Nup84 complex.

Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of nonspecifically interacting proteins. We describe a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. I-DIRT will be of broad value for studying protein complexes in biological systems that can be metabolically labeled.

Time-resolved ChIP-chip can be utilized to monitor the genome-wide dynamics of the GINS complex, yielding quantitative information on replication fork movement.Replication forks progress at remarkably uniform rates across the genome, regardless of location.GINS progression appears to be arrested, albeit with very low frequency, at sites of highly transcribed genes.Comparison of simulation with data leads to novel biological insights regarding the dynamics of replication fork progression

The selectivity filter of K(+) channels comprises four contiguous ion binding sites, S1 through S4. Structural and functional data indicate that the filter contains on average two K(+) ions at any given time and that these ions reside primarily in two configurations, namely to sites S1 and S3 or to sites S2 and S4. Maximum ion flux through the channel is expected to occur when the energy difference between these two binding configurations is zero. In this study, we have used protein semisynthesis to selectively perturb site 1 within the filter of the KcsA channel through use of an amide-to-ester substitution. The modification alters K(+) conduction properties. The structure of the selectivity filter is largely unperturbed by the modification, despite the loss of an ordered water molecule normally located just behind the filter. Introduction of the ester moiety was found to alter the distribution of K(+), Rb(+,) and Cs(+) within the filter, with the most dramatic change found for Rb(+). The redistribution of ions is associated with the appearance of a partially hydrated ion just external to the filter, at a position where no ion is observed in the wild-type channel. The appearance of this new ion-binding site creates a change in the distance between a pair of K(+) ions some fraction of the time, apparently leading to a reduction in the ion conduction rate. Importantly, this finding suggests that the selectivity filter of a potassium channel is optimized both in terms of absolute ion occupancy and in terms of the separation in distance between the conducting ions.

The K ؉ channel-selectivity filter consists of two absolutely conserved glycine residues. Crystal structures show that the first glycine in the selectivity filter, Gly-77 in KcsA, is in a left-handed helical conformation. Although the left-handed helical conformation is not favorable for the naturally occurring L-amino acids, it is favorable for the chirally opposite D-amino acids. Here, we demonstrate that Gly-77 can be replaced by D-Ala with almost complete retention of function. In contrast, substitution with an L-amino acid results in a nonfunctional channel. This finding suggests that glycine is used as a surrogate D-amino acid in the selectivity filter. The absolute conservation of glycine in the K ؉ -selectivity filter can be explained as a result of glycine being the only natural amino acid that can play this role.K ϩ channels ͉ semisynthesis

Selective ion‐conduction properties in lipid membranes have been found for a semisynthetic KcsA K+ channel containing a mutation (A98G) that allows the channel to open. Thus, it is now possible to study ion conduction in a K+ channel with chemical modification of the main‐chain atoms of the selectivity filter. The picture shows the single‐channel responses of the semisynthetic K+ channel and its structure.

Staphylococcus aureus virulence is coordinated through the Agr quorum-sensing system to produce an array of secreted molecules. One important class of secreted virulence factors is the phenol-soluble modulins (PSMs). PSMs are small-peptide toxins that have recently been characterized for their roles in infection, biofilm development, and subversion of the host immune system. In this work, we demonstrate that the signal peptide of the S. aureus quorum-sensing signal, AgrD, shares structural and functional similarities with the PSM family of toxins. The efficacy of this peptide (termed N-AgrD) beyond AgrD propeptide trafficking has never been described before. We observe that N-AgrD, like the PSMs, is found in the amyloid fibrils of S. aureus biofilms and is capable of forming and seeding amyloid fibrils in vitro. N-AgrD displays cytolytic and proinflammatory properties that are abrogated after fibril formation. These data suggest that the N-AgrD leader peptide affects S. aureus biology in a manner similar to that described previously for the PSM peptide toxins. Taken together, our findings suggest that peptide cleavage products can affect cellular function beyond their canonical roles and may represent a class of virulence factors warranting further exploration.