Eduardo Santero

Role of integration host factor in stimulating transcription from the σ54-dependent nifH promoter☆

In a wide variety of nitrogen-fixing organisms among the Purple Bacteria (large division of Gram-negative bacteria) the nitrogen fixation (nif) operons are transcribed by an alternative holoenzyme form of RNA polymerase, sigma 54-holoenzyme. Transcription depends on the activator protein NIFA (nitrogen fixation protein A), which catalyzes isomerization of closed complexes between this polymerase and a promoter to transcriptionally productive open complexes. NIFA-mediated activation of transcription from the nifH promoter of Klebsiella pneumoniae is greatly stimulated by the integration host factor IHF, which binds to a site between the upstream binding site for NIFA and the promoter, and bends the DNA. IHF fails to stimulate activation of transcription from this promoter by another activator of sigma 54-holoenzyme, NTRC (nitrogen regulatory protein C), which lacks a specific binding site in the nifH promoter region. As predicted, if the IHF-induced bend facilitates interaction between NIFA and sigma 54-holoenzyme, substitution of an NTRC-binding site for the NIFA-binding site allowed IHF to stimulate NTRC-mediated activation of transcription from the nifH promoter. The stimulation was of the same order of magnitude as that for NIFA in the native configuration of the promoter-regulatory region (up to 20-fold). With purified NTRC and the substitution construct we could demonstrate that stimulation by IHF in a purified transcription system was comparable to that in a crude coupled transcription-translation system, indicating that the stimulation in the crude system could be accounted for by IHF. The IHF stimulation was observed on linear as well as supercoiled templates, indicating that the geometric requirements are relatively simple. We have attempted to visualize the arrangement of proteins on DNA fragments carrying the nifH promoter-regulatory region of K. pneumoniae by electron microscopy. IHF stimulated NIFA-mediated activation of transcription from the nifH and nifD promoters of Bradyrhizobium japonicum and less so from the nifH promoters of Rhizobium meliloti and Thiobacillus ferrooxidans, consistent with previous observations that stimulation is greatest at promoters that are weak binding sites for sigma 54-holoenzyme in closed complexes.

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.

Transcriptome Analysis of Pseudomonas putida in Response to Nitrogen Availability

This work describes a regulatory network of Pseudomonas putida controlled in response to nitrogen availability. We define NtrC as the master nitrogen regulator and suggest that it not only activates pathways for the assimilation of alternative nitrogen sources but also represses carbon catabolism under nitrogen-limited conditions, possibly to prevent excessive carbon and energy flow in the cell.

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.

Mechanism of translational coupling in the nifLA operon of Klebsiella pneumoniae

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 specialized ribosome system, and the effect of substituting the natural Shine–Dalgarno of nifL or nifA for specialized Shine–Dalgarno sequences was determined. Our results indicate that translational coupling occurs in this operon by a reinitiation mechanism. Additionally, reinitiation at the nifA can happen even in the absence of good Shine–Dalgarno recognition by the reinitiating ribosome, although its efficiency is lower. The effect of a putative translational enhancer sequence (downstream box) on translational coupling efficiency was also determined. Mutations that reduce the homology of the putative downstream box to the consensus had only a minor effect on nifA translation by wild‐type ribosomes. However, they had a significant effect on nifA translation by specialized ribosomes, suggesting that recognition of the downstream box may compensate inefficient ribosomal interactions with the Shine–Dalgarno sequence.

In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20–150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine–converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.

Identification and functional characterization of Sphingomonas macrogolitabida strain TFA genes involved in the first two steps of the tetralin catabolic pathway.

Five genes involved in the two initial steps of the tetralin biodegradation pathway of Sphingomonas macrogolitabida strain TFA have been characterized. ThnA1A2 and ThnA3A4, components of the ring-hydroxylating dioxygenase, were encoded in divergently transcribed operons. ThnA1, ThnA2, and ThnA3 were essential for tetralin ring-hydroxylating dioxygenase activity. ThnB was identified as a dehydrogenase required for tetralin biodegradation.

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.

NtrC-Dependent Regulatory Network for Nitrogen Assimilation in Pseudomonas putida

Pseudomonas putida KT2440 is a model strain for studying bacterial biodegradation processes. However, very little is known about nitrogen regulation in this strain. Here, we show that the nitrogen regulatory NtrC proteins from P. putida and Escherichia coli are functionally equivalent and that substitutions leading to partially active forms of enterobacterial NtrC provoke the same phenotypes in P. putida NtrC. P. putida has only a single P(II)-like protein, encoded by glnK, whose expression is nitrogen regulated. Two contiguous NtrC binding sites located upstream of the sigma(N)-dependent glnK promoter have been identified by footprinting analysis. In vitro experiments with purified proteins demonstrated that glnK transcription was directly activated by NtrC and that open complex formation at this promoter required integration host factor. Transcription of genes orthologous to enterobacterial codB, dppA, and ureD genes, whose transcription is dependent on sigma(70) and which are activated by Nac in E. coli, has also been analyzed for P. putida. Whereas dppA does not appear to be regulated by nitrogen via NtrC, the codB and ureD genes have sigma(N)-dependent promoters and their nitrogen regulation was exerted directly by NtrC, thus avoiding the need for Nac, which is missing in this bacterial species. Based upon these results, we propose a simplified nitrogen regulatory network in P. putida (compared to that in enterobacteria), which involves an indirect-feedback autoregulation of glnK using NtrC as an intermediary.