Patricia Bernal

Solvent tolerance in Gram-negative bacteria

Bacteria have been found in all niches explored on Earth, their ubiquity derives from their enormous metabolic diversity and their capacity to adapt to changes in the environment. Some bacterial strains are able to thrive in the presence of high concentrations of toxic organic chemicals, such as aromatic compounds, aliphatic alcohols and solvents. The extrusion of these toxic compounds from the cell to the external medium represents the most relevant aspect in the solvent tolerance of bacteria, however, solvent tolerance is a multifactorial process that involves a wide range of genetic and physiological changes to overcome solvent damage. These additional elements include reduced membrane permeabilization, implementation of a stress response programme, and in some cases degradation of the toxic compound. We discuss the recent advances in our understanding of the mechanisms involved in solvent tolerance.

Involvement of Cyclopropane Fatty Acids in the Response of Pseudomonas putida KT2440 to Freeze-Drying

ABSTRACT Pseudomonas putida KT2440, a saprophytic soil bacterium that colonizes the plant root, is a suitable microorganism for the removal of pollutants and a stable host for foreign genes used in biotransformation processes. Because of its potential use in agriculture and industry, we investigated the conditions for the optimal preservation of the strain and its derivatives for long-term storage. The highest survival rates were achieved with cells that had reached the stationary phase and which had been subjected to freeze-drying in the presence of disaccharides (trehalose, maltose, and lactose) as lyoprotectants. Using fluorescence polarization techniques, we show that cell membranes of KT2440 were more rigid in the stationary phase than in the exponential phase of growth. This is consistent with the fact that cells grown in the stationary phase exhibited a higher proportion of C17:cyclopropane as a fatty acid than cells in the exponential phase. Mutants for the cfaB gene, which encodes the main C17:cyclopropane synthase, and for the cfaA gene, which encodes a minor C17:cyclopropane synthase, were constructed. These mutants were more sensitive to freeze-drying than wild-type cells, particularly the mutant with a knockout in the cfaB gene that produced less than 2% of the amount of C17:cyclopropane produced by the parental strain.

Insertion of epicatechin gallate into the cytoplasmic membrane of methicillin-resistant Staphylococcus aureus disrupts penicillin-binding protein (PBP) 2a-mediated beta-lactam resistance by delocalizing PBP2.

Epicatechin gallate (ECg) sensitizes methicillin-resistant Staphylococcus aureus (MRSA) to oxacillin and other β-lactam agents; it also reduces the secretion of virulence-associated proteins, prevents biofilm formation, and induces gross morphological changes in MRSA cells without compromising the growth rate. MRSA is resistant to oxacillin because of the presence of penicillin-binding protein 2a (PBP2a), which allows peptidoglycan synthesis to continue after oxacillin-mediated acylation of native PBPs. We show that ECg binds predominantly to the cytoplasmic membrane (CM), initially decreasing the fluidity of the bilayer, and induces changes in gene expression indicative of an attempt to preserve and repair a compromised cell wall. On further incubation, the CM is reorganized; the amount of lysylphosphatidylglycerol is markedly reduced, with a concomitant increase in phosphatidylglycerol, and the proportion of branched chain fatty acids increases, resulting in a more fluid structure. We found no evidence that ECg modulates the enzymatic activity of PBP2a through direct binding to the protein but determined that PBP2 is delocalized from the FtsZ-anchored cell wall biosynthetic machinery at the septal division site following intercalation into the CM. We argue that many features of the ECg-induced phenotype can be explained by changes in the fluid dynamics of the CM.

Cyclopropane fatty acids are involved in organic solvent tolerance but not in acid stress resistance in Pseudomonas putida DOT‐T1E

Bacterial membranes constitute the first physical barrier against different environmental stresses. Pseudomonas putida DOT‐T1E accumulates cyclopropane fatty acids (CFAs) in the stationary phase of growth. In this strain the cfaB gene encodes the main cyclopropane synthase responsible of the synthesis of CFAs, and its expression is mediated by RNA polymerase with sigma factor σ38. We generated a cfaB mutant of P. putida DOT‐T1E and studied its response to solvents, acid pH and other stress conditions such as temperature changes, high osmolarity and the presence of antibiotics or heavy metals in the culture medium. A CfaB knockout mutant was more sensitive to solvent stress than the wild‐type strain, but in contrast to Escherichia coli and Salmonella enterica, the P. putida cfaB mutant was as tolerant to acid shock as the wild‐type strain. The cfaB mutant was also as tolerant as the parental strain to a number of drugs, antibiotics and other damaging agents.

Identification of reciprocal adhesion genes in pathogenic and non-pathogenic Pseudomonas

We used a combination of in silico and large-scale mutagenesis approaches to expand our current knowledge of the genetic determinants used by Pseudomonas putida KT2440 to attach to surfaces. We first identified in silico orthologues that have been annotated in Pseudomonas aeruginosa as potentially involved in attachment. In this search 67 paired-related genes of P. putida KT2440 and P. aeruginosa were identified as associated to adhesion. To test the potential role of the corresponding gene products in adhesion, 37 knockout mutants of KT2440, available in the Pseudomonas Reference Culture Collection, were analysed with regard to their ability to form biofilms in polystyrene microtitre plates; of these, six mutants were deficient in adhesion. Since mutants in all potential adhesion genes were not available, we generated a genome-wide collection of mutants made of 7684 independent mini-Tn5 insertions and tested them for the formation of biofilm on polystyrene microtitre plates. Eighteen clones that exhibited a reduction of at least twofold in biofilm biomass formation were considered candidate mutants in adhesion determinants. DNA sequencing of the insertion site identified five other new genes involved in adhesion. Phenotypic characterization of the mutants showed that 11 of the inactivated proteins were required for attachment to biotic surfaces too. This combined approach allowed us to identify new proteins with a role in P. putida adhesion, including the global regulator RpoN and GacS, PstS that corresponds to one of the paired-related genes for which a mutant was not available in the mutant collection, and a protein of unknown function (PP1633). The remaining mutants corresponded to functions known or predicted to participate in adhesion based on previous evidence, such as the large adhesion proteins LapA, LapF and flagellar proteins. In silico analysis showed this set of genes to be well conserved in all sequenced P. putida strains, and that at least eight reciprocal genes involved in attachment are shared by P. putida and P. aeruginosa.

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

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

Analysis of solvent tolerance in Pseudomonas putida DOT‐T1E based on its genome sequence and a collection of mutants

Pseudomonas putida strains are prevalent in a variety of pristine and polluted environments. The genome of the solvent‐tolerant P. putida strain DOT‐T1E which thrives in the presence of high concentrations of monoaromatic hydrocarbons, contains a circular 6.3 Mbp chromosome and a 133 kbp plasmid. Omics information has been used to identify the genes and proteins involved in solvent tolerance in this bacterium. This strain uses a multifactorial response that involves fine‐tuning of lipid fluidity, activation of a general stress‐response system, enhanced energy generation, and induction of specific efflux pumps that extrude solvents to the medium. Local and global transcriptional regulators participate in a complex network of metabolic functions, acting as the decision makers in the response to solvents.

The Pseudomonas putida T6SS is a plant warden against phytopathogens

Bacterial type VI secretion systems (T6SSs) are molecular weapons designed to deliver toxic effectors into prey cells. These nanomachines have an important role in inter-bacterial competition and provide advantages to T6SS active strains in polymicrobial environments. Here we analyze the genome of the biocontrol agent Pseudomonas putida KT2440 and identify three T6SS gene clusters (K1-, K2- and K3-T6SS). Besides, 10 T6SS effector–immunity pairs were found, including putative nucleases and pore-forming colicins. We show that the K1-T6SS is a potent antibacterial device, which secretes a toxic Rhs-type effector Tke2. Remarkably, P. putida eradicates a broad range of bacteria in a K1-T6SS-dependent manner, including resilient phytopathogens, which demonstrates that the T6SS is instrumental to empower P. putida to fight against competitors. Furthermore, we observed a drastically reduced necrosis on the leaves of Nicotiana benthamiana during co-infection with P. putida and Xanthomonas campestris. Such protection is dependent on the activity of the P. putida T6SS. Many routes have been explored to develop biocontrol agents capable of manipulating the microbial composition of the rhizosphere and phyllosphere. Here we unveil a novel mechanism for plant biocontrol, which needs to be considered for the selection of plant wardens whose mission is to prevent phytopathogen infections.

Transcriptional control of the main aromatic hydrocarbon efflux pump in Pseudomonas

Bacteria of the species Pseudomonas putida are ubiquitous soil inhabitants, and a few strains are able to thrive in the presence of extremely high concentrations of toxic solvents such as toluene and related aromatic hydrocarbons. Toluene tolerance is multifactorial in the sense that bacteria use a wide range of physiological and genetic changes to overcome solvent damage. This includes enhanced membrane impermeabilization through cis to trans isomerization of unsaturated fatty acids, activation of a stress response programme, and induction of efflux pumps that expulse toxic hydrocarbons to the outer medium. The most relevant element in this toluene tolerance arsenal is the TtgGHI efflux pump controlled by the TtgV regulator. We discuss here how TtgV controls expression of this efflux pump in response to solvents.