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 FeCl 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 FeCl 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 rhlI, 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 rhlI 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 rhlI mutant on Fe-limited minimal medium and stimulated the twitching motility of rhlI 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.Pseudomonas aeruginosa is an opportunistic human pathogen that causes debilitating infections, particularly in patients with underlying diseases, such as cystic fibrosis (24, 50), where it can cause chronic infections characterized by the formation of biofilms (24,25,42,50,59,77). These surface-associated communities of sessile bacteria embedded in a polysaccharide matrix are important features of many infectious diseases (25,42,45,59), and their characteristic resistance to antimicrobials (antibiotics and biocides) and host immune responses (4, 20, 22, 32, 57, 60, 66) compromises infection control. Details of in vivo P. aeruginosa biofilm formation, structure, and properties are limited, with most of our understanding of these structures coming from the study of model biofilms formed in vitro (3,15,29,30,56,80,81,84). In vitro biofilm development in P. aeruginosa is characterized by bacterial surface attachment, followed by microcolony formation by clonal expansion or motility-driven cell-to-cell aggregation and subsequent formation of a flat, uniform, confluent biofilm or heterogeneous, structured biofilms characterized by cell aggregates or "mushroom" structures separated by channels or ...

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.The biofilm mode of bacterial growth is a surface-attached state in which cells are closely packed and encased in an extracellular polymeric matrix (10, 27). Biofilms are abundant in nature and are of clinical, environmental, and industrial importance. Biofilm development is known to follow a series of complex but discrete and tightly regulated steps (18, 27), including (i) microbial attachment to the surface, (ii) growth and aggregation of cells into microcolonies, (iii) maturation, and (iv) dissemination of progeny cells that can colonize new niches. Over the last decade, several key processes important for biofilm formation have been identified, including quorum sensing (12) and surface motility (28).One of the best-studied model organisms for biofilm development is the bacterium Pseudomonas aeruginosa (10), a notorious opportunistic pathogen which causes many types of infections, including biofilm-associated chronic lung infections in individuals with cystic fibrosis (10, 24, 41). Like most organisms, P. aeruginosa requires iron for growth, as iron serves as a cofactor for enzymes that are involved in many basic cellular functions and metabolic pathways. Recent work has shown that at iron concentrations that are not limiting for growth, this metal serves as a signal for biofilm development (40). Iron limitation imposed, for example, by the mammalian iron chelator lactoferrin blocks the ability of P. aeruginosa biofilms to mature from thin layers of cells attached to a surface into large multicellular mushroom-like biofilm structures (40). By chelating iron, lactoferrin induces twitching motility (a specialized form of surface motility), which causes the cells to move across the surface instead of settling down to form structured communities (39,40). In a recent paper, Berlutti et al. (5) provided further support for the role of iron in cell aggregation and biofilm formation. They reported that in t...

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 coli (FecAE.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 Mg2+ 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 Fe2+ and resultant oxidative stress. Consistent with citrate delivering Fe to cells as Fe2+, a P. aeruginosa mutant lacking the FeoB Fe2+ 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 Fe3+ 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 Fe2+ 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 Fe2+, probably in the periplasm, prior to its transport into cells via Fe transport components.

A null mutation in the mexS gene of Pseudomonas aeruginosa yielded an increased level of expression of a 3-gene operon containing a gene, xenB, whose product is highly homologous to a xenobiotic reductase in Pseudomonas fluorescens shown previously to remove nitro groups from trinitrotoluene and nitroglycerin (D. S. Blehert, B. G. Fox, and G. H. Chambliss, J. Bacteriol. 181:6254, 1999). This expression, which paralleled an increase in mexEF-oprN expression in the same mutant, was, like mexEF-oprN, dependent on the MexT LysR family positive regulator previously implicated in mexEF-oprN expression. As nitration is a well-known result of nitrosative stress, a role for xenB (and the coregulated mexEF-oprN) in a nitrosative stress response was hypothesized and tested. Using s-nitrosoglutathione (GSNO) as a source of nitrosative stress, the expression of xenB and mexEF-oprN was shown to be GSNO inducible, although in the case of xenB, this was seen only for a mutant lacking MexEF-OprN. In both instances, this GSNO-inducible expression was dependent upon MexT. Chloramphenicol, a nitroaromatic antimicrobial that is a substrate for MexEF-OprN, was shown to induce mexEF-oprN but not xenB, again dependent upon the MexT regulator, possibly because it resembles a nitrosated nitrosative stress product accommodated by MexEF-OprN.Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobials (15), a resistance that is increasingly attributable to the operation of broadly specific, tripartite multidrug efflux systems of the resistance-nodulation-division (RND) family (39,40). Several RND family multidrug efflux systems have been described for P. aeruginosa, although the major systems contributing to intrinsic and/or acquired multidrug resistance include MexAB-OprM, MexXY-OprM, 40). Unlike MexAB-OprM and MexXYOprM, which contribute to intrinsic resistance (1,7,26,32), the MexEF-OprN and MexCD-OprJ systems are typically quiescent in wild-type cells (under usual laboratory growth conditions) (19,28,50), with expression and, therefore, a contribution to antimicrobial resistance following a mutational upregulation of the efflux genes (13,20,30,41,48). In the case of MexEF-OprN, this involves the reversion of mutations present in the mexT gene of a number of so-called wild-type strains (29, 30) or a mutation of the mexS gene (also known as qrh [25]), encoding an oxidoreductase of unknown function (48). mexT, which occurs upstream of mexEF-oprN and downstream of mexS, encodes a LysR family positive regulator that promotes mexEF-oprN (25,28,36) and mexS (25) expression. mexEF-oprN expression and modest multidrug resistance have also been reported for a mutant disrupted in the mvaT gene (53), encoding a global regulator of virulence gene expression (8).Originally identified as a determinant of fluoroquinolone resistance (12, 13), MexEF-OprN accommodates a variety of antimicrobials, including trimethoprim and chloramphenicol (28), with chloramphenicol readily selecting multidrug-...

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Expression of the mexXY multidrug efflux operon in wild type Pseudomonas aeruginosa is substantially enhanced by the ribosome-targeting antimicrobial spectinomycin (18-fold) and this is wholly dependent upon the product of the PA5471 gene. In a mutant strain lacking the mexZ gene encoding a repressor of mexXY gene expression, expression of the efflux operon increases modestly (5-fold) and is still responsive (18-fold) to spectinomycin. Spectinomycin induction of mexXY expression in the mexZ mutant is, however, independent of PA5471 suggesting that PA5471 functions as an anti-repressor (dubbed ArmZ for anti-repressor MexZ) that serves only to modulate MexZ's repressor activity, with additional gene(s)/gene product(s) providing for the bulk of the antimicrobial-inducible mexXY expression. Consistent with PA5471/ArmZ functioning as a MexZ anti-repressor, an interaction between MexZ and ArmZ was confirmed using a bacterial 2-hybrid assay. Mutations compromising this interaction (P68S, G76S, R216C, R221W, R221Q, G231D and G252S) were identified and localized to one region of an ArmZ structural model that may represent a MexZ-interacting domain. Introduction of representative mutations into the chromosome of P. aeruginosa reduced (P68S, G76S) or obviated (R216C, R2211W) antimicrobial induction of mexXY gene expression, rendering the mutants pan-aminoglycoside-susceptible. These data confirm the importance of an ArmZ-MexZ interaction for antimicrobial-inducible mexXY expression and intrinsic aminoglycoside resistance in P. aeruginosa.

Screening of a transposon insertion mutant library of Pseudomonas aeruginosa for increased susceptibility to paromomycin identified a number of genes whose disruption enhanced susceptibility of this organism to multiple aminoglycosides, including tobramycin, amikacin, and gentamicin. These included genes associated with lipid biosynthesis or metabolism (lptA, faoA), phosphate uptake (pstB), and two-component regulators (amgRS, PA2797-PA2798) and a gene of unknown function (PA0392). Deletion mutants lacking these showed enhanced panaminoglycoside susceptibility that was reversed by the cloned genes, confirming their contribution to intrinsic panaminoglycoside resistance. None of these mutants showed increased aminoglycoside permeation of the cell envelope, indicating that increased susceptibility was not related to enhanced aminoglycoside uptake owing to a reduced envelope barrier function. Several mutants (pstB, faoA, PA0392, amgR) did, however, show increased cytoplasmic membrane depolarization relative to wild type following gentamicin exposure, consistent with the membranes of these mutants being more prone to perturbation, likely by gentamicin-generated mistranslated polypeptides. Mutants lacking any two of these resistance genes in various combinations invariably showed increased aminoglycoside susceptibility relative to single-deletion mutants, confirming their independent contribution to resistance and highlighting the complexity of the intrinsic aminoglycoside resistome in P. aeruginosa. Deletion of these genes also compromised the high-level panaminoglycoside resistance of clinical isolates, emphasizing their important contribution to acquired resistance.

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