The acetoin assimilation pathway of Pseudomonas putida KT2440 is regulated by overlapping global regulatory elements that respond to nutritional cues.

Many microorganisms produce and excrete acetoin (3-hydroxy-2-butanone) when growing in environments that contain glucose or other fermentable carbon sources. This excreted compound can then be assimilated by other bacterial species such as pseudomonads. This work shows that acetoin is not a preferred carbon source of Pseudomonas putida, and that the induction of genes required for its assimilation is down-modulated by different, independent, global regulatory systems when succinate, glucose or components of the LB medium are also present. The expression of the acetoin degradation genes was found to rely on the RpoN alternative sigma factor and to be modulated by the Crc/Hfq, Cyo and PTSNtr regulatory elements, with the impact of the latter three varying according to the carbon source present in addition to acetoin. Pyruvate, a poor carbon source for P. putida, did not repress acetoin assimilation. Indeed, the presence of acetoin significantly improved growth on pyruvate, revealing these compounds to have a synergistic effect. This would provide a clear competitive advantage to P. putida when growing in environments in which all the preferred carbon sources have been depleted and pyruvate and acetoin remain as leftovers from the fermentation of sugars by other microorganisms.

Expression of the ISPpu9 transposase of Pseudomonas putida KT2440 is regulated by two small RNAs and the secondary structure of the mRNA 5'-untranslated region.

Insertion sequences (ISs) are mobile genetic elements that only carry the information required for their own transposition. Pseudomonas putida KT2440, a model bacterium, has seven copies of an IS called ISPpu9 inserted into repetitive extragenic palindromic sequences. This work shows that the gene for ISPpu9 transposase, tnp, is regulated by two small RNAs (sRNAs) named Asr9 and Ssr9, which are encoded upstream and downstream of tnp, respectively. The tnp mRNA has a long 5'-untranslated region (5'-UTR) that can fold into a secondary structure that likely includes the ribosome-binding site (RBS). Mutations weakening this structure increased tnp mRNA translation. Asr9, an antisense sRNA complementary to the 5'-UTR, was shown to be very stable. Eliminating Asr9 considerably reduced tnp mRNA translation, suggesting that it helps to unfold this secondary structure, exposing the RBS. Ectopic overproduction of Asr9 increased the transposition frequency of a new ISPpu9 entering the cell by conjugation, suggesting improved tnp expression. Ssr9 has significant complementarity to Asr9 and annealed to it in vitro forming an RNA duplex; this would sequester it and possibly facilitate its degradation. Thus, the antisense Asr9 sRNA likely facilitates tnp expression, improving transposition, while Ssr9 might counteract Asr9, keeping tnp expression low.

Influence of the Hfq and Crc global regulators on the control of iron homeostasis in Pseudomonas putida.

Metabolically versatile bacteria use catabolite repression control to select their preferred carbon sources, thus optimizing carbon metabolism. In pseudomonads, this occurs through the combined action of the proteins Hfq and Crc, which form stable tripartite complexes at target mRNAs, inhibiting their translation. The activity of Hfq/Crc is antagonised by small RNAs of the CrcZ family, the amounts of which vary according to carbon availability. The present work examines the role of Pseudomonas putida Hfq protein under conditions of low-level catabolite repression, in which Crc protein would have a minor role since it is sequestered by CrcZ/CrcY. The results suggest that, under these conditions, Hfq remains operative and plays an important role in iron homeostasis. In this scenario, Crc appears to participate indirectly by helping CrcZ/CrcY to control the amount of free Hfq in the cell. Iron homeostasis in pseudomonads relies on regulatory elements such as the Fur protein, the PrrF1-F2 sRNAs, and several extracytoplasmic sigma factors. Our results show that the absence of Hfq is paralleled by a reduction in PrrF1-F2 small RNAs. Hfq thus provides a regulatory link between iron and carbon metabolism, coordinating the iron supply to meet the needs of the enzymes operational under particular nutritional regimes.

Effect of Crc and Hfq proteins on the transcription, processing, and stability of the Pseudomonas putida CrcZ sRNA.

In Pseudomonas putida, the Hfq and Crc proteins regulate the expression of many genes in response to nutritional and environmental cues, by binding to mRNAs that bear specific target motifs and inhibiting their translation. The effect of these two proteins is antagonized by the CrcZ and CrcY small RNAs (sRNAs), the levels of which vary greatly according to growth conditions. The crcZ and crcY genes are transcribed from promoters PcrcZ and PcrcY, respectively, a process that relies on the CbrB transcriptional activator and the RpoN σ factor. Here we show that crcZ can also be transcribed from the promoter of the immediate upstream gene, cbrB, a weak constitutive promoter. The cbrB-crcZ transcript was processed to render a sRNA very similar in size to the CrcZ produced from promoter PcrcZ The processed sRNA, termed CrcZ*, was able to antagonize Hfq/Crc because, when provided in trans, it relieved the deregulated Hfq/Crc-dependent hyperrepressing phenotype of a ΔcrcZΔcrcY strain. CrcZ* may help in attaining basal levels of CrcZ/CrcZ* that are sufficient to protect the cell from an excessive Hfq/Crc-dependent repression. Since a functional sRNA can be produced from PcrcZ, an inducible strong promoter, or by cleavage of the cbrB-crcZ mRNA, crcZ can be considered a 3'-untranslated region of the cbrB-crcZ mRNA. In the absence of Hfq, the processed form of CrcZ was not observed. In addition, we show that Crc and Hfq increase CrcZ stability, which supports the idea that these proteins can form a complex with CrcZ and protect it from degradation by RNases.

Transcriptional and translational control through the 5'-leader region of the dmpR master regulatory gene of phenol metabolism.

Expression of pathways for dissimilation of toxic aromatic compounds such as (methyl)phenols interfaces both stress-response and carbon catabolite repression control cascades. In Pseudomonas putida, carbon catabolite repression is mediated by the protein Crc - a translational repressor that counteracts utilization of less-preferred carbon sources as growth substrates until they are needed. In this work we dissect the regulatory role of the 5'-leader region (5'-LR) of the dmpR gene that encodes the master regulator of (methyl)phenol catabolism. Using deletion and substitution mutants combined with artificial manipulations of Crc availability in P. putida, we present evidence that a DNA motif within the 5'-leader region is critical for inhibition of the output from the Pr promoter that drives transcription of dmpR, while the RNA chaperone Hfq facilitates Crc-mediated translation repression through the 5'-leader region of the dmpR mRNA. The results are discussed in the light of a model in which Hfq assists Crc to target a sequence within a loop formed by secondary structure of the 5'-LR mRNA. Our results support the idea that Crc functions as a global translational inhibitor to co-ordinate hierarchical carbon utilization in Pseudomonads.

The Crc and Hfq proteins of Pseudomonas putida cooperate in catabolite repression and formation of ribonucleic acid complexes with specific target motifs.

The Crc protein is a global regulator that has a key role in catabolite repression and optimization of metabolism in Pseudomonads. Crc inhibits gene expression post-transcriptionally, preventing translation of mRNAs bearing an AAnAAnAA motif [the catabolite activity (CA) motif] close to the translation start site. Although Crc was initially believed to bind RNA by itself, this idea was recently challenged by results suggesting that a protein co-purifying with Crc, presumably the Hfq protein, could account for the detected RNA-binding activity. Hfq is an abundant protein that has a central role in post-transcriptional gene regulation. Herein, we show that the Pseudomonas putida Hfq protein can recognize the CA motifs of RNAs through its distal face and that Crc facilitates formation of a more stable complex at these targets. Crc was unable to bind RNA in the absence of Hfq. However, pull-down assays showed that Crc and Hfq can form a co-complex with RNA containing a CA motif in vitro. Inactivation of the hfq or the crc gene impaired catabolite repression to a similar extent. We propose that Crc and Hfq cooperate in catabolite repression, probably through forming a stable co-complex with RNAs containing CA motifs to result in inhibition of translation initiation.

Interrogating Social Sustainability in the Biofuels Sector in Latin America: Tensions Between Global Standards and Local Experiences in Mexico, Brazil, and Colombia.

Across the Americas, biofuels production systems are diverse due to geographic conditions, historical patterns of land tenure, different land use patterns, government policy frameworks, and relations between the national state and civil society, all of which shape the role that biofuels play in individual nations. Although many national governments throughout the Americas continue to incentivize growth of the biofuels industry, one key challenge for biofuels sustainability has been concern about its social impacts. In this article, we discuss some of the key social issues and tensions related to the recent expansion of biofuels production in Mexico, Colombia, and Brazil. We argue that a process of "simplification" of ecological and cultural diversity has aided the expansion of the biofuels frontier in these countries, but is also undermining their viability. We consider the ability of governments and non-state actors in multi-stakeholder initiatives (MSI) to address social and environmental concerns that affect rural livelihoods as a result of biofuels expansion. We analyze the tensions between global sustainability standards, national level policies for biofuels development, and local level impacts and visions of sustainability. We find that both government and MSI efforts to address sustainability concerns have limited impact, and recommend greater incorporation of local needs and expertise to improve governance.

What are the signals that control catabolite repression in Pseudomonas?

Metabolically versatile bacteria exhibit a global regulatory response known as carbon catabolite repression (CCR), which prioritizes some carbon sources over others when all are present in sufficient amounts. This optimizes growth by distributing metabolite fluxes, but can restrict yields in biotechnological applications. The molecular mechanisms and preferred substrates for CCR vary between bacterial groups. Escherichia coli prioritizes glucose whereas Pseudomonas sp. prefer certain organic acids or amino acids. A significant issue in understanding (and potentially bypassing) CCR is the lack of information about the signals that trigger this regulatory response. In E. coli, several key compounds act as flux sensors, governing the flow of metabolites through catabolic pathways and preventing imbalances. These flux sensors can also modulate the CCR response. It has been suggested that the order of substrate preference is determined by carbon uptake flux rather than substrate identity. For Pseudomonas, much less information is available, as the signals that induce CCR are poorly understood. This article briefly discusses the available evidence on the signals that trigger CCR and the questions that remain to be answered in Pseudomonas.

Inactivation of Pseudomonas putida KT2440 pyruvate dehydrogenase relieves catabolite repression and improves the usefulness of this strain for degrading aromatic compounds.

Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl-CoA, which feeds the tricarboxylic acid cycle. We investigated how the loss of PDH affects metabolism in Pseudomonas putida. PDH inactivation resulted in a strain unable to utilize compounds whose assimilation converges at pyruvate, including sugars and several amino acids, whereas compounds that generate acetyl-CoA supported growth. PDH inactivation also resulted in the loss of carbon catabolite repression (CCR), which inhibits the assimilation of non-preferred compounds in the presence of other preferred compounds. Pseudomonas putida can degrade many aromatic compounds, most of which produce acetyl-CoA, making it useful for biotransformation and bioremediation. However, the genes involved in these metabolic pathways are often inhibited by CCR when glucose or amino acids are also present. Our results demonstrate that the PDH-null strain can efficiently degrade aromatic compounds even in the presence of other preferred substrates, which the wild-type strain does inefficiently, or not at all. As the loss of PDH limits the assimilation of many sugars and amino acids and relieves the CCR, the PDH-null strain could be useful in biotransformation or bioremediation processes that require growth with mixtures of preferred substrates and aromatic compounds.

The contribution of proteomics to the unveiling of the survival strategies used by Pseudomonas putida in changing and hostile environments.

Pseudomonas putida is a ubiquitous, metabolically very versatile, Gram-negative bacterium adapted to habitats as diverse as soil, water and the rhizosphere. Most strains are nonpathogenic, many are used as experimental models, and many others have biotechnological applications in the areas of agriculture, bioremediation, biocatalysis, and the production of bioplastics. This review summarizes the contribution of proteomic technologies to our understanding of how P. putida responds to different carbon sources, how it adapts to living at suboptimal temperatures or attached to surfaces, and how it responds to the presence of toxic compounds such as aromatic molecules and heavy metals. The examples described illustrate the value of proteomics in furthering our knowledge of the physiology and behavior of bacteria, knowledge that is important for understanding how they behave in their natural habitats and for optimizing their behavior in biotechnological applications.

Two small RNAs, CrcY and CrcZ, act in concert to sequester the Crc global regulator in Pseudomonas putida, modulating catabolite repression.

The Crc protein is a translational repressor that recognizes a specific target at some mRNAs, controlling catabolite repression and co-ordinating carbon metabolism in pseudomonads. In Pseudomonas aeruginosa, the levels of free Crc protein are controlled by CrcZ, a sRNA that sequesters Crc, acting as an antagonist. We show that, in Pseudomonas putida, the levels of free Crc are controlled by CrcZ and by a novel 368 nt sRNA named CrcY. CrcZ and CrcY, which contain six potential targets for Crc, were able to bind Crc specifically in vitro. The levels of CrcZ and CrcY were low under conditions generating a strong catabolite repression, and increased strongly when catabolite repression was absent. Deletion of either crcZ or crcY had no effect on catabolite repression, but the simultaneous absence of both sRNAs led to constitutive catabolite repression that compromised growth on some carbon sources. Overproduction of CrcZ or CrcY significantly reduced repression. We propose that CrcZ and CrcY act in concert, sequestering and modulating the levels of free Crc according to metabolic conditions. The CbrA/CbrB two-component system activated crcZ transcription, but had little effect on crcY. CrcY was detected in P. putida, Pseudomonas fluorescens and Pseudomonas syringae, but not in P. aeruginosa.

Pseudomonas putida growing at low temperature shows increased levels of CrcZ and CrcY sRNAs, leading to reduced Crc-dependent catabolite repression.

The Crc protein of Pseudomonas inhibits the expression of genes involved in the transport and assimilation of a number of non-preferred carbon sources when preferred substrates are available, thus coordinating carbon metabolism. Crc acts by binding to target mRNAs, inhibiting their translation. In Pseudomonas putida, the amount of free Crc available is controlled by two sRNAs, CrcY and CrcZ, which bind to and sequester Crc. The levels of these sRNAs vary according to metabolic conditions. Pseudomonas putida grows optimally at 30°C, but can also thrive at 10°C. The present work shows that when cells grow exponentially at 10°C, the repressive effect of Crc on many genes is significantly reduced compared with that seen at 30°C. Total Crc levels were similar at both temperatures, but those of CrcZ and CrcY were significantly higher at 10°C. Therefore, Crc-mediated repression may, at least in part, be reduced at 10°C because the fraction of Crc protein sequestered by CrcZ and CrcY is larger, reducing the amount of free Crc available to bind its targets. This may help P. putida to face cold stress. The results reported might help understanding the behaviour of this bacterium in bioremediation or rhizoremediation strategies at low temperatures.

The translational repressor Crc controls the Pseudomonas putida benzoate and alkane catabolic pathways using a multi-tier regulation strategy.

Metabolically versatile bacteria usually perceive aromatic compounds and hydrocarbons as non-preferred carbon sources, and their assimilation is inhibited if more preferable substrates are available. This is achieved via catabolite repression. In Pseudomonas putida, the expression of the genes allowing the assimilation of benzoate and n-alkanes is strongly inhibited by catabolite repression, a process controlled by the translational repressor Crc. Crc binds to and inhibits the translation of benR and alkS mRNAs, which encode the transcriptional activators that induce the expression of the benzoate and alkane degradation genes respectively. However, sequences similar to those recognized by Crc in benR and alkS mRNAs exist as well in the translation initiation regions of the mRNA of several structural genes of the benzoate and alkane pathways, which suggests that Crc may also regulate their translation. The present results show that some of these sites are functional, and that Crc inhibits the induction of both pathways by limiting not only the translation of their transcriptional activators, but also that of genes coding for the first enzyme in each pathway. Crc may also inhibit the translation of a gene involved in benzoate uptake. This multi-tier approach probably ensures the rapid regulation of pathway genes, minimizing the assimilation of non-preferred substrates when better options are available. A survey of possible Crc sites in the mRNAs of genes associated with other catabolic pathways suggested that targeting substrate uptake, pathway induction and/or pathway enzymes may be a common strategy to control the assimilation of non-preferred compounds.

The Crc global regulator inhibits the Pseudomonas putida pWW0 toluene/xylene assimilation pathway by repressing the translation of regulatory and structural genes.

In Pseudomonas putida, the expression of the pWW0 plasmid genes for the toluene/xylene assimilation pathway (the TOL pathway) is subject to complex regulation in response to environmental and physiological signals. This includes strong inhibition via catabolite repression, elicited by the carbon sources that the cells prefer to hydrocarbons. The Crc protein, a global regulator that controls carbon flow in pseudomonads, has an important role in this inhibition. Crc is a translational repressor that regulates the TOL genes, but how it does this has remained unknown. This study reports that Crc binds to sites located at the translation initiation regions of the mRNAs coding for XylR and XylS, two specific transcription activators of the TOL genes. Unexpectedly, eight additional Crc binding sites were found overlapping the translation initiation sites of genes coding for several enzymes of the pathway, all encoded within two polycistronic mRNAs. Evidence is provided supporting the idea that these sites are functional. This implies that Crc can differentially modulate the expression of particular genes within polycistronic mRNAs. It is proposed that Crc controls TOL genes in two ways. First, Crc inhibits the translation of the XylR and XylS regulators, thereby reducing the transcription of all TOL pathway genes. Second, Crc inhibits the translation of specific structural genes of the pathway, acting mainly on proteins involved in the first steps of toluene assimilation. This ensures a rapid inhibitory response that reduces the expression of the toluene/xylene degradation proteins when preferred carbon sources become available.

The Crc global regulator binds to an unpaired A-rich motif at the Pseudomonas putida alkS mRNA coding sequence and inhibits translation initiation.

Crc is a key global translational regulator in Pseudomonads that orchestrates the hierarchy of induction of several catabolic pathways for amino acids, sugars, hydrocarbons or aromatic compounds. In the presence of amino acids, which are preferred carbon sources, Crc inhibits translation of the Pseudomonas putida alkS and benR mRNAs, which code for transcriptional regulators of genes required to assimilate alkanes (hydrocarbons) and benzoate (an aromatic compound), respectively. Crc binds to the 5'-end of these mRNAs, but the sequence and/or structure recognized, and the way in which it inhibits translation, were unknown. We have determined the secondary structure of the alkS mRNA 5'-end through its sensitivity to several ribonucleases and chemical reagents. Footprinting and band-shift assays using variant alkS mRNAs have shown that Crc specifically binds to a short unpaired A-rich sequence located adjacent to the alkS AUG start codon. This interaction is stable enough to prevent formation of the translational initiation complex. A similar Crc-binding site was localized at benR mRNA, upstream of the Shine-Dalgarno sequence. This allowed predicting binding sites at other Crc-regulated genes, deriving a consensus sequence that will help to validate new Crc targets and to discriminate between direct and indirect effects of this regulator.

The global regulator Crc modulates metabolism, susceptibility to antibiotics and virulence in Pseudomonas aeruginosa.

The capacity of a bacterial pathogen to produce a disease in a treated host depends on the former's virulence and resistance to antibiotics. Several scattered pieces of evidence suggest that these two characteristics can be influenced by bacterial metabolism. This potential relationship is particularly important upon infection of a host, a situation that demands bacteria adapt their physiology to their new environment, making use of newly available nutrients. To explore the potential cross-talk between bacterial metabolism, antibiotic resistance and virulence, a Pseudomonas aeruginosa model was used. This species is an important opportunistic pathogen intrinsically resistant to many antibiotics. The role of Crc, a global regulator that controls the metabolism of carbon sources and catabolite repression in Pseudomonas, was analysed to determine its contribution to the intrinsic antibiotic resistance and virulence of P. aeruginosa. Using proteomic analyses, high-throughput metabolic tests and functional assays, the present work shows the virulence and antibiotic resistance of this pathogen to be linked to its physiology, and to be under the control (directly or indirectly) of Crc. A P. aeruginosa strain lacking the Crc regulator showed defects in type III secretion, motility, expression of quorum sensing-regulated virulence factors, and was less virulent in a Dictyostelium discoideum model. In addition, this mutant strain was more susceptible to beta-lactams, aminoglycosides, fosfomycin and rifampin. Crc might therefore be a good target in the search for new antibiotics.

The Pseudomonas putida Crc global regulator controls the hierarchical assimilation of amino acids in a complete medium: evidence from proteomic and genomic analyses.

The Crc protein is a global translational regulator involved in catabolite repression of catabolic pathways for several non-preferred carbon sources in Pseudomonads when other preferred substrates are present. Using proteomic and transcriptomic approaches, we have analyzed the influence of Crc in cells growing in a complete medium, where amino acids are the main carbon source. Inactivation of the crc gene modified the expression of at least 134 genes. Most of them were involved in the transport and assimilation of amino acids or sugars. This allowed envisioning which amino acids are preferentially used. Crc did not inhibit the pathways for proline, alanine, glutamate, glutamine and histidine. These amino acids are good carbon sources for P. putida. In the case of arginine, lysine, aspartate and asparagine, which can be assimilated through several pathways, Crc favored one particular route, inhibiting other alternatives. Finally, Crc-inhibited genes needed to assimilate valine, isoleucine, leucine, tyrosine, phenylalanine, threonine, glycine and serine, amino acids that provide a less efficient growth. Crc has therefore a key role in coordinating metabolism, controlling the sequential assimilation of amino acids when cells grow in a complete medium. Inactivation of crc reduced growth rate, suggesting that Crc optimizes metabolism.

The target for the Pseudomonas putida Crc global regulator in the benzoate degradation pathway is the BenR transcriptional regulator.

Crc protein is a global regulator involved in catabolite repression control of several pathways for the assimilation of carbon sources in pseudomonads when other preferred substrates are present. In Pseudomonas putida cells growing exponentially in a complete medium containing benzoate, Crc strongly inhibits the expression of the benzoate degradation genes. These genes are organized into several transcriptional units. We show that Crc directly inhibits the expression of the peripheral genes that transform benzoate into catechol (the ben genes) but that its effect on genes corresponding to further steps of the pathway (the cat and pca genes of the central catechol and beta-ketoadipate pathways) is indirect, since these genes are not induced because the degradation intermediates, which act as inducers, are not produced. Crc inhibits the translation of target genes by binding to mRNA. The expression of the ben, cat, and pca genes requires the BenR, CatR, and PcaR transcriptional activators, respectively. Crc significantly reduced benABCD mRNA levels but did not affect those of benR. Crc bound to the 5' end of benR mRNA but not to equivalent regions of catR and pcaR mRNAs. A translational fusion of the benR and lacZ genes was sensitive to Crc, but a transcriptional fusion was not. We propose that Crc acts by reducing the translation of benR mRNA, decreasing BenR levels below those required for the full expression of the benABCD genes. This strategy provides great metabolic flexibility, allowing the hierarchical assimilation of different structurally related compounds that share a common central pathway by selectively regulating the entry of each substrate into the central pathway.

Genomic analysis of the role of RNase R in the turnover of Pseudomonas putida mRNAs.

RNase R is a 3'-5' highly processive exoribonuclease that can digest RNAs with extensive secondary structure. We analyzed the global effect of eliminating RNase R on the Pseudomonas putida transcriptome and the expression of the rnr gene under diverse conditions. The absence of RNase R led to increased levels of many mRNAs, indicating that it plays an important role in mRNA turnover.