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Roberto Kolter

Publications and source records attributed to Roberto Kolter.

32 records · Page 2Linked to original sources
PubMed2005

Pseudomonas-Saccharomyces interactions: influence of fungal metabolism on bacterial physiology and survival.

Fungal-bacterial interactions are ubiquitous, yet their molecular basis is only poorly understood. In this study, a novel beneficial interaction between a strain of Pseudomonas putida and the fungus Saccharomyces cerevisiae was identified. When the bacteria were incubated alone in grape juice or in synthetic medium containing various concentrations of glucose, they lost viability rapidly during stationary phase. However, when the bacteria were incubated in these media in the presence of the fungus, their stationary phase survival improved dramatically. On agar plates containing glucose, the beneficial effects of the fungus were manifested in robust bacterial growth and exopolysaccharide production that led to visible mucoidy. In contrast, bacteria grew poorly and were nonmucoid in such media in the absence of the fungus. By using the available S. cerevisiae deletion library, yeast mutants that were unable to mediate this beneficial interaction were identified. These mutants revealed that the beneficial effect on bacterial physiology and survival was mediated by the ability of the fungus to metabolize the available glucose and consequent effects on the medium's pH. In natural environments where the concentration of glucose is high, it is likely that the presence of fungi has had profound beneficial effects on the physiology and survival of certain P. putida strains throughout their natural history.

Coculture Techniques↗
PubMed2004

The SAT1 flipper, an optimized tool for gene disruption in Candida albicans.

The construction of Candida albicans mutants by targeted gene disruption usually depends on the use of nutritional markers for the selection of prototrophic transformants from auxotrophic host strains, but it is becoming increasingly evident that this strategy may cause difficulties in the interpretation of mutant phenotypes. Here, we describe a new method for inactivating both alleles of a target gene in C. albicans wild-type strains to obtain homozygous null mutants. The SAT1 flipping method relies on the use of a cassette that contains a dominant nourseothricin resistance marker (caSAT1) for the selection of integrative transformants and a C. albicans-adapted FLP gene that allows the subsequent excision of the cassette, which is flanked by FLP target sequences, from the genome. Two rounds of integration/excision generate homozygous mutants that differ from the wild-type parent strain only by the absence of the target gene, and reintegration of an intact gene copy for complementation of mutant phenotypes is performed in the same way. Transformants are obtained after only 1 day of growth on a selective medium, and integration into the target locus occurs with high specificity after adding homologous flanking sequences on both sides of the cassette. FLP-mediated excision of the SAT1 flipper cassette is achieved by simply growing the transformants for a few hours in medium without selective pressure, and nourseothricin-sensitive (NouS) derivatives can easily be identified by their slower growth on indicator plates containing a low concentration of nourseothricin. We demonstrate the use of the system by deleting the OPT1 gene, which encodes an oligopeptide transporter, in the C. albicans model strain SC5314. The null mutants became resistant to the toxic peptide KLLEth, and reintroduction of an intact OPT1 copy restored susceptibility. The SAT1 flipping method provides a highly efficient method for gene disruption in C. albicans wild-type strains, which eliminates currently encountered problems in the genetic analysis of this important human fungal pathogen.

Candida albicans↗
PubMed2004

Escherichia coli evolution during stationary phase.

The process of evolution by natural selection has been known for a century and a half, yet the mechanics of selection are still poorly understood. In most cases where natural selection has been studied, the genetic and physiological bases of fitness variation that result in population changes were not identified, leaving only a partial understanding of selection. Starved cultures of the bacterium Escherichia coli present a model system with which to address the genetic and physiological bases of natural selection. This is a model system that also reflects the prevalent state of bacteria in the natural world; due to intense competition for nutrients, microorganisms spend the majority of their lives under starvation conditions. Genetic analyses of a single survivor of starvation identified four adaptive mutations(1). Investigation of these mutations has revealed insights into the molecular and physiological bases of evolution during prolonged starvation stress.

Adaptation, Biological↗
PubMed2004

Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms.

Pseudomonas aeruginosa forms diverse matrix-enclosed surface-associated multicellular assemblages (biofilms) that aid in its survival in a variety of environments. One such biofilm is the pellicle that forms at the air-liquid interface in standing cultures. We screened for transposon insertion mutants of P. aeruginosa PA14 that were unable to form pellicles. Analysis of these mutants led to the identification of seven adjacent genes, named pel genes, the products of which appear to be involved in the formation of the pellicle's extracellular matrix. In addition to being required for pellicle formation, the pel genes are also required for the formation of solid surface-associated biofilms. Sequence analyses predicted that three pel genes encode transmembrane proteins and that five pel genes have functional homologues involved in carbohydrate processing. Microscopic and macroscopic observations revealed that wild-type P. aeruginosa PA14 produces a cellulase-sensitive extracellular matrix able to bind Congo red; no extracellular matrix was produced by the pel mutants. A comparison of the carbohydrates produced by the wild-type strain and pel mutants suggested that glucose was a principal component of the matrix material. Together, these results suggest that the pel genes are responsible for the production of a glucose-rich matrix material required for the formation of biofilms by P. aeruginosa PA14.

Bacterial Proteins↗
PubMed2004

A Pseudomonas aeruginosa quorum-sensing molecule influences Candida albicans morphology.

Candida albicans is an opportunistic pathogen that is commonly found as a member of the human microflora. The ability of C. albicans to alter its cellular morphology has been associated with its virulence; yeast cells are more prevalent in commensal interactions whereas filamentous cells appear important in opportunistic infections. C. albicans encounters a multitude of other microbial species in the host environment and it is likely that they impact the C. albicans transition between virulent and non-virulent states. Here, we report that C. albicans morphology is significantly affected by the presence of Pseudomonas aeruginosa, another opportunistic pathogen. In a screen using a C. albicans HWP1-lacZ strain to indicate regions of filamentous growth, we identified P. aeruginosa mutants incapable of inhibiting C. albicans filamentation. Through these studies, we found that 3-oxo-C12 homoserine lactone, a cell-cell signalling molecule produced by P. aeruginosa, was sufficient to inhibit C. albicans filamentation without affecting fungal growth rates. Both microscopic analysis and real-time reverse transcription polymerase chain reaction analysis of morphology-specific markers confirmed that filamentation was suppressed by 200 microM 3-oxo-C12 homoserine lactone. Structurally related compounds with a 12-carbon chain length, e.g. C12-acyl homoserine lactone and dodecanol also affected C. albicans filamentation at similar concentrations. In contrast, other acylated homoserine lactones of different chain lengths did not affect fungal morphology. The activity of 3OC12HSL is compared with that of farnesol, a C. albicans-produced molecule also with a C12-backbone. The effects that bacteria have on the morphology of C. albicans represents one of the ways by which bacteria can influence the behaviour of fungal cells.

4-Butyrolactone↗
PubMed2004

Genes involved in formation of structured multicellular communities by Bacillus subtilis.

The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization. Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruiting-body-like aerial projections whose tips serve as preferential sites for sporulation. To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B. subtilis genome. This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation. This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media. In this way we identified six genes that are involved in the development of multicellular communities. These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function). Further analysis revealed that these six genes play different roles in B. subtilis community development.

Bacillus subtilis↗
PubMed2004

Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix.

Pseudomonas aeruginosa forms biofilms, which are cellular aggregates encased in an extracellular matrix. Molecular genetics studies of three common autoaggregative phenotypes, namely wrinkled colonies, pellicles, and solid-surface-associated biofilms, led to the identification of two loci, pel and psl, that are involved in the production of carbohydrate-rich components of the biofilm matrix. The pel gene cluster is involved in the production of a glucose-rich matrix material in P. aeruginosa strain PA14 (L. Friedman and R. Kolter, Mol. Microbiol. 51:675-690, 2004). Here we investigate the role of the pel gene cluster in P. aeruginosa strain ZK2870 and identify a second genetic locus, termed psl, involved in the production of a mannose-rich matrix material. The 11 predicted protein products of the psl genes are homologous to proteins involved in carbohydrate processing. P. aeruginosa is thus able to produce two distinct carbohydrate-rich matrix materials. Either carbohydrate-rich matrix component appears to be sufficient for mature biofilm formation, and at least one of them is required for mature biofilm formation in P. aeruginosa strains PA14 and ZK2870.

Bacterial Proteins↗
PubMed2004

Burkholderia spp. alter Pseudomonas aeruginosa physiology through iron sequestration.

Pseudomonas aeruginosa and members of the Burkholderia cepacia complex often coexist in both the soil and the lungs of cystic fibrosis patients. To gain an understanding of how these different species affect each other's physiology when coexisting, we performed a screen to identify P. aeruginosa genes that are induced in the presence of Burkholderia: A random gene fusion library was constructed in P. aeruginosa PA14 by using a transposon containing a promoterless lacZ gene. Fusion strains were screened for their ability to be induced in the presence of Burkholderia strains in a cross-streak assay. Three fusion strains were induced specifically by Burkholderia species; all three had transposon insertions in genes known to be iron regulated. One of these fusion strains, containing a transposon insertion in gene PA4467, was used to characterize the inducing activity from Burkholderia: Biochemical and genetic evidence demonstrate that ornibactin, a siderophore produced by nearly all B. cepacia strains, can induce P. aeruginosa PA4467. Significantly, PA4467 is induced early in coculture with an ornibactin-producing but not an ornibactin-deficient B. cepacia strain, indicating that ornibactin can be produced by B. cepacia and detected by P. aeruginosa when the two species coexist.

Artificial Gene Fusion↗
PubMed2003

Bacterial evolution through the selective loss of beneficial Genes. Trade-offs in expression involving two loci.

The loss of preexisting genes or gene activities during evolution is a major mechanism of ecological specialization. Evolutionary processes that can account for gene loss or inactivation have so far been restricted to one of two mechanisms: direct selection for the loss of gene activities that are disadvantageous under the conditions of selection (i.e., antagonistic pleiotropy) and selection-independent genetic drift of neutral (or nearly neutral) mutations (i.e., mutation accumulation). In this study we demonstrate with an evolved strain of Escherichia coli that a third, distinct mechanism exists by which gene activities can be lost. This selection-dependent mechanism involves the expropriation of one gene's upstream regulatory element by a second gene via a homologous recombination event. Resulting from this genetic exchange is the activation of the second gene and a concomitant inactivation of the first gene. This gene-for-gene expression tradeoff provides a net fitness gain, even if the forfeited activity of the first gene can play a positive role in fitness under the conditions of selection.

Adaptation, Physiological↗
PubMed2003

Identification and characterization of a Vibrio cholerae gene, mbaA, involved in maintenance of biofilm architecture.

The formation of biofilms is thought to play a key role in the environmental survival of the marine bacterium Vibrio cholerae. Although the factors involved in V. cholerae attachment to abiotic surfaces have been extensively studied, relatively little is known about the mechanisms involved in the subsequent maturation of the biofilms. Here we report the identification of a novel gene, which we have named mbaA (for maintenance of biofilm architecture), that plays a role in the formation and maintenance of the highly organized three-dimensional architecture of V. cholerae El Tor biofilms. We demonstrate that although the absence of mbaA does not significantly affect the initial attachment of cells onto the surface, it leads to the formation of biofilms that lack the typical structure, including the pillars of cells separated by fluid-filled channels that are evident in mature wild-type biofilms. Microscopic analysis indicates that the absence of mbaA leads to an increase in the amount of extracellular matrix material in the biofilms. The predicted mbaA product is a member of a family of regulatory proteins, containing GGDEF and EAL domains, suggesting that MbaA regulates the synthesis of some component of the biofilm matrix.

Bacterial Proteins↗
PubMed2002

Pseudomonas-Candida interactions: an ecological role for virulence factors.

Bacterial-fungal interactions have great environmental, medical, and economic importance, yet few have been well characterized at the molecular level. Here, we describe a pathogenic interaction between Pseudomonas aeruginosa and Candida albicans, two opportunistic pathogens. P. aeruginosa forms a dense biofilm on C. albicans filaments and kills the fungus. In contrast, P. aeruginosa neither binds to nor kills yeast-form C. albicans. Several P. aeruginosa virulence factors that are important in disease are involved in the killing of C. albicans filaments. We propose that many virulence factors studied in the context of human infection may also have a role in bacterial-fungal interactions.

Antibiosis↗
PubMed2002

Why are bacteria refractory to antimicrobials?

The incidence of antibiotic resistance in pathogenic bacteria is rising. Antibiotic resistance can be achieved via three distinct routes: inactivation of the drug, modification of the target of action, and reduction in the concentration of drug that reaches the target. It has long been recognized that specific antibiotic resistance mechanisms can be acquired through mutation of the bacterial genome or by gaining additional genes through horizontal gene transfer. Recent attention has also brought to light the importance of different physiological states for the survival of bacteria in the presence of antibiotics. It is now apparent that bacteria have complex, intrinsic resistance mechanisms that are often not detected in the standard antibiotic sensitivity tests performed in clinical laboratories. The development of resistance in bacteria found in surface-associated aggregates or biofilms, owing to these intrinsic mechanisms, is paramount.

Anti-Bacterial Agents↗
PubMed2002

Alcohol-induced delay of viability loss in stationary-phase cultures of Escherichia coli.

During prolonged incubation in stationary phase Escherichia coli undergoes starvation-induced differentiation, resulting in highly resistant cells. In rich medium with high amino acid content further incubation of cultures at high cell density leads to the generation of a population of cells no longer able to form colonies. The viability loss is due to some component of spent medium, active at high pH and high cell density, and can be prevented either by keeping the pH close to neutrality, by washing off the nonsalt components of the medium, or by keeping the saturating cell density low. Exposure to short-chain n-alcohols within a specific time window in stationary phase also prevents viability loss, in an rpoS-dependent fashion. The development of stress resistance, a hallmark of stationary-phase cells, is affected following alcohol treatment, as is the response to extracellular factors in spent medium. Alcohols seem to block cells in an early phase of starvation-induced differentiation, most likely by interfering with processes important for regulation of sigma(s) such as cell density signals and sensing the nutrient content of the medium.

Adaptation, Physiological↗