C-di-GMP and biofilm are regulated in Pseudomonas putida by the CfcA/CfcR two-component system in response to salts.

In Pseudomonas putida KT2440, cfcR encodes an orphan multidomain response regulator with diguanylate cyclase activity, which is responsible for the synthesis of c-di-GMP, a second messenger key in the transition from planktonic to sessile bacterial lifestyles. When overexpressed, cfcR enhances biofilm formation and causes other phenotype alterations. The cfcA gene, encoding a membrane-anchored multisensory CHASE3/GAF hybrid histidine kinase (HK), is required to develop this pleiotropic phenotype. Here we show autophosphorylation of CfcA through HisKA/HATPase_c domains and then transfer of the phosphoryl group to an internal receiver (REC) domain. CfcA REC domains are nonessential for phosphotransfer from CfcA~P to the REC domain of CfcR. CfcA~P also phosphorylates the REC domain of CfcD, a second HK encoded in the same gene cluster as CfcA, which negatively regulates the CfcA/CfcR pathway. To evaluate the impact of CfcA domains on CfcR activity, a battery of mutants with in-frame domain deletions was generated, whose CfcA protein locations were also examined. CfcA membrane anchorage contributes to protein stability and CfcR activation. Salt enhances c-di-GMP levels through CfcR, a response which is hampered by alteration of a presumed ligand-binding motif in the CHASE3 sensor domain. Thus, in P. putida, c-di-GMP is salt-regulated through the CfcA/CfcR/CfcD system.

Effluent decontamination by the ibuprofen-mineralizing strain, Sphingopyxis granuli RW412: Metabolic processes.

The high global consumption of ibuprofen and its limited elimination by wastewater treatment plants (WWTPs), has led to the contamination of aquatic systems by this common analgesic and its metabolites. The potentially negative environmental and public health effects of this emerging contaminant have raised concerns, driving the demand for treatment technologies. The implementation of bacteria which mineralize organic contaminants in biopurification systems used to decontaminate water or directly in processes in WWTPs, is a cheap and sustainable means for complete elimination before release into the environment. In this work, an ibuprofen-mineralizing bacterial strain isolated from sediments of the River Elbe was characterized and assayed to remediate different ibuprofen-polluted media. Strain RW412, which was identified as Sphingopyxis granuli, has a 4.48 Mb genome which includes plasmid sequences which harbor the ipf genes that encode the first steps of ibuprofen mineralization. Here, we confirm that these genes encode enzymes which initiate CoA ligation to ibuprofen, followed by aromatic ring activation by a dioxygenase and retroaldol cleavage to unequivocally produce 4-isobutylcatechol and propionyl-CoA which then undergo further degradation. In liquid mineral salts medium, the strain eliminated more than 2 mM ibuprofen within 74 h with a generation time of 16 h. Upon inoculation into biopurification systems, it eliminated repeated doses of ibuprofen within a few days. Furthermore, in these systems the presence of RW412 avoided the accumulation of ibuprofen metabolites. In ibuprofen-spiked effluent from a municipal WWTP, ibuprofen removal by this strain was 7 times faster than by the indigenous microbiota. These results suggest that this strain can persist and remain active under environmentally relevant conditions, and may be a useful innovation to eliminate this emerging contaminant from urban wastewater treatment systems.

Characterization of the bacteriocins and the PrtR regulator in a plant-associated Pseudomonas strain.

The emergence of antibiotic resistant bacterial strains demands the development of new antimicrobial agents. In the last decades, bacteriocins have gained significant interest due to their potential application as biopreservatives in the food industry and as therapeutic agents in medicine. Recent studies project the use of these antimicrobials in agriculture as biocontrol agents. The characterization of bacteriocins and their genetic regulation, however, have been scarcely studied in plant-associated bacteria. In this report, an in-silico and proteomic analysis was performed to identify the bacteriocins produced by Pseudomonas fluorescens SF4c. More than one functional bacteriocin was detected in this strain (S-type bacteriocins and phage-tail-like bacteriocins [tailocins]). It is known that the regulator PrtR represses bacteriocin production in P. aeruginosa under normal condition. However, the mechanism for tailocin regulation remains unknown in plant-associated pseudomonads. In this work, an orthologue of the prtR of P. aeruginosa was identified in the SF4c-tailocin cluster and a prtR null mutant constructed. The expression and production of tailocins was abolished in this mutant; thus evidencing that, unlike P. aeruginosa, PrtR is a positive regulator of tailocins expression in P. fluorescens.

Draft Genome Sequences of Isolates from Sediments of the River Elbe That Are Highly Tolerant to Diclofenac.

Here, we report the genome sequences of one Achromobacter and four Pseudomonas strains isolated from sediments of the River Elbe which are highly tolerant toward the xenobiotic target compound diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) and emerging contaminant.

Transcriptional response of Erwinia amylovora to copper shock: in vivo role of the copA gene.

Fire blight is a devastating plant disease caused by the bacterium Erwinia amylovora, and its control is frequently based on the use of copper-based compounds whose mechanisms of action are not well known. Consequently, in this article, we investigate the response of E. amylovora to copper shock by a whole-genome microarray approach. Transcriptional analyses showed that, in the presence of copper, 23 genes were increased in expression; these genes were classified mainly into the transport and stress functional categories. Among them, the copA gene was strongly induced and regulated in a finely tuned manner by copper. Mutation of copA, soxS, arcB, yjcE, ygcF, yhhQ, galF and EAM_3469 genes revealed that tolerance to copper in E. amylovora can be achieved by complex physiological mechanisms, including: (i) the control of copper homeostasis through, at least, the extrusion of Cu(I) by a P-type ATPase efflux pump CopA; and (ii) the overcoming of copper toxicity caused by oxidative stress by the expression of several reactive oxygen species (ROS)-related genes, including the two major transcriptional factors SoxS and ArcB. Furthermore, complementation analyses demonstrated the important role of copA for copper tolerance in E. amylovora, not only in vitro, but also in inoculated pear shoots.

Rhizosphere selection of Pseudomonas putida KT2440 variants with increased fitness associated to changes in gene expression.

As the interface between plant roots and soil, the rhizosphere is a complex environment where nutrients released by the plant promote microbial growth. Increasing evidences indicate that the plant also exerts a selective pressure on microbial populations in the rhizosphere, favouring colonization by certain groups. In this work, we have designed an experimental setup to begin analysing the evolution of a specific bacterial population in the rhizosphere, using Pseudomonas putida KT2440 as model organism. After several rounds of selection without passage through laboratory growth conditions, derivatives of this strain with increased fitness in the rhizosphere were isolated. Detailed analysis of one of these clones indicated that this effect is specific for rhizosphere conditions and derives from changes in its transcriptional profile in this environment, with 43 genes being differentially expressed with respect to the parental strain. Several of these genes belong to functional categories which could affect stress adaptation and availability of particular nutrients. By inactivating two genes identified as upregulated in the selected clone (coding for a stress-response protein and a rRNA modifying protein), these functions were shown to contribute to rhizosphere fitness. Our data also suggest the existence of different evolutionary pathways leading to increased rhizosphere fitness.

Interplay between extracellular matrix components of Pseudomonas putida biofilms.

The extracellular matrix of bacterial biofilms has at least two key functions: to serve as a structural scaffold for the multicellular community, and to play a protective role against external stress. In this work, we report a compensatory effect whereby Pseudomonas putida reacts to the lack of either of the two main surface proteins involved in biofilm formation, LapA and LapF, by increasing expression and production of a species-specific EPS. Elevated levels of the second messenger molecule cyclic di-GMP alter the balance of extracellular matrix components, and the phenotypes of lapA and lapF mutants under these conditions are indicative of direct interactions taking place between large secreted proteins and exopolysaccharides. Our data suggest the existence of a mechanism by which bacteria would sense alterations in the composition of the extracellular matrix, leading to changes in expression of the different elements.

Characterization of a phage-like pyocin from the plant growth-promoting rhizobacterium Pseudomonas fluorescens SF4c.

R-type and F-type pyocins are high-molecular-mass bacteriocins produced by Pseudomonas aeruginosa that resemble bacteriophage tails. They contain no head structures and no DNA, and are used as defence systems. In this report, we show that Pseudomonas fluorescens SF4c, a strain isolated from the wheat rhizosphere, produces a high-molecular-mass bacteriocin which inhibits the growth of closely related bacteria. A mutant deficient in production of this antimicrobial compound was obtained by transposon mutagenesis. Sequence analysis revealed that the transposon had disrupted a gene that we have named ptm, since it is homologous to that encoding phage tape-measure protein in P. fluorescens Pf0-1, a gene belonging to a prophage similar to phage-like pyocin from P. aeruginosa PAO1. In addition, we have identified genes from the SF4c pyocin cluster that encode a lytic system and regulatory genes. We constructed a non-polar ptm mutant of P. fluorescens SF4c. Heterologous complementation of this mutation restored the production of bacteriocin. Real-time PCR was used to analyse the expression of pyocin under different stress conditions. Bacteriocin was upregulated by mitomycin C, UV light and hydrogen peroxide, and was downregulated by saline stress. This report constitutes, to our knowledge, the first genetic characterization of a phage tail-like bacteriocin in a rhizosphere Pseudomonas strain.