Search

The fundamental transformation of American politics can be summed up by the recent history of a single Senate seat. In 1991, Pennsylvania's three-term senator John Heinz was killed in a light plane accident. A Republican, he compiled a moderate record as his party's leading supporter of environmental and labor union causes. In the special election that followed, the Republicans ran another relatively moderate candidate, Richard Thornburgh a former governor and US attorney general, against Harris Wofford, the interim senator. Wofford, who began his career as the first associate director of the Peace Corps, was significantly more liberal than Heinz or Thornburgh was conservative. In a campaign orchestrated by the then relatively unknown James Carville, Wofford ran a platform of fundamental reform of the U.S. Healthcare system. This electoral strategy was wildly successful as Thornburgh was beaten easily and healthcare became the "hot" issue going into the 1992 presidential elections.

A general approach to least-squares unidimensional scaling is presented. Ordering information contained in the parameters is used to transform the standard squared error loss function into a discrete rather than continuous form. Monte Carlo tests with 38,094 ratings of 261 senators, and 1,258 representatives demonstrate the procedure's application. (SLD)

Iron is an essential element for life but also serves as an environmental signal for biofilm development in the opportunistic human pathogen Pseudomonas aeruginosa. Under iron-limiting conditions, P. aeruginosa displays enhanced twitching motility and forms flat unstructured biofilms. In this study, we present evidence suggesting that iron-regulated production of the biosurfactant rhamnolipid is important to facilitate the formation of flat unstructured biofilms. We show that under iron limitation the timing of rhamnolipid expression is shifted to the initial stages of biofilm formation (versus later in biofilm development under iron-replete conditions) and results in increased bacterial surface motility. In support of this observation, an rhlAB mutant defective in biosurfactant production showed less surface motility under iron-restricted conditions and developed structured biofilms similar to those developed by the wild type under iron-replete conditions. These results highlight the importance of biosurfactant production in determining the mature structure of P. aeruginosa biofilms under iron-limiting conditions. doi: 10.1128/JB.01601-09

The PA5471 gene required for induction of the MexXY multidrug efflux system in response to ribosometargeting antimicrobials was itself shown to be inducible by ribosome-targeting antimicrobials (Y. Morita, M. L. Sobel, and K. Poole, J. Bacteriol. 188:1847-1855, 2006). Using a lacZ transcriptional reporter, drug inducibility of PA5471 was shown to require the entirety of the 367-bp PA5472-PA5471 intergenic region. A constitutive promoter activity was, however, localized to the first 75 bp of this region, within which a single PA5471 transcription initiation site was mapped. That 3' sequences of the intergenic region blocked PA5471 expression and made it antibiotic dependent was suggestive of an attenuation mechanism of control. A 13-amino-acid leader peptide (LP)-encoding open reading frame preceded by a Shine-Dalgarno sequence was identified ca. 250 bp upstream of the PA5471 coding sequence, and its expression and translation were confirmed using a lacZ translational reporter. Alteration of the initiation codon (M1T) or introduction of translational stop signals at codons 3 (Q3Am) and 8 (C8Op) of this LP sequence (PA5471.1) yielded high-level constitutive expression of PA5471, suggesting that interference with LP translation was linked to PA5471 gene expression. Consistent with this, a Q3K mutation in the LP sequence maintained the drug inducibility of PA5471 expression. Introduction of the LP Q3Am mutation into the chromosome of Pseudomonas aeruginosa yielded stronger expression of PA5471 than did antibiotic (chloramphenicol) exposure of wild-type P. aeruginosa, in agreement with lacZ transcriptional fusion data. Still, the Q3Am mutation yielded modest expression of mexXY, less than that seen for antibiotic-treated wild-type P. aeruginosa. These data suggest that PA5471 is not sufficient for MexXY recruitment in response to antibiotic exposure and that additional antibiotic-dependent effects are needed.

In an attempt to identify components of a ferric citrate uptake system in Pseudomonas aeruginosa, a mutant library of a siderophore-deficient strain (IA614) was constructed and screened for defects in citrate-promoted growth in an Fe-restricted medium. A mutant disrupted in gene PA3901, encoding a homologue of the outer-membrane ferric citrate receptor, FecA, of Escherichia coil ([FecA.sub.E.c.]), was recovered and shown to be deficient in citrate-promoted growth and citrate-mediated Fe uptake. A mutant disrupted in gene PA4825, encoding a homologue of the MgtA/MgtB [Mg.sup.2+] transporters in Salmonella enterica, was similarly deficient in citrate-promoted growth, though this was due to a citrate sensitivity of the mutant apparently resulting from citrate-promoted acquisition of [Fe.sup.2+] and resultant oxidative stress. Consistent with citrate delivering Fe to cells as [Fe.sup.2+], a P. aeruginosa mutant lacking the FeoB [Fe.sup.2+] transporter homologue, PA4358, was compromised for citrate-promoted growth in Fe-restricted medium and showed markedly reduced citrate-mediated Fe uptake. Subsequent elimination of two [Fe.sup.3+] transporter homologues, PA5216 and PA4687, in the feoB mutant failed to further compromise citrate-promoted growth or Fe uptake, though the additional loss of pcoA, encoding a periplasmic ferroxidase implicated in [Fe.sup.2+] acquisition, completely abrogated citrate-mediated Fe uptake. Fe acquisition mediated by other siderophores (e.g. pyoverdine) was, however, unaffected in the quadruple knockout strain. These data indicate that Fe delivered to P. aeruginosa by citrate is released as [Fe.sup.2+], probably in the periplasm, prior to its transport into cells via Fe transport components.

Reducing iron (Fe) levels in a defined minimal medium reduced the growth yields of planktonic and biofilm Pseudomonas aeruginosa, though biofilm biomass was affected to the greatest extent and at Fe[Cl.sub.3] concentrations where planktonic cell growth was not compromised. Highlighting this apparently greater need for Fe, biofilm growth yields were markedly reduced in a mutant unable to produce pyoverdine (and, so, deficient in pyoverdine-mediated Fe acquisition) at concentrations of Fe[Cl.sub.3] that did not adversely affect biofilm yields of a pyoverdine-producing wild-type strain. Concomitant with the reduced biofilm yields at low Fe concentrations, P. aeruginosa showed enhanced twitching motility in Fe-deficient versus Fe-replete minimal media. A mutant deficient in low-Fe-stimulated twitching motility but normal as regards twitching motility on Fe-rich medium was isolated and shown to be disrupted in rhll, whose product is responsible for synthesis of the N-butanoyl homoserine lactone (C4-HSL) quorum-sensing signal. In contrast to wild-type cells, which formed thin, flat, undeveloped biofilms in Fe-limited medium, the rhll mutant formed substantially developed though not fully mature biofilms under Fe limitation. C4-HSL production increased markedly in Fe-limited versus Fe-rich P. aeruginosa cultures, and cell-free low-Fe culture supernatants restored the twitching motility of the rhll mutant on Fe-limited minimal medium and stimulated the twitching motility of rhll and wild-type P. aeruginosa on Fe-rich minimal medium. Still, addition of exogenous C4-HSL did not stimulate the twitching motility of either strain on Fe-replete medium, indicating that some Fe-regulated and RhlI/C4-HSL-dependent extracellular product(s) was responsible for the enhanced twitching motility (and reduced biofilm formation) seen in response to Fe limitation.