The LuxR Regulators PcoR and RfiA Co-regulate Antimicrobial Peptide and Alginate Production in Pseudomonas corrugata.

Cyclic lipopeptides (CLPs) are considered as some of the most important secondary metabolites in different plant-associated bacteria, thanks to their antimicrobial, cytotoxic, and surfactant properties. In this study, our aim was to investigate the role of the Quorum Sensing (QS) system, PcoI/PcoR, and the LuxR-type transcriptional regulator RfiA in CLP production in the phytopatogenic bacterium, Pseudomonas corrugata based on our previous work where we reported that the pcoR and rfiA mutants were devoid of the CLPs cormycin and corpeptin production. Due to the close genetic link between the QS system and the RfiA (rfiA is co-transcribed with pcoI), it was difficult to ascertain the specific regulatory role in the expression of target genes. A transcriptional approach was undertaken to identify the specific role of the PcoR and RfiA transcriptional regulators for the expression of genes involved in CLP production. The RNA-seq-based transcriptional analysis of the wild-type (WT) strain CFBP 5454 in comparison with GL2 (pcoR mutant) and GLRFIA (rfiA mutant) was performed in cultural conditions favoring CLP production. Differential gene expression revealed that 152 and 130 genes have significantly different levels of expression in the pcoR and rfiA mutants, respectively. Of these, the genes linked to the biosynthesis of CLPs and alginate were positively controlled by both PcoR and RfiA. Blast homology analysis showed that 19 genes in a large CLP biosynthetic cluster involved in the production of three antimicrobial peptides, which span approximately 3.5% of the genome, are strongly over-expressed in the WT strain. Thus, PcoR and RfiA function mainly as activators in the production of bioactive CLPs, in agreement with phenotype analysis of mutants. RNA-seq also revealed that almost all the genes in the structural/biosynthetic cluster of alginate exopolysaccharide (EPS) are under the control of the PcoR-RfiA regulon, as supported by the 10-fold reduction in total EPS yield isolated in both mutants in comparison to the parent strain. A total of 68 and 38 gene expressions was independently regulated by PcoR or RfiA proteins, respectively, but at low level. qPCR experiments suggest that growth medium and plant environment influence the expression of CLP and alginate genes.

Influence of Host Plant on Thaumetopoea pityocampa Gut Bacterial Community.

Microbial communities associated to the gut of insects are attracting an increasing interest, mainly because of their role in influencing several host life-traits. The characterization of the gut microbial community is pivotal for understanding insect ecology and, thus, to develop novel pest management strategies. The pine processionary moth, Thaumetopoea pytiocampa (Denis & Schiff.) (Lepidoptera: Thaumetopoeidae), is a severe defoliator of pine forests, able to feed on several pine species. In this work, we performed a metabarcoding analysis to investigate, for the first time, the diversity of the gut bacterial community of pine processionary larvae associated with three different host pine species (Pinus halepensis, Pinus nigra subsp. laricio, and Pinus pinaster). We found that the gut microbial community of T. pityocampa larvae collected on P. halapensis was different from that associated with larvae collected from P. nigra and P. pinaster. Moreover, the high presence of bacteria belonging to the genera Modestobacter, Delftia, and unidentified Methylobacteriaceae retrieved in larvae feeding on P. halapensis suggested that specific interactions can occur. Our results provide the evidence that different host plant differently impact on the microbiota diversity of T. pityocampa larvae, contributing to the general knowledge of this pest with information that could be useful in shaping the next generation of pest control strategies.

A novel GIS-based approach to assess beekeeping suitability of Mediterranean lands.

Honeybees are critically important for the environment and to the economy. However, there are in substantial decline worldwide, leading to serious threat to the stability and yield of food crops. Beekeeping is of pivotal importance, combining the wide economical aspect of honey production and the important ecological services provided by honeybees. In this scenario, the prompt identification of beekeeping areas is strategic, since it maximised productivity and lowered the risks of colony losses. Fuzzy logic is an ideal approach for problem-solving tasks, as it is specifically designed to manage problems with a high degree of uncertainty. This research tested a novel GIS-based approach to assess beekeeping suitability of lands located in Calabria (Southern Italy), without relying to Analytic Hierarchy Process - Multiple Criteria Decision Making (AHP-MCDM), thus avoiding the constraints due to the technique and decision makers' influences. Furthermore, the data used here were completely retrieved from open access sources, highlighting that our approach is characterized by low costs and can be easily reproduced for a wide arrays of geographical contexts. Notably, the results obtained by our experiments were validated by the actual beekeeping reality. Besides beekeeping, the use of this system could not only be applied in beekeeping land suitability evaluations, but may be successfully extended to other types of land suitability evaluations.

Comparative genomic analysis of multiple strains of two unusual plant pathogens: Pseudomonas corrugata and Pseudomonas mediterranea.

The non-fluorescent pseudomonads, Pseudomonas corrugata (Pcor) and P. mediterranea (Pmed), are closely related species that cause pith necrosis, a disease of tomato that causes severe crop losses. However, they also show strong antagonistic effects against economically important pathogens, demonstrating their potential for utilization as biological control agents. In addition, their metabolic versatility makes them attractive for the production of commercial biomolecules and bioremediation. An extensive comparative genomics study is required to dissect the mechanisms that Pcor and Pmed employ to cause disease, prevent disease caused by other pathogens, and to mine their genomes for genes that encode proteins involved in commercially important chemical pathways. Here, we present the draft genomes of nine Pcor and Pmed strains from different geographical locations. This analysis covered significant genetic heterogeneity and allowed in-depth genomic comparison. All examined strains were able to trigger symptoms in tomato plants but not all induced a hypersensitive-like response in Nicotiana benthamiana. Genome-mining revealed the absence of type III secretion system and known type III effector-encoding genes from all examined Pcor and Pmed strains. The lack of a type III secretion system appears to be unique among the plant pathogenic pseudomonads. Several gene clusters coding for type VI secretion system were detected in all genomes. Genome-mining also revealed the presence of gene clusters for biosynthesis of siderophores, polyketides, non-ribosomal peptides, and hydrogen cyanide. A highly conserved quorum sensing system was detected in all strains, although species specific differences were observed. Our study provides the basis for in-depth investigations regarding the molecular mechanisms underlying virulence strategies in the battle between plants and microbes.

N-acyl-homoserine-lactone quorum sensing in tomato phytopathogenic Pseudomonas spp. is involved in the regulation of lipodepsipeptide production.

Pseudomonas corrugata and Pseudomonas mediterranea are two closely related phytopathogenic bacteria both causal agents of tomato pith necrosis. P. corrugata produces phytotoxic and antimicrobial cationic lipodepsipeptides (LDPs) which are thought to act as major virulence factors. Previous studies have demonstrated that P. corrugata CFBP 5454 has an N-acyl homoserine lactone (AHL) quorum sensing (QS) system PcoI/PcoR and that LDP production occurs at high population densities. No molecular studies on virulence have thus far been reported for P. mediterranea. In this study, we show that P. mediterranea also produces LDPs as well as possessing an AHL-dependent QS system, designated PmeI/PmeR, which is highly homologous to the PcoI/R system of P. corrugata producing and responding to C(6)-AHL. Downstream of pmeI, a partial DNA sequence revealed the presence of a homolog of the rfiA gene of P. corrugata which encodes a transcriptional regulator involved in bacterial virulence. Pathogenicity tests and MALDI-TOF spectra of wild-type strains of both bacterial species and their respective QSs and rfiA derivative mutants revealed that, in the absence of LDPs, the strains induce very weak symptoms indicating that LDPs may act as major virulence factors. Mutational analysis of both QS systems suggests that their mode of action is in places different.