Fernando Govantes

Nitrogen control of atrazine utilization in pseudomonas sp. strain ADP

ABSTRACT 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 sp. strain ADP compared to cells cultivated on growth-limiting nitrogen sources. The presence of atrazine in addition to the nitrogen sources did not stimulate degradation. High degradation rates obtained in the presence of ammonium plus the glutamine synthetase inhibitor MSX and also with an Nas− mutant derivative grown on nitrate suggest that nitrogen regulation operates by sensing intracellular levels of some key nitrogen-containing metabolite. Nitrate amendment in soil microcosms resulted in decreased atrazine mineralization by the wild-type strain but not by the Nas− mutant. This suggests that, although nitrogen repression of the atrazine catabolic pathway may have a strong impact on atrazine biodegradation in nitrogen-fertilized soils, the use of selected mutant variants may contribute to overcoming this limitation.

Interplay between three global regulatory proteins mediates oxygen regulation of the Escherichia coli cytochrome d oxidase (cydAB) operon

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 antagonize H‐NS action to render cydAB expression insensitive to H‐NS repression in anaerobiosis. The targets for H‐NS‐mediated aerobic repression are the four oxygen‐regulated promoters, designated P1, P2, P3 and P4. H‐NS control is the result of H‐NS binding to an extended region within the cydAB promoter element, including sequences upstream from and overlapping the four regulated promoters. We propose a regulatory model in which oxygen control of cydAB transcription is mediated by three alternative protein–DNA complexes that are assembled sequentially on the promoter region as the cells are shifted from aerobic to microaerobic and to anaerobic conditions. According to this model, ArcA‐P plays a central role in cydAB regulation by antagonizing H‐NS repression of cydAB transcription when oxygen becomes limiting. This allows peak gene expression and subsequent repression by Fnr under fully anaerobic conditions.

Regulation of the Pseudomonas sp. Strain ADP Cyanuric Acid Degradation Operon

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 Pseudomonas putida showed that atzDEF is subjected to dual regulation in response to nitrogen limitation and cyanuric acid. The gene adjacent to atzD, orf99 (renamed here atzR), encoding a LysR-like regulator, was found to be required for both responses. Expression of atzR-lacZ was induced by nitrogen limitation and repressed by AtzR. Nitrogen regulation of atzD-lacZ and atzR-lacZ expression was dependent on the alternative sigma factor sigmaN and NtrC, suggesting that the cyanuric acid degradation operon may be subject to general nitrogen control. However, while atzR is transcribed from a sigmaN-dependent promoter, atzDEF transcription appears to be driven from a sigma70-type promoter. Expression of atzR from a heterologous promoter revealed that although NtrC regulation of atzD-lacZ requires the AtzR protein, it is not the indirect result of NtrC-activated AtzR synthesis. We propose that expression of the cyanuric acid degradation operon atzDEF is controlled by means of a complex regulatory circuit in which AtzR is the main activator. AtzR activity is in turn modulated by the presence of cyanuric acid and by a nitrogen limitation signal transduced by the Ntr system.

The LysR-type regulator AtzR binding site: DNA sequences involved in activation, repression and cyanuric acid-dependent repositioning

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 the cis‐acting sequences at the atzR–atzDEF divergent promoter region required for AtzR‐dependent regulation. AtzR binds a single site overlapping both the PatzR and PatzDEF promoters and induces a DNA bend immediately upstream from PatzDEF. Interaction of AtzR with the inducer cyanuric acid shortens the protein–DNA interaction region and relaxes the DNA bend. The AtzR binding site contains a strong binding determinant, the repression binding site (RBS), centred at position −65 relative to the atzDEF transcriptional start, containing the LTTR binding consensus motif. Integrity of the RBS is essential for high‐affinity AtzR binding, activation and autorepression. A second, weaker binding determinant, the activation binding site (ABS), is present between the RBS and PatzDEF. Deletion of the ABS only provokes a modest decrease in AtzR affinity for the promoter region in vitro, but abolishes repression of PatzR in vivo. Involvement of the ABS in autorepression has not been previously reported.

Atrazine biodegradation in the lab and in the field: enzymatic activities and gene regulation

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 restrict atrazine elimination in nitrogen‐fertilized soils by indigenous organisms or in bioaugmentation approaches. The mechanisms of nitrogen control have been investigated in the model strain Pseudomonas sp. ADP. Expression of atzA, atzB ad atzC, involved in the conversion of atrazine in cyanuric acid, is constitutive. The atzDEF operon, encoding the enzymes responsible for cyanuric acid mineralization, is a target for general nitrogen control. Regulation of atzDEF involves a complex interplay between the global regulatory elements of general nitrogen control and the pathway‐specific LysR‐type regulator AtzR. In addition, indirect evidence suggests that atrazine transport may also be a target for nitrogen regulation in this strain. The knowledge about regulatory mechanisms may allow the design of rational bioremediation strategies such as biostimulation using carbon sources or the use of mutant strains impaired in the assimilation of nitrogen sources for bioaugmentation.

Lack of CbrB in Pseudomonas putida affects not only amino acids metabolism but also different stress responses and biofilm development

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 CbrAB system in Pseudomonas putida. Our results show that CbrB is involved in coordination with the nitrogen control system activator, NtrC, in the uptake and assimilation of several amino acids. In addition, CbrB affects other carbon utilization pathways and a number of apparently unrelated functions, such as chemotaxis, stress tolerance and biofilm development. Based on these new findings, we propose that CbrB is a high-ranked element in the regulatory hierarchy of P. putida that directly or indirectly controls a variety of metabolic and behavioural traits required for adaptation to changing environmental conditions.

Regulation of the atrazine-degradative genes in Pseudomonas sp. strain ADP.

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 focus on the recent advances in the understanding of the mechanisms of genetic regulation operating on the atrazine-degradative genes. The Pseudomonas sp. strain ADP atrazine-degradation pathway is encoded by two sets of genes: the constitutively expressed atzA, atzB and atzC, and the strongly regulated atzDEF operon. A complex cascade-like circuit is responsible for the integrated regulation of atzDEF expression in response to nitrogen availability and cyanuric acid. Mechanistic studies have revealed several unusual traits, such as the upstream activating sequence-independent regulation and repression by competition with sigma(54)-RNA polymerase for DNA binding occurring at the sigma(54)-dependent PatzR promoter, and the dual mechanism of transcriptional regulation of the PatzDEF promoter by the LysR-type regulator AtzR in response to two dissimilar signals. These findings have provided new insights into the regulation of the atrazine-biodegradative pathway that are also relevant to widespread bacterial regulatory phenomena, such as global nitrogen control and transcriptional activation by LysR-type transcriptional regulators.

The c-di-GMP phosphodiesterase BifA regulates biofilm development in Pseudomonas putida

We previously showed the isolation of biofilmpersistent 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 Pseudomon asaeruginosa BifA. Here we show the phenotypic characterization of a ΔbifA mutant in P. putida KT2442.This mutant displayed increased biofilm and pellicle formation, cell aggregation in liquid medium and decreased starvation-induced biofilm dispersal relative to the wild type. Unlike its P. aeruginosa counterpart, P. putida BifA did not affect swarming motility. The hyperadherent phenotype of the ΔbifA mutant correlates with a general increase in cyclic diguanylate (c-di-GMP) levels, Congo Red-binding exopolyaccharide production and transcription of the adhesin-encoding lapA gene. Integrity of the EAL motif and a modified GGDEF motif (altered to GGDQF)were crucial for BifA activity, and c-di-GMP depletion by overexpression of a heterologous c-di-GMP phosphodiesterase in the ΔbifA mutant restored wild-type biofilm dispersal and lapA expression.Our results indicate that BifA is a phosphodiesterase involved in the regulation of the c-di-GMP pool and required for the generation of the low c-di-GMP signal that triggers starvation-induced biofilm dispersal.

Complex interplay between the LysR‐type regulator AtzR and its binding site mediates atzDEF activation in response to two distinct signals

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, nitrogen limitation and cyanuric acid. In the present work we analyse the structure and function of the cis‐acting elements involved in AtzR activation of atzDEF. Hydroxyl radical footprinting assays revealed that the ABS is composed of three functional subsites spaced at one helix‐turn intervals. Two modes of interaction with the ABS are detected in vitro: AtzR binds at the ABS‐2 and ABS‐3 subsites in the absence of inducer, and relocates to interact with the ABS‐1 and ABS‐2 subsites in the presence of cyanuric acid. In vivo mutational analysis indicates that ABS‐1 and ABS‐2 are required for full PatzDEF activation in all conditions. In contrast, ABS‐3 acts as a ‘subunit trap’ that hinders productive AtzR interactions with ABS‐1 and ABS‐2. Our results strongly suggest an activation model in which cyanuric acid and nitrogen limitation cooperate to reposition AtzR from an inactive, ABS‐3 bound configuration to an active, ABS‐1‐ and ABS‐2‐bound configuration.

Activation and repression of a σN-dependent promoter naturally lacking upstream activation sequences

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 σN‐dependent promoter PatzR, activated by NtrC and repressed by AtzR. Here we use in vivo and in vitro approaches to address the mechanisms of PatzR activation and repression. Activation by NtrC did not require any promoter sequences other than the σN recognition motif both in vivo and in vitro, suggesting that NtrC activates PatzR in an upstream activation sequences‐independent fashion. Regarding AtzR‐dependent autorepression, our in vitro transcription experiments show that the concentration of AtzR required for repression of the PatzR promoter in vitro correlates with AtzR affinity for its binding site. In addition, AtzR prevents transcription from PatzR when added to a preformed E‐σN–PatzR closed complex, but isomerization to an open complex prevents repression. Gel mobility shift and DNase I footprint assays indicate that DNA‐bound AtzR and E‐σN are mutually exclusive. Taken together, these results strongly support the notion that AtzR represses transcription from PatzR by competing with E‐σN for their overlapping binding sites. There are no previous reports of a similar mechanism for repression of σN‐dependent transcription.