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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 PTS

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.

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.

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., S.H-A. and D.S-H. received predoctoral fellowships from the Spanish Ministry of Science and Competitiveness (MINECO). Work was supported by grants BFU2012-32797 (MINECO, Spain) and BIO2015-66203-P (MINECO/FEDER).

Summary 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 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 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.

In its natural habitats (soil, water and rhizosphere), Pseudomonas putida can suffer frequent and long-term changes in temperature that affect its growth and survival. Pseudomonas putida KT2440, a well-characterized model strain, grows optimally at 30°C but can proliferate at temperatures as low as 4°C. However, little information is available on the physiological changes that occur when P. putida grows at low temperatures. To investigate this area, the transcriptome and proteome profiles of cells exponentially growing in a complex medium at 10°C were compared with those of cells exponentially growing at 30°C. Low temperature modified the expression of at least 266 genes (some 5% of the genome). Many of the genes showing differential expression were involved in energy metabolism or in the transport and binding of substrates, although genes implicated in other cellular functions were also affected. Several changes seemed directed towards neutralizing problems created by low temperature, such as increased protein misfolding, the increased stability of DNA/RNA secondary structures, reduced membrane fluidity and a reduced growth rate. The present results improve our understanding of the P. putida lifestyle at low temperature, which may be relevant for its applications in bioremediation and in promotion of plant growth.

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 β-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.

A total of 12 non-epidemiologically related clinical isolates of Streptococcus mitis that showed different levels of resistance to penicillin were studied. Membrane-protein profiles and penicillin-binding protein (PBP) patterns showed a great polymorphism; and patterns of 4-7 PBPs, with sizes that ranged from approximately 101 kDa to approximately 40 kDa, were detected in each strain. No association could be found between PBP pattern and resistance level to penicillin among these isolates. Arbitrarily primed PCR confirmed the genetic diversity among this group of streptococci. One of the isolates of intermediate level of resistance to penicillin, which showed a PBP pattern similar to that of the high-resistance strains, was used as a laboratory model to analyse the mechanism underlying high-resistance acquisition by these strains. A 14-fold increase in penicillin resistance was obtained after a single selection step, which resulted in a decrease in penicillin affinity for PBP1. The size of this PBP (92 kDa) and the differences in PBP profiles of the penicillin-resistant clinical isolates suggest the existence in S. mitis of PBP-mediated mechanisms to acquire high-level resistance to penicillin, among which alterations in PBP1 seem to play a main role, in contrast to the PBP2X mediated mechanism described for other streptococci.

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.

The Crc protein is a global regulator that controls the hierarchical assimilation of carbon sources in Pseudomonads by inhibiting expression of several catabolic pathways. Crc does not bind DNA and its mechanism of action has remained elusive. Among other genes, Crc inhibits expression of alkS, the transcriptional activator of the Pseudomonas putida OCT plasmid alkane degradation pathway. AlkS activates expression of its own gene. In the presence of saturating AlkS levels, translational fusions of alkS to the lacZ reporter gene were responsive to Crc, but transcriptional fusions were not. In translational fusions, the first 33 nt of alkS mRNA, which includes up to position +3 relative to the translation start site, were sufficient to confer an efficient response to Crc. In vitro, purified Crc could bind specifically to an alkS mRNA fragment spanning positions +1 to +43, comprising the translation initiation region. We have previously shown that Crc has little effect on the stability of alkS mRNA. We conclude that Crc modulates AlkS levels by binding to the translation initiation region of alkS mRNA, thereby inhibiting translation. Because AlkS is an unstable protein present in limiting amounts, reducing its levels leads to decreased expression of all genes in the pathway.

The expression of an antisense RNA revealed that an Mn-catalase was required in Thermus thermophilus for aerobic but not for anaerobic growth. The antisense system is based on the constitutive expression of a "bicistronic" transcript consisting of the kanamycin resistance gene mRNA followed by the antisense RNA against the selected target, The financial support of project BIO2001-1627 to J. Berenguer and projects MATINOES G5RD-CT-2002-00752 from EC and PPQ2002-011231 from Spanish CICYT to J. M. Guisán are acknowledged. A. Hidalgo and R. Moreno are recipients of fellowships from Gobierno Vascco and Biotools B & M, respectively. An Institutional Grant from Fundación Ramón Areces to the CBMSO is also acknowledged

The presence of a periplasmic space within the cell envelope of Thermus thermophilus was analyzed in a mutant (HB8(Delta)UTR1) defective in the regulation of its S-layer (surface crystalline layer). This mutant forms round multicellular bodies (MBs) surrounded by a common envelope as the culture approaches the stationary phase. Confocal microscopy revealed that the origin of the MBs is the progressive detachment of the external layers coupled with the accumulation of NH(2)-containing material between the external envelopes and the peptidoglycan. A specific pattern of proteins was found as soluble components of the intercellular space of the MBs by a single fractionation procedure, suggesting that they are periplasmic-like components. To demonstrate this, we cloned a gene ( phoA) from T. thermophilus HB8 encoding a signal peptide-wearing alkaline phosphatase (AP), and engineered it to be overexpressed in the mutant from a shuttle vector. Most of the AP activity (>80%) was found as a soluble component of the MBs' intercellular fraction. All these data indicate that Thermus thermophilus actually has a periplasmic space which is functionally similar to that of Proteobacteria. The potential application of the HB8(Delta)UTR1 mutant for the overexpression of periplasmic thermophilic proteins is discussed.

Producción Científica, Thermus thermophilus HB8 can grow anaerobically by using a membrane-bound nitrate reductase to catalyze the reduction of nitrate as a final electron acceptor in respiration. In contrast to other denitrifiers, the nitrite produced does not continue the reduction pathway but accumulates in the growth medium after its active extrusion from the cell. We describe the presence of two genes,narK1 and narK2, downstream of the nitrate reductase-encoding gene cluster (nar) that code for two homologues to the major facilitator superfamily of transporters. The sequences of NarK1 and NarK2 are 30% identical to each other, but whereas NarK1 clusters in an average-distance tree with putative nitrate transporters, NarK2 does so with putative nitrite exporters. To analyze whether this differential clustering was actually related to functional differences, we isolated derivatives with mutations of one or both genes. Analysis revealed that single mutations had minor effects on growth by nitrate respiration, whereas a double narK1 narK2 mutation abolished this capability. Further analysis allowed us to confirm that the double mutant is completely unable to excrete nitrite, while single mutants have a limitation in the excretion rates compared with the wild type. These data allow us to propose that both proteins are implicated in the transport of nitrate and nitrite, probably acting as nitrate/nitrite antiporters. The possible differential roles of these proteins in vivo are discussed., Comisión Interministerial de Ciencia y Tecnologı́a (Project BIO98-0183), Ministerio de Educación, Cultura y Deporte y Unión Europea (Proyect 2FD97-0127- C02-01)