Open Access
Pseudomonas savastanoi pv. savastanoi: some like it knot
(Wiley, 2012) Ramos, Cayo; Matas Casado, Isabel María; Bardají Goikoetxea, Leire; Aragón, Isabel M.; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena
Pseudomonas savastanoi pv. savastanoi is the causal agent of olive (Olea europaea) knot disease and an unorthodox member of the P. syringae complex, causing aerial tumours instead of the foliar necroses and cankers characteristic of most members of this complex. Olive knot is present wherever olive is grown; although losses are difficult to assess, it is assumed that olive knot is one of the most important diseases of the olive crop. The last century has witnessed a good deal of scientific articles describing the biology, epidemiology and control of this pathogen. However, most P. savastanoi pv. savastanoi strains are highly recalcitrant to genetic manipulation, which has effectively left the pathogen out of the scientific progress in molecular biology that has elevated the foliar pathogens of the P. syringae complex to supermodels. A series of studies in the last years have made significant advances in the biology, ecology and genetics of P. savastanoi pv. savastanoi, paving the way for the molecular dissection of its interaction with other non-pathogenic bacteria and their woody hosts. The selection of a genetically pliable model strain was soon followed by the development of rapid methods for virulence assessment with micropropagated olive plants and the analysis of cellular interactions with the plant host. The generation of a draft genome of strain NCPPB 3335 and the closed sequence of its three native plasmids has allowed for functional and comparative genomic analyses for the identification of its pathogenicity gene complement. This includes 34 putative type III effector genes and genomic regions, shared with other pathogens of woody hosts, that encode metabolic pathways associated with the degradation of lignin-derived compounds. Now, the time is right to explore the molecular basis of the P. savastanoi pv. savastanoi-olive interaction and to get insights into why some pathovars like it necrotic and why some like it knot. Synonyms: Pseudomonas syringae pv. savastanoi Taxonomy: Kingdom Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Family Pseudomonadaceae; Genus Pseudomonas; included in genomospecies 2 together with at least P. amygdali, P. ficuserectae, P. meliae and 16 other pathovars from the P. syringae complex (aesculi, ciccaronei, dendropanacis, eriobotryae, glycinea, hibisci, mellea, mori, myricae, phaseolicola, photiniae, sesami, tabaci, ulmi, and certain strains of lachrymans and morsprunorum); when a formal proposal is made for the unification of these bacteria, the species name P. amygdali would take priority over P. savastanoi. Microbiological properties: Gram-negative rods, 0.4-0.8 by 1.0-3.0 µm, aerobic. Motile by one to four polar flagella, rather slow growing, optimal temperatures for growth of 25–30 °C, oxidase negative, arginine dihydrolase negative, elicits the hypersensitive response on tobacco, most isolates are fluorescent and levan negative although some isolates are non-fluorescent and levan positive. Host range: P. savastanoi pv. savastanoi causes tumours in cultivated and wild olive and ash (Fraxinus excelsior). Although strains from olive were reported to infect oleander (Nerium oleander), this is generally not the case; however, strains of P. savastanoi pv. nerii can infect olive. Pathovars fraxini and nerii differentiate from pv. savastanoi mostly in their host range, and were not formally recognized until 1996. Literature previous to about 1996 generally name strains of the three pathovars as P. syringae subsp. savastanoi or P. savastanoi subsp. savastanoi, contributing to confusion about host range and biological properties. Disease symptoms: Symptoms of infected trees include hyperplastic growths (tumorous galls or knots) on the stems and branches of the host plant and, occasionally, on leaves and fruits. Epidemiology: The pathogen can survive and multiply on aerial plant surfaces, as well as in knots, from where it can be dispersed by rain, wind, insects and human activities, entering the plant through wounds. Populations are very unevenly distributed in the plant, and suffer drastic fluctuations throughout the year, with maximum numbers of bacteria occurring during rainy and warm months. Populations of P. savastanoi pv. savastanoi are normally associated to non-pathogenic bacteria, both epiphytically and endophytically, and were demonstrated to form mutualistic consortia with Erwinia toletana and Pantoea agglomerans that could result in increased bacterial populations and disease symptoms. Disease control: Based on preventive measures, mostly sanitary and cultural practices. Integrated control programs benefit from regular applications of copper formulations, which should be maintained at least a few years for maximum benefit. Olive cultivars vary in their susceptibility to olive knot, but there are no known cultivars with full resistance to the pathogen. Useful websites: http://www.pseudomonas-syringae.org/; http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl; ASAP access to the P. savastanoi pv. savastanoi NCPPB 3335 genome sequence https://asap.ahabs.wisc.edu/asap/logon.php.
Open Access
Characterisation of the mgo operon in Pseudomonas syringae pv. syringae UMAF0158 that is required for mangotoxin production
(BioMed Central, 2012) Arrebola, Eva; Carrión, Víctor J.; Cazorla, Francisco M.; Pérez García, Alejandro; Murillo Martínez, Jesús; Vicente, Antonio de; Producción Agraria; Nekazaritza Ekoizpena
Background: Mangotoxin is an antimetabolite toxin that is produced by strains of Pseudomonas syringae pv. syringae; mangotoxin-producing strains are primarily isolated from mango tissues with symptoms of bacterial apical necrosis. The toxin is an oligopeptide that inhibits ornithine N-acetyl transferase (OAT), a key enzyme in the biosynthetic pathway of the essential amino acids ornithine and arginine. The involvement of a putative nonribosomal peptide synthetase gene (mgoA) in mangotoxin production and virulence has been reported. Results: In the present study, we performed a RT-PCR analysis, insertional inactivation mutagenesis, a promoter expression analysis and terminator localisation to study the gene cluster containing the mgoA gene. Additionally, we evaluated the importance of mgoC, mgoA and mgoD in mangotoxin production. A sequence analysis revealed an operon-like organisation. A promoter sequence was located upstream of the mgoB gene and was found to drive lacZ transcription. Two terminators were located downstream of the mgoD gene. RT-PCR experiments indicated that the four genes (mgoBCAD) constitute a transcriptional unit. This operon is similar in genetic organisation to those in the three other P. syringae pathovars for which complete genomes are available (P. syringae pv. syringae B728a, P. syringae pv. tomato DC3000 and P. syringae pv. phaseolicola 1448A). Interestingly, none of these three reference strains is capable of producing mangotoxin. Additionally, extract complementation resulted in a recovery of mangotoxin production when the defective mutant was complemented with wild-type extracts. Conclusions: The results of this study confirm that mgoB, mgoC, mgoA and mgoD function as a transcriptional unit and operon. While this operon is composed of four genes, only the last three are directly involved in mangotoxin production.
Open Access
Detection by multiplex PCR and characterization of nontoxigenic strains of Pseudomonas syringae pv. phaseolicola from different places in Spain. Short communication
(Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), 2006) Rico, A.; Erdozáin García, María; Ortiz Barredo, Amaia; Ruiz de Galarreta, José Ignacio; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena
El control eficiente de la grasa de la judía causada por Pseudomonas syringae pv. phaseolicola se basa principalmente en la utilización de semilla libre del patógeno. La detección del patógeno en semilla se efectúa mediante métodos altamente sensibles basados en la detección por PCR de los genes responsables de la biosíntesis de la faseolotoxina, la cual, hasta ahora, se consideraba que era sintetizada por todas las cepas del patógeno con importancia epidemiológica. Sin embargo, en la Comunidad de Castilla y León, España, las epidemias de grasa de la judía en campo se asocian frecuentemente con cepas no toxigénicas de P. syringae pv. phaseolicola, que no pueden ser detectadas con los métodos moleculares y serológicos actuales. Los resultados presentados en este trabajo demuestran la existencia de aislados no toxigénicos de P. syringae pv. phaseolicola en zonas distintas de Castilla y León, lo que implica la necesidad de establecer una metodología fiable para la certificación de semillas de judía. Con este propósito, se presenta un sencillo protocolo en dos fases que permite la identificación de los dos tipos de aislados, y que se basa en una PCR multiplex con enriquecimiento a partir de extractos de semilla y en ensayos de patogenicidad.
Open Access
Two homologues of the global regulator Csr/Rsm redundantly control phaseolotoxin biosynthesis and virulence in the plant pathogen Pseudomonas amygdali pv. phaseolicola 1448A
(MDPI, 2020) Ramírez Zapata, Diana; Ramos, Cayo; Aguilera, Selene; Bardají Goikoetxea, Leire; Martínez Gil, Marta; Murillo Martínez, Jesús; Institute for Multidisciplinary Research in Applied Biology - IMAB
The widely conserved Csr/Rsm (carbon storage regulator/repressor of stationary-phase metabolites) post-transcriptional regulatory system controls diverse phenotypes involved in bacterial pathogenicity and virulence. Here we show that Pseudomonas amygdali pv. phaseolicola 1448A contains seven rsm genes, four of which are chromosomal. In RNAseq analyses, only rsmE was thermoregulated, with increased expression at 18 °C, whereas the antagonistic sRNAs rsmX1, rsmX4, rsmX5 and rsmZ showed increased levels at 28 °C. Only double rsmA-rsmE mutants showed significantly altered phenotypes in functional analyses, being impaired for symptom elicitation in bean, including in planta growth, and for induction of the hypersensitive response in tobacco. Double mutants were also non-motile and were compromised for the utilization of different carbon sources. These phenotypes were accompanied by reduced mRNA levels of the type III secretion system regulatory genes hrpL and hrpA, and the flagellin gene, fliC. Biosynthesis of the phytotoxin phaseolotoxin by mutants in rsmA and rsmE was delayed, occurring only in older cultures, indicating that these rsm homologues act as inductors of toxin synthesis. Therefore, genes rsmA and rsmE act redundantly, although with a degree of specialization, to positively regulate diverse phenotypes involved in niche colonization. Additionally, our results suggest the existence of a regulatory molecule different from the Rsm proteins and dependent on the GacS/GacA (global activator of antibiotic and cyanide production) system, which causes the repression of phaseolotoxin biosynthesis at high temperatures.
Open Access
The mangotoxin biosynthetic operon (mbo) is specifically distributed within Pseudomonas syringae genomospecies 1 and was acquired only once during evolution
(American Society for Microbiology, 2013) Carrión, Víctor J.; Gutiérrez Barranquero, José Antonio; Arrebola, Eva; Bardají Goikoetxea, Leire; Codina, Juan Carlos; Vicente, Antonio de; Cazorla, Francisco M.; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Mangotoxin production was ﬁrst described in Pseudomonas syringae pv. syringae strains. A phenotypic characterization of 94 P. syringae strains was carried out to determine the genetic evolution of the mangotoxin biosynthetic operon (mbo). We designed a PCR primer pair speciﬁc for the mbo operon to examine its distribution within the P. syringae complex. These primers ampliﬁed a 692-bp DNA fragment from 52 mangotoxin-producing strains and from 7 non-mangotoxin-producing strains that harbor the mbo operon, whereas 35 non-mangotoxin-producing strains did not yield any ampliﬁcation. This, together with the analysis of draft genomes, allowed the identiﬁcation of the mbo operon in ﬁve pathovars (pathovars aptata, avellanae, japonica, pisi, and syringae), all of which belong to genomospecies 1, suggesting a limited distribution of the mbo genes in the P. syringae complex. Phylogenetic analyses using partial sequences from housekeeping genes differentiated three groups within genomospecies 1. All of the strains containing the mbo operon clustered in groups I and II, whereas those lacking the operon clustered in group III; however, the relative branching order of these three groups is dependent on the genes used to construct the phylogeny. The mbo operon maintains synteny and is inserted in the same genomic location, with high sequence conservation around the insertion point, for all the strains in groups I and II. These data support the idea that the mbo operon was acquired horizontally and only once by the ancestor of groups I and II from genomospecies 1 within the P. syringae complex.
Open Access
The influence of the accessory genome on bacterial pathogen evolution
(Taylor & Francis, 2011) Jackson, Robert W.; Vinatzer, Boris; Arnold, Dawn L.; Dorus, Steve; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena
Bacterial pathogens exhibit significant variation in their genomic content of virulence factors. This reflects the abundance of strategies pathogens evolved to infect host organisms by suppressing host immunity. Molecular arms-races have been a strong driving force for the evolution of pathogenicity, with pathogens often encoding overlapping or redundant functions, such as type III protein secretion effectors and hosts encoding ever more sophisticated immune systems. The pathogens’ frequent exposure to other microbes, either in their host or in the environment, provides opportunities for the acquisition or interchange of mobile genetic elements. These DNA elements accessorize the core genome and can play major roles in shaping genome structure and altering the complement of virulence factors. Here, we review the different mobile genetic elements focusing on the more recent discoveries and highlighting their role in shaping bacterial pathogen evolution.