Extraordinary long-stem confers resistance of intrinsic terminators to processive antitermination.

Many prokaryotic operons encode a processive antitermination (P-AT) system. Transcription complexes associated with an antitermination factor can bypass multiple transcription termination signals regardless of their sequences. However, to avoid compromising transcriptional regulation of downstream regions, the terminator at the end of the operon needs to be resistant to antitermination. So far, no studies on the mechanism of resistance to antitermination have been reported. The recently discovered conAn P-AT system is composed of two components that are encoded at the start of many conjugation operons on plasmids of Gram-positive bacteria. Here we report the identification of a conAn-resistant terminator, named TerR, in the conjugation operon of the Bacillus subtilis plasmid pLS20, re-defining the end of the conjugation operon. We investigated the various characteristics of TerR and show that its extraordinary long stem is the determining feature for resistance to antitermination. This is the first P-AT resistance mechanism to be reported.

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.

A complex genetic switch involving overlapping divergent promoters and DNA looping regulates expression of conjugation genes of a gram-positive plasmid.

Plasmid conjugation plays a significant role in the dissemination of antibiotic resistance and pathogenicity determinants. Understanding how conjugation is regulated is important to gain insights into these features. Little is known about regulation of conjugation systems present on plasmids from Gram-positive bacteria. pLS20 is a native conjugative plasmid from the Gram-positive bacterium Bacillus subtilis. Recently the key players that repress and activate pLS20 conjugation have been identified. Here we studied in detail the molecular mechanism regulating the pLS20 conjugation genes using both in vivo and in vitro approaches. Our results show that conjugation is subject to the control of a complex genetic switch where at least three levels of regulation are integrated. The first of the three layers involves overlapping divergent promoters of different strengths regulating expression of the conjugation genes and the key transcriptional regulator RcoLS20. The second layer involves a triple function of RcoLS20 being a repressor of the main conjugation promoter and an activator and repressor of its own promoter at low and high concentrations, respectively. The third level of regulation concerns formation of a DNA loop mediated by simultaneous binding of tetrameric RcoLS20 to two operators, one of which overlaps with the divergent promoters. The combination of these three layers of regulation in the same switch allows the main conjugation promoter to be tightly repressed during conditions unfavorable to conjugation while maintaining the sensitivity to accurately switch on the conjugation genes when appropriate conditions occur. The implications of the regulatory switch and comparison with other genetic switches involving DNA looping are discussed.

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.

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.

Deep eutectic solvent-assisted synthesis of biodegradable polyesters with antibacterial properties.

Bacterial infection related to the implantation of medical devices represents a serious clinical complication, with dramatic consequences for many patients. In past decades, numerous attempts have been made to develop materials with antibacterial and/or antifouling properties by the incorporation of antibiotic and/or antiseptic compounds. In this context, deep eutectic solvents (DESs) are acquiring increasing interest not only as efficient carriers of active principle ingredients (APIs) but also as assistant platforms for the synthesis of a wide repertoire of polymer-related materials. Herein, we have successfully prepared biodegradable poly(octanediol-co-citrate) polyesters with acquired antibacterial properties by the DES-assisted incorporation of quaternary ammonium or phosphonium salts into the polymer network. In the resulting polymers, the presence of these salts (i.e., choline chloride, tetraethylammonium bromide, hexadecyltrimethylammonium bromide, and methyltriphenylphosphonium bromide) inhibits bacterial growth in the early postimplantation steps, as tested in cultures of Escherichia coli on solid agar plates. Later, positive polymer cytocompatibility is expected to support cell colonization, as anticipated from in vitro preliminary studies with L929 fibroblasts. Finally, the attractive elastic properties of these polyesters permit matching those of soft tissues such as skin. For all of these reasons, we envisage the utility of some of these antibacterial, biocompatible, and biodegradable polyesters as potential candidates for the preparation of antimicrobial wound dressings. These results further emphasize the enormous versatility of DES-assisted synthesis for the incorporation, in the synthesis step, of a wide palette of APIs into polymeric networks suitable for biomedical applications.

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.

Differential expression of the three Alcanivorax borkumensis SK2 genes coding for the P450 cytochromes involved in the assimilation of hydrocarbons.

Alcanivorax borkumensis, a marine bacterium highly specialized in degrading linear and branched alkanes, plays a key ecological role in the removal of marine oil spills. It contains several alternative enzyme systems for terminal hydroxylation of alkanes, including three P450 cytochromes (P450-1, P450-2 and P450-3). The present work shows cytochrome P450-1 to be expressed from the promoter of the upstream gene fdx. Promoter Pfdx was more active when C8 -C18 n-alkanes or pristane were assimilated than when pyruvate was available. The product of ABO_0199 (named CypR) was identified as a transcriptional activator of Pfdx . The inactivation of cypR impaired growth on tetradecane, showing the importance of the fdx-P450-1 and/or cypR genes. P450-2 expression was low-level and constitutive under all conditions tested, while that of P450-3 from promoter P450-3 was much higher when cells assimilated pristane than when n-alkanes or pyruvate were available. However, the inactivation of P450-3 had no visible impact on pristane assimilation. Cyo terminal oxidase, a component of the electron transport chain, was found to stimulate promoter PP450-3 activity, but it did not affect promoters Pfdx or PP450-2 . A. borkumensis, therefore, appears to carefully coordinate the expression of its multiple hydrocarbon degradation genes using both specific and global regulatory systems.

Green Synthesis of Hierarchically Structured Silver-Polymer Nanocomposites with Antibacterial Activity.

The in situ formation of silver nanoparticles (AgNPs) aided by chondroitin sulfate and the preparation of a hierarchically structured silver-polymer nanocomposite with antimicrobial activity is shown. Green synthesis of AgNPs is carried out by thermal treatment (80 and 90 °C) or UV irradiation of a chondroitin sulfate solution containing AgNO3 without using any further reducing agents or stabilizers. Best control of the AgNPs size and polydispersity was achieved by UV irradiation. The ice-segregation-induced self-assembly (ISISA) process, in which the polymer solution containing the AgNPs is frozen unidirectionally, and successively freeze-drying were employed to produce the chondroitin sulfate 3D scaffolds. The scaffolds were further crosslinked with hexamethylene diisocyanate vapors to avoid water solubility of the 3D structures in aqueous environments. The antimicrobial activity of the scaffolds was tested against Escherichia coli. The minimum inhibitory concentration (MIC) found for AgNPs-CS (chondroitin sulfate) scaffolds was ca. 6 ppm.

Marine hydrocarbonoclastic bacteria as whole-cell biosensors for n-alkanes.

Whole-cell biosensors offer potentially useful, cost-effective systems for the in-situ monitoring of seawater for hydrocarbons derived from accidental spills. The present work compares the performance of a biosensor system for the detection of alkanes in seawater, hosted in either Escherichia coli (commonly employed in whole-cell biosensors but not optimized for alkane assimilation) or different marine bacteria specialized in assimilating alkanes. The sensor system was based on the Pseudomonas putida AlkS regulatory protein and the PalkB promoter fused to a gene encoding the green fluorescent protein. While the E. coli sensor provided the fastest response to pure alkanes (25-fold induction after 2 h under the conditions used), a sensor based on Alcanivorax borkumensis was slower, requiring 3-4 h to reach similar induction values. However, the A. borkumensis sensor showed a fourfold lower detection threshold for octane (0.5 µM), and was also better at sensing the alkanes present in petrol. At petrol concentrations of 0.0125%, the A. borkumensis sensor rendered a sevenfold induction, while E. coli sensor showed no response. We discuss possible explanations to this behaviour in terms of the cellular adaptations to alkane uptake and the basal fluorescence produced by each bacterial strain, which was lowest for A. borkumensis.

The Pseudomonas putida Crc global regulator is an RNA binding protein that inhibits translation of the AlkS transcriptional regulator.

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.

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.

Levels and activity of the Pseudomonas putida global regulatory protein Crc vary according to growth conditions.

The global regulatory protein Crc is involved in the repression of several catabolic pathways for sugars, hydrocarbons, and nitrogenated and aromatic compounds in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolite repression), therefore modulating carbon metabolism. We have analyzed whether the levels or the activity of Crc is regulated. Crc activity was followed by its ability to inhibit the induction by alkanes of the P. putida OCT plasmid alkane degradation pathway when cells grow in a complete medium, where the effect of Crc is very strong. The abundance of crc transcripts and the amounts of Crc protein were higher under repressing conditions than under nonrepressing conditions. The presence of crc on a high-copy-number plasmid considerably increased Crc levels, but this impaired its ability to inhibit the alkane degradation pathway. Crc shows similarity to a family of nucleases that have highly conserved residues at their catalytic sites. Mutation of the corresponding residues in Crc (Asp220 and His246) led to proteins that can inhibit induction of the alkane degradation pathway when present at normal or elevated levels in the cell. Repression by these mutant proteins occurred only under repressing conditions. These results suggest that both the amounts and the activity of Crc are modulated and support previous proposals that Crc may form part of a signal transduction pathway. Furthermore, the activity of the mutant proteins suggests that Crc is not a nuclease.

Differential expression of the components of the two alkane hydroxylases from Pseudomonas aeruginosa.

Oxidation of n-alkanes in bacteria is normally initiated by an enzyme system formed by a membrane-bound alkane hydroxylase and two soluble proteins, rubredoxin and rubredoxin reductase. Pseudomonas aeruginosa strains PAO1 and RR1 contain genes encoding two alkane hydroxylases (alkB1 and alkB2), two rubredoxins (alkG1 and alkG2), and a rubredoxin reductase (alkT). We have localized the promoters for these genes and analyzed their expression under different conditions. The alkB1 and alkB2 genes were preferentially expressed at different moments of the growth phase; expression of alkB2 was highest during the early exponential phase, while alkB1 was induced at the late exponential phase, when the growth rate decreased. Both genes were induced by C(10) to C(22)/C(24) alkanes but not by their oxidation derivatives. However, the alkG1, alkG2, and alkT genes were expressed at constant levels in both the absence and presence of alkanes.