María A. Llamas

The Heterologous Siderophores Ferrioxamine B and Ferrichrome Activate Signaling Pathways in Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes two siderophores, pyoverdine and pyochelin, under iron-limiting conditions. These siderophores are recognized at the cell surface by specific outer membrane receptors, also known as TonB-dependent receptors. In addition, this bacterium is also able to incorporate many heterologous siderophores of bacterial or fungal origin, which is reflected by the presence of 32 additional genes encoding putative TonB-dependent receptors. In this work, we have used a proteomic approach to identify the inducing conditions for P. aeruginosa TonB-dependent receptors. In total, 11 of these receptors could be discerned under various conditions. Two of them are only produced in the presence of the hydroxamate siderophores ferrioxamine B and ferrichrome. Regulation of their synthesis is affected by both iron and the presence of a cognate siderophore. Analysis of the P. aeruginosa genome showed that both receptor genes are located next to a regulatory locus encoding an extracytoplasmic function sigma factor and a transmembrane sensor. The involvement of this putative regulatory locus in the specific induction of the ferrioxamine B and ferrichrome receptors has been demonstrated. These results show that P. aeruginosa has evolved multiple specific regulatory systems to allow the regulation of TonB-dependent receptors.

Mutations in each of the tol genes of Pseudomonas putida reveal that they are critical for maintenance of outer membrane stability.

The outer membrane of gram-negative bacteria functions as a permeability barrier that protects cells against a large number of antibacterial agents. OprL protein of Pseudomonas putida has been shown to be crucial to maintain the stability of this cell component (J. J. Rodríguez-Herva, M.-I. Ramos-González, and J. L. Ramos. J. Bacteriol. 178:1699-1706, 1996). In the present study we cloned and mutagenized the orf1, tolQ, tolR, tolA, and tolB genes from P. putida KT2440, which were located upstream of the oprL gene. Polar and nonpolar mutations of the P. putida tolQ, tolR, tolA, and tolB genes were generated in vitro by using the omega-Km(r) interposon, which carries two transcriptional stop signals, or a promoterless xylE cassette, lacking any transcriptional stop signal, respectively. The mutant constructs were used to inactivate, by reverse genetics procedures, the corresponding chromosomal copies of the genes. The phenotype of each mutant strain was analyzed and compared with those of the wild-type strain and the previously characterized P. putida oprL::xylE mutant. All mutant strains exhibited a similar phenotype: altered cell morphology, bleb formation at the cell surface, release of periplasmic and outer membrane proteins to the extracellular medium, increased sensitivity to a variety of compounds (i.e., EDTA, sodium dodecyl sulfate, deoxycholate, and some antibiotics), filament formation, and severely reduced cell motility. Altogether, these results demonstrate the importance of the Tol-OprL system for the maintenance of outer membrane integrity in P. putida and suggest a possible role of these proteins in assembling outer membrane components.

A Novel Extracytoplasmic Function (ECF) Sigma Factor Regulates Virulence in Pseudomonas aeruginosa

Next to the two-component and quorum sensing systems, cell-surface signaling (CSS) has been recently identified as an important regulatory system in Pseudomonas aeruginosa. CSS systems sense signals from outside the cell and transmit them into the cytoplasm. They generally consist of a TonB-dependent outer membrane receptor, a sigma factor regulator (or anti-sigma factor) in the cytoplasmic membrane, and an extracytoplasmic function (ECF) sigma factor. Upon perception of the extracellular signal by the receptor the ECF sigma factor is activated and promotes the transcription of a specific set of gene(s). Although most P. aeruginosa CSS systems are involved in the regulation of iron uptake, we have identified a novel system involved in the regulation of virulence. This CSS system, which has been designated PUMA3, has a number of unusual characteristics. The most obvious difference is the receptor component which is considerably smaller than that of other CSS outer membrane receptors and lacks a β-barrel domain. Homology modeling of PA0674 shows that this receptor is predicted to be a bilobal protein, with an N-terminal domain that resembles the N-terminal periplasmic signaling domain of CSS receptors, and a C-terminal domain that resembles the periplasmic C-terminal domains of the TolA/TonB proteins. Furthermore, the sigma factor regulator both inhibits the function of the ECF sigma factor and is required for its activity. By microarray analysis we show that PUMA3 regulates the expression of a number of genes encoding potential virulence factors, including a two-partner secretion (TPS) system. Using zebrafish (Danio rerio) embryos as a host we have demonstrated that the P. aeruginosa PUMA3-induced strain is more virulent than the wild-type. PUMA3 represents the first CSS system dedicated to the transcriptional activation of virulence functions in a human pathogen.

Characterization of five novel Pseudomonas aeruginosa cell-surface signalling systems

Cell‐surface signalling is a sophisticated regulatory mechanism used by Gram‐negative bacteria to sense signals from outside the cell and transmit them into the cytoplasm. This regulatory system consists of an outer membrane‐localized TonB‐dependent receptor (TonB‐dependent transducer), a cytoplasmic membrane‐localized antisigma factor and an extracytoplasmic function (ECF) sigma factor. Pseudomonas aeruginosa contains 13 potential surface signalling systems of which only six have been studied in detail. In this work we have identified the regulons of five novel P. aeruginosa signalling systems. For that, the ECF sigmas PA0149, PA1912, PA2050, PA2093 and PA4896 have been overexpressed and their target gene candidates have been identified using DNA microarray, proteomic analysis, and/or lacZ reporter construct. All five ECF sigma factors control the production of one TonB‐dependent transducer. Interestingly, two sigma factors, PA2050 and PA2093, regulate the synthesis of a second transducer. Furthermore, we show that although all these sigma factors seem to control putative (metal) transport systems, one of them also regulates the expression of P. aeruginosa pyocins. Finally, we also show that the PA1912‐PA1911‐PA1910 (designated FemI‐FemR‐FemA in this work) signalling system responds to the presence of the Mycobacterium siderophores mycobactin and carboxymycobactin and is involved in the utilization of these heterologous siderophores.

Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane.

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.

Cell-surface signaling in Pseudomonas: stress responses, iron transport, and pathogenicity

Membrane-spanning signaling pathways enable bacteria to alter gene expression in response to extracytoplasmic stimuli. Many such pathways are cell-surface signaling (CSS) systems, which are tripartite molecular devices that allow Gram-negative bacteria to transduce an extracellular stimulus into a coordinated transcriptional response. Typically, CSS systems are composed of the following: (1) an outer membrane receptor, which senses the extracellular stimulus; (2) a cytoplasmic membrane-spanning protein involved in signal transduction from the periplasm to the cytoplasm; and (3) an extracytoplasmic function (ECF) sigma factor that initiates expression of the stimulus-responsive gene(s). Members of genus Pseudomonas provide a paradigmatic example of how CSS systems contribute to the global control of gene expression. Most CSS systems enable self-regulated uptake of iron via endogenous (pyoverdine) or exogenous (xenosiderophores, heme, and citrate) carriers. Some are also implicated in virulence, biofilm formation, and cell-cell interactions. Incorporating insights from the well-characterized alginate regulatory circuitry, this review will illustrate common themes and variations at the level of structural and functional properties of Pseudomonas CSS systems. Control of the expression and activity of ECF sigma factors are central to gene regulation via CSS, and the variety of intrinsic and extrinsic factors influencing these processes will be discussed.

Characterization of the integrated filamentous phage Pf5 and its involvement in small-colony formation

Bacteriophages play an important role in bacterial virulence and phenotypic variation. It has been shown that filamentous bacteriophage Pf4 of Pseudomonas aeruginosa strain PAO1 mediates the formation of small-colony variants (SCVs) in biofilms. This morphology type is associated with parameters of poor lung function in cystic fibrosis patients, and SCVs are often more resistant to antibiotics than wild-type cells. P. aeruginosa strain PA14 also contains a Pf1-like filamentous prophage, which is designated Pf5, and is highly homologous to Pf4. Since P. aeruginosa PA14 produces SCVs very efficiently in biofilms grown in static cultures, the role of Pf5 in SCV formation under these conditions was investigated. The presence of the Pf5 replicative form in total DNA from SCVs and wild-type cells was detected, but it was not possible to detect the Pf5 major coat protein by immunoblot analysis in PA14 SCV cultures. This suggests that the Pf5 filamentous phage is not present at high densities in the PA14 SCVs. Consistent with these results, we were unable to detect coaB expression in SCV cultures and SCV colonies. The SCV variants formed under static conditions were not linked to Pf5 phage activity, since Pf5 insertion mutants with decreased or no production of the Pf5 RF produced SCVs as efficiently as the wild-type strain. Finally, analysis of 48 clinical P. aeruginosa isolates showed no association between the presence of Pf1-like filamentous phages and the ability to form SCVs under static conditions; this suggests that filamentous phages are generally not involved in the emergence of P. aeruginosa SCVs.

Promising biotechnological applications of antibiofilm exopolysaccharides.

Polysaccharides are polymers of carbohydrates with anenormous structural diversity, from long linear repetition ofthe same monomer to highly branched structures of dif-ferent sugars. This high structural diversity reflects thefunctional diversity of these molecules. There are twotypes of polysaccharides, storage polysaccharides (i.e.glycogen) and structural polysaccharides, which are nor-mally secreted by the cell and form different cell structures(i.e. cellulose, chitin). Extracellular polysaccharides orexopolysaccharides belong to this last group.Exopolysaccharides are produced not only by microor-ganisms, but also by algae, plants and animals (Suther-land, 2005). Bacterial exopolysaccharides are a majorcomponent of the extracellular polymeric substance(EPS) or matrix of biofilms, and mediate most of thecell-to-cell and cell-to-surface interactions required forbiofilm formation and stabilization (Flemming and Win-gender, 2010). The matrices of biofilms from natural envi-ronments, such as marine and fresh water, soil, or chronicinfections, contain a ubiquitous composition of polysac-charides. More than 30 different matrix polysaccharideshave been characterized so far. Several are homopoly-saccharides (i.e. glucans, fructans, cellulose), but most ofthese are heteropolysaccharides consisting on a mixtureof sugar residues. Exopolysaccharides can even differbetween strains of single species, as exemplified bystrains of

Transcriptional Organization of the Pseudomonas putida tol-oprL Genes

Proteins of the Tol system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. In Pseudomonas putida, the seven genes, orf1, tolQ, tolR, tolA, tolB, oprL, and orf2, which encode the proteins of this complex, are clustered in a 5.8-kb region of chromosomal DNA. Analysis of polar mutations, reverse transcriptase PCR assays, and transcriptional fusion constructs with a promoterless lacZ gene revealed that the genes are arranged in two operons: orf1 tolQ tolR tolA tolB and oprL orf2. We were also able to find a transcript that was initiated at the orf1 promoter and covered the two operons in a single mRNA. On the basis of the OprL protein level, we surmised that this transcript contributed only about 10 to 15% of the total OprL protein. Primer extension analysis identified the oprL orf2 operon promoter within the tolB gene, and the -10 and -35 regions exhibited some similarity to those of sigma(70)-recognized promoters. The transcription start point of orf1 was located 91 bp upstream of the orf1 start codon, and the -10/-35 region also exhibited sigma(70) -10/-35 recognition sequences. The expression from both promoters in rich and minimal media was constitutive and was very little influenced by the growth phase or iron-deficient conditions. In addition, analyses of the beta-galactosidase activities of different translational fusion constructs revealed that translation of tolA and orf2 genes was dependent on the translation of their corresponding upstream genes (tolR and oprL, respectively).

Antibiotic adjuvants: identification and clinical use

The discovery of penicillin by Alexander Fleming in 1928 changed the course of medicine. Since then, antibiotics have represented virtually the only effective treatment option for bacterial infections. However, their efficacy has been seriously compromised by over-use and misuse of these drugs, which have led to the emergence of bacteria that are resistant to many commonly used antibiotics. Bacteria present three general categories of antibiotic resistance: acquired, intrinsic and adaptive (Alekshun and Levy, 2007). Acquired resistance is the result of mutations in chromosomal genes or the incorporation of new genetic material (plasmids, transposons, integrons, naked DNA) by horizontal gene transfer. It provides selective advantage in the presence of antimicrobial compounds and it is passed on to progeny resulting in the emergence of antibiotic-resistant strains. Bacteria have an extraordinary ability to acquire antibiotic resistance, which is best understood from an evolutionary perspective. Thus, while the use of antibiotics as therapeutics started less than 70 years ago, bacterial resistance mechanisms have co-evolved with natural antimicrobial compounds for billions of years (D’Costa et al., 2011). Bacterial intrinsic resistance to antibiotics is, in contrast to acquired resistance, not related to antibiotic selection but to the specific characteristics of the bacteria. Gram-negative bacteria are for example resistant to many antibiotics due to the presence of a lipopolysaccharide-containing outer membrane with low permeability that functions as an extra barrier preventing the entrance of antibiotics into the cell. Furthermore, many bacteria contain efflux pumps that pump antibiotics out of the cell and thereby decrease their effectiveness. Finally, adaptive resistance involves a temporary increase in the ability of a bacterium to survive an antibiotic, mainly as the result of alterations in gene and/or protein expression triggered by environmental conditions (i.e. stress, nutrient conditions, growth state, subinhibitory levels of the antibiotic) (Poole, 2012). In contrast to intrinsic and acquired resistance mechanisms, which are stable and can be transmitted on to the progeny, adaptive resistance is transient and usually reverts upon the removal of the inducing condition. The combination of these antibiotic resistance mechanisms has led to the emergence of multidrug-resistant pathogens, which are a serious threat for medical care. Among other strategies, the discovery or development of new antibiotic agents had been thought to be a solution to overcome the deficiencies of the existing ones. However, development and marketing approval of new antibiotics have not kept pace with the increasing public health threat of bacterial drug resistance. An alternative to the development of new antibiotics is to find potentiators of the already existing ones, a less expensive alternative to the problem (Ejim et al., 2011; Kalan and Wright, 2011). Potentiators of antibiotic activity are known as antibiotic adjuvants. These compounds are active molecules, preferably with non-antibiotic activity, that in combination with antibiotics enhance the antimicrobial activity of the latter. Combinations of two antibiotics are also considered adjuvants when their effect is synergistic (i.e. the coadministration of the two drugs has a significantly greater effect than that of each antibiotic alone). Antibiotic adjuvants can function either by reversing resistance mechanisms in naturally sensitive pathogens or by sensitizing intrinsic resistant strains. Identification of new molecules that can function as adjuvants is currently an important topic of research. In this context, Taylor and colleagues have recently published a work aimed to identify molecules that potentiate the antimicrobial activity of antibiotics commonly used against Gram-positive bacteria but that have, however, little or no effect on Gramnegative pathogens (Taylor et al., 2012). Using the Gram-negative bacterium Escherichia coli as model in combination with the aminocoumarin antibiotic novobiocin, the authors set up and performed a forward chemical genetic screen with a library of 30 000 small molecules. Three rounds of selection in which molecules that did not enhance novobiocin activity, that had intrinsic antibacterial activity, or that had undesirable secondary *For correspondence. E-mail marian.llamas@eez.csic.es; Tel. (+34) 958181600 ext. 309; Fax (+34) 958129600. Microbial Biotechnology (2013) 6(5), 445–449 doi:10.1111/1751-7915.12044 Funding Information PB acknowledges financial support from the Spanish Ministry of Economy through a Juan de la Cierva postdoctoral-fellowship (JCI-2010-06615), and (MAL through a Ramon&Cajal grant (RYC-2011-08874). bs_bs_banner