The Regulatory Hierarchy Following Signal Integration by the CbrAB Two-Component System: Diversity of Responses and Functions.

CbrAB is a two-component system, unique to bacteria of the family Pseudomonaceae, capable of integrating signals and involved in a multitude of physiological processes that allow bacterial adaptation to a wide variety of varying environmental conditions. This regulatory system provides a great metabolic versatility that results in excellent adaptability and metabolic optimization. The two-component system (TCS) CbrA-CbrB is on top of a hierarchical regulatory cascade and interacts with other regulatory systems at different levels, resulting in a robust output. Among the regulatory systems found at the same or lower levels of CbrAB are the NtrBC nitrogen availability adaptation system, the Crc/Hfq carbon catabolite repression cascade in Pseudomonas, or interactions with the GacSA TCS or alternative sigma ECF factor, such as SigX. The interplay between regulatory mechanisms controls a number of physiological processes that intervene in important aspects of bacterial adaptation and survival. These include the hierarchy in the use of carbon sources, virulence or resistance to antibiotics, stress response or definition of the bacterial lifestyle. The multiple actions of the CbrAB TCS result in an important competitive advantage.

Unraveling the role of the CbrA histidine kinase in the signal transduction of the CbrAB two-component system in Pseudomonas putida.

The histidine kinase CbrA of the CbrAB two-component system of Pseudomonas putida is a key element to recognise the activating signal and mediate auto- and trans-phosphorylation of the response element CbrB. CbrA is encoded by the gene cbrA which is located downstream of a putative open reading frame we have named cbrX. We describe the role of the CbrX product in the expression of CbrA and show there is translational coupling of the genes. We also explore the role of the transmembrane (TM) and PAS domains of CbrA in the signal recognition. A ΔcbrXA mutant lacking its TM domains is uncoupled in its growth in histidine and citrate as carbon sources, but its overexpression restores the ability to grow in such carbon sources. In these conditions ΔTM-CbrA is able to respond to carbon availability, thus suggesting an intracellular nature for the signal sensed.

The CbrB Regulon: Promoter dissection reveals novel insights into the CbrAB expression network in Pseudomonas putida.

CbrAB is a high ranked global regulatory system exclusive of the Pseudomonads that responds to carbon limiting conditions. It has become necessary to define the particular regulon of CbrB and discriminate it from the downstream cascades through other regulatory components. We have performed in vivo binding analysis of CbrB in P. putida and determined that it directly controls the expression of at least 61 genes; 20% involved in regulatory functions, including the previously identified CrcZ and CrcY small regulatory RNAs. The remaining are porines or transporters (20%), metabolic enzymes (16%), activities related to protein translation (5%) and orfs of uncharacterised function (38%). Amongst the later, we have selected the operon PP2810-13 to make an exhaustive analysis of the CbrB binding sequences, together with those of crcZ and crcY. We describe the implication of three independent non-palindromic subsites with a variable spacing in three different targets; CrcZ, CrcY and operon PP2810-13 in the CbrAB activation. CbrB is a quite peculiar σN-dependent activator since it is barely dependent on phosphorylation for transcriptional activation. With the depiction of the precise contacts of CbrB with the DNA, the analysis of the multimerisation status and its dependence on other factors such as RpoN o IHF, we propose a model of transcriptional activation.

Evolutionary Cell Biology of Division Mode in the Bacterial Planctomycetes-Verrucomicrobia- Chlamydiae Superphylum.

Bacteria from the Planctomycetes, Verrucomicrobia, and Chlamydiae (PVC) superphylum are exceptions to the otherwise dominant mode of division by binary fission, which is based on the interaction between the FtsZ protein and the peptidoglycan (PG) biosynthesis machinery. Some PVC bacteria are deprived of the FtsZ protein and were also thought to lack PG. How these bacteria divide is still one of the major mysteries of microbiology. The presence of PG has recently been revealed in Planctomycetes and Chlamydiae, and proteins related to PG synthesis have been shown to be implicated in the division process in Chlamydiae, providing important insights into PVC mechanisms of division. Here, we review the historical lack of observation of PG in PVC bacteria, its recent detection in two phyla and its involvement in chlamydial cell division. Based on the detection of PG-related proteins in PVC proteomes, we consider the possible evolution of the diverse division mechanisms in these bacteria. We conclude by summarizing what is known and what remains to be understood about the evolutionary cell biology of PVC division modes.

Development of Genetic Tools for the Manipulation of the Planctomycetes.

Bacteria belonging to the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) superphylum are of interest for biotechnology, evolutionary cell biology, ecology, and human health. Some PVC species lack a number of typical bacterial features while others possess characteristics that are usually more associated to eukaryotes or archaea. For example, the Planctomycetes phylum is atypical for the absence of the FtsZ protein and for the presence of a developed endomembrane system. Studies of the cellular and molecular biology of these infrequent characteristics are currently limited due to the lack of genetic tools for most of the species. So far, genetic manipulation in Planctomycetes has been described in Planctopirus limnophila only. Here, we show a simple approach that allows mutagenesis by homologous recombination in three different planctomycetes species (i.e., Gemmata obscuriglobus, Gimesia maris, and Blastopirellula marina), in addition to P. limnophila, thus extending the repertoire of genetically modifiable organisms in this superphylum. Although the Planctomycetes show high resistance to most antibiotics, we have used kanamycin resistance genes in G. obscuriglobus, P. limnophila, and G. maris, and tetracycline resistance genes in B. marina, as markers for mutant selection. In all cases, plasmids were introduced in the strains by mating or electroporation, and the genetic modification was verified by Southern Blotting analysis. In addition, we show that the green fluorescent protein (gfp) is expressed in all four backgrounds from an Escherichia coli promoter. The genetic manipulation achievement in four phylogenetically diverse planctomycetes will enable molecular studies in these strains, and opens the door to developing genetic approaches not only in other planctomycetes but also other species of the superphylum, such as the Lentisphaerae.

A Pseudomonas putida cbrB transposon insertion mutant displays a biofilm hyperproducing phenotype that is resistant to dispersal.

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 characterize a miniTn5-luxAB-Km transposon insertion mutant in cbrB (MPO406) in Pseudomonas putida leading to a biofilm overproducing phenotype that is not dispersed when nutrients are depleted. Comparison with a cbrB deletion mutant revealed that all phenotypes previously attributed to CbrB in P. putida correlated in both strains, with the exception of biofilm overproduction and absence of dispersal. We show that in the insertion mutant, the expression of the downstream regulatory RNA CrcZ is upregulated, and also show the presence of a truncated form of CbrB. Also, two additional point mutations in lapG and lapD have been detected in MPO406 by whole genome sequencing. Combination of these effects provides a robust biofilm overproducing phenotype. We present the mutant strain MPO406 as a good candidate to perform bio-production of substances of biotechnological interest or other processes such as bioremediation, which take advantage of immobilized cells on solid surfaces.

Hierarchical management of carbon sources is regulated similarly by the CbrA/B systems in Pseudomonas aeruginosa and Pseudomonas putida.

The CbrA/B system in pseudomonads is involved in the utilization of carbon sources and carbon catabolite repression (CCR) through the activation of the small RNAs crcZ in Pseudomonas aeruginosa, and crcZ and crcY in Pseudomonas putida. Interestingly, previous works reported that the CbrA/B system activity in P. aeruginosa PAO1 and P. putida KT2442 responded differently to the presence of different carbon sources, thus raising the question of the exact nature of the signal(s) detected by CbrA. Here, we demonstrated that the CbrA/B/CrcZ(Y) signal transduction pathway is similarly activated in the two Pseudomonas species. We show that the CbrA sensor kinase is fully interchangeable between the two species and, moreover, responds similarly to the presence of different carbon sources. In addition, a metabolomics analysis supported the hypothesis that CCR responds to the internal energy status of the cell, as the internal carbon/nitrogen ratio seems to determine CCR and non-CCR conditions. The strong difference found in the 2-oxoglutarate/glutamine ratio between CCR and non-CCR conditions points to the close relationship between carbon and nitrogen availability, or the relationship between the CbrA/B and NtrB/C systems, suggesting that both regulatory systems sense the same sort or interrelated signal.

Transcriptional activation of the CrcZ and CrcY regulatory RNAs by the CbrB response regulator in Pseudomonas putida.

The CbrAB two-component system has been described as a high-ranked element in the regulatory hierarchy of Pseudomonas putida that controls a variety of metabolic and behavioural traits required for adaptation to changing environmental conditions. We show that the response regulatory protein CbrB, an activator of σ(N) -dependent promoters, directly controls the expression of the small RNAs CrcZ and CrcY in P. putida. These two RNAs sequester the protein Crc, which is a translational repressor of multiple pathways linked to carbon catabolite repression. We characterized the in vivo and in vitro activation by CbrB at both crcZ and crcY promoters, and identified new DNA sequences where the protein binds. IHF, a co-activator at many σ(N) -dependent promoters, also binds to the promoter regions and contributes to the activation of the sRNAs. CbrB phosphorylation is necessary at physiological activation conditions, but a higher dose of the protein allows in vitro transcriptional activation in its non-phosphorylated form. We also show there is some production of CrcY coming from an upstream promoter independent of CbrB. Thus, CbrAB constitute a global signal transduction pathway integrated in a higher regulatory network that also controls catabolite repression through the expression of the two regulatory RNAs CrcZ and CrcY.

Regulation of glutamate dehydrogenase expression in Pseudomonas putida results from its direct repression by NtrC under nitrogen-limiting conditions.

Nitrogen-regulated genes in enterobacteria are positively controlled by the transcriptional activator of σ(N) -dependent promoters NtrC, either directly or indirectly, through the dual regulator Nac. Similar to enterobacteria, gdhA encoding glutamate dehydrogenase from Pseudomonas putida is one of the few genes that is induced by excess nitrogen. In P. putida, the binding of NtrC to the gdhA promoter region and in vitro transcription suggest that, unlike its enterobacterial homologue that is repressed by Nac, gdhA is directly repressed by NtrC. Footprinting analyses demonstrated that NtrC binds to four distinct sites in the gdhA promoter. NtrC dimers bind cooperatively, and those bound closer to the promoter interact with the dimers bound further upstream, thus producing a proposed repressor loop in the DNA. The formation of the higher-order complex and the repressor loop appears to be important for repression but not absolutely essential. Both the phosphorylated and the non-phosphorylated forms of NtrC efficiently repressed gdhA transcription in vitro and in vivo. Therefore, NtrC repression of gdhA under nitrogen-limiting conditions does not depend on the phosphorylation of the regulator; rather, it relies on an increase in the repressor concentration under these conditions.

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.

Distinct roles for NtrC and GlnK in nitrogen regulation of the Pseudomonas sp. strain ADP cyanuric acid utilization operon.

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 encoded LysR-type transcriptional regulator AtzR. Here, we take advantage of the poor induction of atzDEF in Escherichia coli to identify Pseudomonas factors involved in nitrogen control of atzDEF expression. Simultaneous production of P. putida NtrC and GlnK, along with AtzR, restored the normal atzDEF regulatory pattern. Gene expression analysis in E. coli and P. putida indicated that NtrC activates atzR expression, while the role of GlnK is to promote AtzR activation of atzDEF under nitrogen limitation. Activation of atzDEF in a mutant background deficient in GlnK uridylylation suggests that post-translational modification is not strictly required for transduction of the nitrogen limitation signal to AtzR. The present data and our previous results are integrated in a regulatory circuit that describes all the known responses of the atzDEF operon.

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.

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.

Proteomic and transcriptional characterization of aromatic degradation pathways in Rhodoccocus sp. strain TFB.

Rhodococcus sp. strain TFB is a versatile gram-positive bacterium able to grow on a wide variety of aromatic compounds as carbon and energy sources. Since the strain is refractory to genetic analysis, a proteomic approach was used to study the metabolic pathways involved in the catabolism of such compounds by analyzing differentially induced proteins. The most marked difference was observed when the proteome profiles of phthalate-grown cells were compared with those cultured in the presence of tetralin- or naphthalene, suggesting that different metabolic pathways are involved in the degradation of mono- and polyaromatic compounds. Comparison with the proteome of glucose-grown cells indicated that each pathway was specifically induced by the corresponding aromatic compound. A combination of proteomics and molecular biology led to the identification of 14 proteins (65-80% identical to known Pht proteins) that describe a complete pathway for the catabolism of phthalate to central metabolites via intradiol cleavage of protochatechuic acid. Chaperonins were also induced in phthalate-grown cells, indicating that growth on this compound induces a stress response. Absence of catabolite repression by glucose was observed by both transcriptional and proteome analysis, suggesting that Rhodococcus sp. strain TFB may have advantages over other tightly regulated strains in bioremediation.

Growth phase-dependent expression of the Pseudomonas putida KT2440 transcriptional machinery analysed with a genome-wide DNA microarray.

Bacterial transcriptional networks are built on a hierarchy of regulators, on top of which lie the components of the RNA polymerase (in particular the sigma factors) and the global control elements, which play a pivotal role. We have designed a genome-wide oligonucleotide-based DNA microarray for Pseudomonas putida KT2440. In combination with real-time reverse transcription polymerase chain reaction (RT-PCR), we have used it to analyse the expression pattern of the genes encoding the RNA polymerase subunits (the core enzyme and the 24 sigma factors), and various proteins involved in global regulation (Crc, Lrp, Fur, Anr, Fis, CsrA, IHF, HupA, HupB, HupN, BipA and several MvaT-like proteins), during the shift from exponential growth in rich medium into starvation and stress brought about by the entry into stationary phase. Expression of the genes encoding the RNA polymerase core and the vegetative sigma factor decreased in stationary phase, while that of sigma(S) increased. Data obtained for sigma(N), sigma(H), FliA and for the 19 extracytoplasmic function (ECF)-like sigma factors suggested that their mRNA levels change little upon entry into stationary phase. Expression of Crc, BipA, Fis, HupB, HupN and the MvaT-like protein PP3693 decreased in stationary phase, while that of HupA and the MvaT-like protein PP3765 increased significantly. Expression of IHF was indicative of post-transcriptional control. These results provide the first global study of the expression of the transcriptional machinery through the exponential stationary-phase shift in P. putida.

Synapsis and strand exchange in the resolution and DNA inversion reactions catalysed by the beta recombinase.

In the presence of a sequence-independent chromatin-associated protein, such as Hbsu or HMGB, the beta recombinase catalyses resolution between two directly oriented recombination sites (six sites) and both resolution and DNA inversion between two inversely oriented six sites. Assembly of the synaptic complex requires binding of the beta recombinase to the six sites and the presence of Hbsu. Whether resolution or inversion will take place depends on the relative orientation of the two six sites, the level of DNA supercoiling and the amounts of Hbsu. In this work, the topologies of the products of the resolution and inversion reactions were analysed. The resolution reaction generated mainly singly catenated DNA circles, while DNA inversion gave rise to unknotted circles and small amounts of DNA molecules containing 3- or 5-noded knots. In spite of the distinctive features of the beta system, the topology of synapsis and strand exchange during the resolution reaction is similar to that of Tn3 and gammadelta resolvases. The ability of the beta recombinase to catalyse both inversion and resolution reactions probably reflects different possible architectures of the synaptic complex, which to a large extent depends on Hbsu.