Fernando Govantes

• Ph.D in Biology
• Professor at Pablo de Olavide University

Pseudomonas putida biofilm growth is associated to nutrient-sufficient conditions and biofilm dispersal is induced by nutrient starvation, signaled by the stringent response-associated nucleotide alarmone (p)ppGpp. We have used transcriptomic analysis to show that (p)ppGpp regulates the hsbAR-hptB gene cluster, encoding components of a phosphorelay...

Here, employing computer simulation tools, we present a study on the development of a bacterial biofilm from a single starter cell on a flat inert surface overlaid by an aqueous solution containing nutrients. In our simulations, surface colonization involves an initial stage of two-dimensional cell proliferation to eventually transition to three-di...

Background Microglial dysfunction plays a causative role in Alzheimer’s disease (AD) pathogenesis. Here we focus on a germline insertion/deletion variant mapping SIRPβ1, a surface receptor that triggers amyloid-β(Aβ) phagocytosis via TYROBP. Objective To analyze the impact of this copy-number variant in SIRPβ1 expression and how it affects AD mole...

The Gram-negative bacterium Pseudomonas putida bears a tuft of flagella at a single cell pole. New flagella must be assembled de novo every cell cycle to secure motility of both daughter cells. Here we show that the coordinated action of FimV, FlhF and FleN sets the location, timing and number of flagella assembled. The polar landmark proteins FimV...

The second messenger cyclic di-GMP (c-di-GMP) controls the transition between motility and sessility in many bacterial species by a variety of mechanisms, including the production of multiple exopolysaccharides. Pseudomonas syringae pv. tomato (Pto) DC3000 is a plant pathogenic bacteria able to synthesize acetylated cellulose under high c-di-GMP le...

Microglia play an important role in the maintenance of brain homeostasis, and microglial dysfunction plays a causative role in Alzheimer disease pathogenesis. Here we focus on the signal regulatory protein SIRPβ1, a surface receptor expressed on the myeloid cells that triggers amyloidβ and cell debris phagocytosis via TYROBP. We found that a common...

Structural predictions have matched the accuracy of experimental structures in the case of close homologues, outperformed docking methods for multimeric complexes and helped sampling the conformational landscape of transporters and receptors. Such successes prompt the question whether predictions can be used to relate experimental structures in the...

A single region of the Pseudomonas putida genome, designated the flagellar cluster, includes 59 genes potentially involved in the biogenesis and function of the flagellar system. Here, we combine bioinformatics and in vivo gene expression analyses to clarify the transcriptional organization and regulation of the flagellar genes in the cluster. We h...

A single region of the Pseudomonas putida genome, designated the flagellar cluster, includes 59 genes potentially involved in the biogenesis and function of the flagellar system. Here we combine bioinformatics and in vivo gene expression analyses to clarify the transcriptional organization and regulation of the flagellar genes in the cluster. We ha...

Microscopic organisms, such as bacteria, have the ability of colonizing surfaces and developing biofilms that can determine diseases and infections. Most bacteria secrete a significant amount of extracellular polymer substances that are relevant for biofilm stabilization and growth. In this work, we apply computer simulation and perform experiments...

Microscopic organisms, such as bacteria, have the ability of colonizing surfaces and developing biofilms that can determine diseases and infections. Most bacteria secrete a significant amount of extracellular polymer substances that are relevant for biofilm stabilization and growth. In this work, we apply computer simulation and perform experiments...

The Pseudomonas putida flhA-flhF-fleN-fliA cluster encodes a component of the flagellar export gate and three regulatory elements potentially involved in flagellar biogenesis and other functions. Here we show that these four genes form an operon, whose transcription is driven from the upstream PflhA promoter. A second promoter, PflhF, provides addi...

Flagellation patterns of the flhF and fleN mutants. Flagellar stain of the wild-type KT2442, the ΔflhF mutant MRB69, the ΔfleN mutant MRB71 and the ΔflhF-fleN mutant MRB78 strains. (PDF)

Predicted FleQ-binding sites at the PlapA and PbcsD promoter regions. (PDF)

Near surface flagellar motility of the wild-type strain KT2442. (MOV)

Gel mobility shift assays on the PbcsD and PlapA promoters. Panels A, B and C: Autoradiograph of a representative PAGE gel containing the indicated probe, FleQ and/or FleN at the indicated concentrations. Assays were performed in the absence (-) or in the presence (+) of c-di-GMP and in the absence (-) or in the presence (+) of ATP. Closed arrowhea...

Near surface flagellar motility of the ΔflhF-fleN mutant. (MOV)

Flagellar motility tracks of wild-type and mutant P. putida strains. Five cells from each video sequence of the wild-type, ΔflhF, ΔfleN and ΔflhF-fleN strains were monitored over time. Tracks were generated using the MtrackJ plugin of ImageJ. (PDF)

SDS-PAGE gel showing FleN overproduction and purification. Lane 1: uninduced overproducing strain, whole cells; lane 2: induced overproducing strain, whole cells; lane 3: induced overproducing strain, clarified soluble extract; lane 4: chitin affinity resin flow-through; lane 5: wash buffer eluate; lane 6: chitin-bound protein prior to cleavage; la...

Near surface flagellar motility of the ΔflhF mutant. (MOV)

Near surface flagellar motility of the ΔfleN mutant. (MOV)

Bacteria swim and swarm using rotating flagella that are driven by a membrane-spanning motor complex. Performance of the flagella motility apparatus is modulated by the chemosensory signal transduction system to allow navigation through physico-chemical gradients - a process that can be fine-tuned by the bacterial second messenger c-di-GMP. We have...

Most bacteria form organized sessile communities, known as biofilms. Their ubiquity and relevance have stimulated the development of efficient mathematical models able to predict biofilm evolution and characteristics at different conditions. Here we present a study of the early stages of bacterial biofilm formation modeled by means of individual ce...

The ability to form stable surface-attached communities called biofilms is of paramount importance to both beneficial and harmful interactions between microbes and microbial, plant or animal partners. Assessment of biofilm formation ability is often performed by growing the organisms in microtiter plate wells and staining the well-attached material...

Biofilm dispersal is a genetically programmed response enabling bacterial cells to exit the biofilm in response to particular physiological or environmental conditions. In Pseudomonas putida biofilms, nutrient starvation triggers c-di-GMP hydrolysis by phosphodiesterase BifA, releasing inhibition of protease LapG by the c-di-GMP effector protein La...

Most bacteria alternate between a free living planktonic lifestyle and the formation of struc-tured surface-associated communities named biofilms. The transition between these two lifestyles requires a precise and timely regulation of the factors involved in each of the stages that has been likened to a developmental process. Here we characterize t...

PatzT is an internal promoter of the atzRSTUVW operon directing the synthesis of AtzT, AtzU, AtzV and AtzW, components of an ABC-type cyanuric acid transport system. PatzT is σN-dependent, activated by the general nitrogen control regulator NtrC with the assistance of IHF, and repressed by the LysR-type transcriptional regulator (LTTR) AtzR. We hav...

Microorganisms are rich repositories of genetic material encoding many activities of potential interest. Recent advances make identifying and exploiting the metabolic treasures of uncultured microbes an easier proposition. Improved expression vectors and metagenomic screening techniques make it easier to identify activities of interest. Synthetic b...

Out of 8000 candidates from a genetic screening for Pseudomonas putida KT2442 mutants showing defects in biofilm formation, 40 independent mutants with diminished levels of biofilm were analyzed. Most of these mutants carried insertions in genes of the lap cluster, whose products are responsible for synthesis, export and degradation of the adhesin...

The CbrAB two-component system in the Pseudomonads controls a variety of metabolic and behavioural traits required for its adaptation to changing environmental conditions, including the uptake or assimilation of certain carbon sources, and processes such as chemotaxis or stress tolerance. In this work we characterise a miniTn5-luxAB-Km transposon i...

We previously showed the isolation of biofilm-persistent Pseudomonas putida mutants that fail to undergo biofilm dispersal upon entry in stationary phase. Two such mutants were found to bear insertions in PP0914, encoding a GGDEF/EAL domain protein with high similarity to Pseudomonas aeruginosa BifA. Here we show the phenotypic characterization of...

The Pseudomonas sp. ADP plasmid pADP-1 encodes the activities involved in the hydrolytic degradation of the s-triazine herbicide atrazine. Here we explore the presence of a specific transport system for the central intermediate of the atrazine utilization pathway, cyanuric acid, in Pseudomonas sp. ADP. Growth in fed-batch cultures containing limiti...

Here we describe two new methods for the genetic characterization of bacterial biofilm development. First, we have designed a microtitre dish-based approach for high-throughput screening of Pseudomonas putida mutants showing increased biofilm under dispersal conditions. Using this method, nine such biofilm-persistent mutants, bearing transposon ins...

The LysR-type regulator AtzR activates the Pseudomonas sp. ADP atzDEF operon in response to nitrogen limitation and cyanuric acid. Activation involves repositioning of the AtzR tetramer on the PatzDEF promoter and relaxation of an AtzR-induced DNA bend. Here we examine the in vivo and in vitro contribution of an A5 -tract present at the PatzDEF pro...

The atzS-atzT-atzU-atzV-atzW gene cluster of the Pseudomonas sp. strain ADP atrazine-degradative plasmid pADP-1, which carries genes for an outer membrane protein and the components of a putative ABC-type solute transporter, is located downstream from atzR, which encodes the LysR-type transcriptional regulator of the cyanuric acid-degradative opero...

The CbrAB two-component system has been described in certain species of Pseudomonads as a global regulatory system required for the assimilation of several amino acids (e.g. histidine, proline or arginine) as carbon or carbon and nitrogen sources. In this work, we used global gene expression and phenotypic analyses to characterize the roles of the...

The Gram-negative bacterium Pseudomonas sp. strain ADP is the best-characterized organism able to mineralize the s-triazine herbicide atrazine. This organism has been the subject of extensive biochemical and genetic characterization that has led to its use in bioremediation programs aimed at the decontamination of atrazine-polluted sites. Here, we...

AtzR is a LysR-type regulator responsible for activation of the cyanuric acid utilization operon atzDEF. AtzR binds the PatzDEF promoter region at a strong recognition element, designated the repressor binding site, and a weaker binding determinant, the activator binding site (ABS). AtzR activates transcription in response to two dissimilar signals...

The Pseudomonas sp. strain ADP atzDEF operon encodes the enzymes involved in cyanuric acid mineralization, the final stage of the s-triazine herbicide atrazine degradative pathway. We have previously shown that atzDEF is under nitrogen control in both its natural host and Pseudomonas putida KT2442. Expression of atzDEF requires the divergently enco...

The Pseudomonas sp. strain ADP protein AtzR is a LysR-type transcriptional regulator required for activation of the atzDEF operon in response to nitrogen limitation and cyanuric acid. Transcription of atzR is directed by the sigma(N)-dependent promoter PatzR, activated by NtrC and repressed by AtzR. Here we use in vivo and in vitro approaches to ad...

Atrazine is an herbicide of the s-triazine family that is used primarily as a nitrogen source by degrading microorganisms. While many catabolic pathways for xenobiotics are subjected to catabolic repression by preferential carbon sources, atrazine utilization is repressed in the presence of preferential nitrogen sources. This phenomenon appears to...

The LysR-type transcriptional regulator (LTTR) AtzR of Pseudomonas sp. strain ADP activates the cyanuric acid-utilization atzDEF operon in response to low nitrogen availability and the presence of cyanuric acid. AtzR also represses expression of its own gene, atzR, transcribed divergently from atzDEF. Here we identify and functionally characterize...

In recent times, the use of the s-triazine herbicide atrazine has become a major concern, due to increasing evidence of severe ecotoxicological effects. The development of strategies for bioremediation of contaminated soils and waters requires the isolation and development of strains that harbor an appropriate catabolic pathway, are competitive in...

Pseudomonas sp. strain ADP is the model strain for studying bacterial degradation of the s-triazine herbicide atrazine. In this work, we focused on the expression of the atzDEF operon, involved in mineralization of the central intermediate of the pathway, cyanuric acid. Expression analysis of atzD-lacZ fusions in Pseudomonas sp. strain ADP and Pseu...

Pseudomonas sp. strain ADP uses the herbicide atrazine as the sole nitrogen source. We have devised a simple atrazine degradation assay to determine the effect of other nitrogen sources on the atrazine degradation pathway. The atrazine degradation rate was greatly decreased in cells grown on nitrogen sources that support rapid growth of Pseudomonas...

The Escherichia coli cydAB operon, encoding the subunits of the high-affinity cytochrome d oxidase, is maximally transcribed in microaerobiosis as a result of the combined action of the oxygen-responsive regulators Fnr and ArcA. Here, we report that the histone-like protein H-NS is an aerobic repressor of cydAB expression. ArcA is shown to antagoni...

The Escherichia coli cydAB operon encodes the high-affinity terminal oxidase of the oxygen respiratory chain, cytochrome d oxidase. The sensor-regulator pair, ArcB-ArcA, is responsible for the microaerobic activation of the cydAB operon, whereas the anaerobic regulator Fnr represses its expression in the absence of oxygen. Fnr binds in vitro at two...

Salmonella typhimurium in vivo-induced (ivi) genes were grouped by their coordinate behavior in response to a wide variety of environmental and genetic signals, including pH, Mg2+, Fe2+, and PhoPQ. All of the seven ivi fusions that are induced by both low pH and low Mg2+ (e.g., iviVI-A) are activated by the PhoPQ regulatory system. Iron-responsive...

The nifLA operon of Klebsiella pneumoniae encodes the sensor-activator pair involved in the regulation of other nif genes. Balanced synthesis of both proteins, which is required for correct regulation, is achieved by coupling translation of nifA to that of nifL. The mechanism of translational coupling at the nifLA operon was analysed using a specia...

The nifLA operon of Klebsiella pneumoniae codes for the two antagonistic regulatory proteins which control expression of all other nitrogen fixation genes. NifA is a transcriptional activator, and NifL inhibits NifA. The importance of a correct NifL-NifA stoichiometry for efficient regulation of nitrogen fixation genes has been investigated by cons...

The effect of premature stop codons in the nifL gene on the expression of nifA-lacZ operon and protein fusions in Klebsiella pneumoniae was analysed in detail. Our results revealed transcriptional polarity in this operon. By dissecting the operon, intragenic regions containing Rho-dependent transcription terminators have been identified. As shown f...

The Klebsiella pneumoniae nifH promoter is very strictly controlled by nitrogen availability and highly dependent on sigma 54 and integration host factor (IHF) for expression. This promoter region has been used to examine the role of IHF in the activation of transcription from sigma 54-dependent promoters and to analyze the positional restrictions...