Contribution of the non-effector members of the HrpL regulon, iaaL and matE, to the virulence of Pseudomonas syringae pv. tomato DC3000 in tomato plants

Incluye 4 ficheros de datosBackground: The phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear. Results: A conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants. Conclusions: Our results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence.This research was supported by the Spanish Plan Nacional I+D+i grants AGL2011-30343-CO2-01, AGL2014-53242-C2-1-R and BIO2007-67874-C02-02 as well as by grants ref. P08-CVI-03475 and P12-AGR-0370 from the Junta de Andalucía (Spain)

FleQ, FleN and c-di-GMP coordinately regulate cellulose production in Pseudomonas syringae pv. tomato DC3000

The second messenger cyclic di-GMP (c-di-GMP) controls the transition between motility and sessility in many bacterial species by a variety of mechanisms, including the production of multiple exopolysaccharides. Pseudomonas syringae pv. tomato (Pto) DC3000 is a plant pathogenic bacteria able to synthesize acetylated cellulose under high c-di-GMP levels thanks to the expression of the wssABCDEFGHI operon. Increased cellulose production enhances air-liquid biofilm formation and generates a wrinkled colony phenotype on solid media. We previously showed that under low levels of c-di-GMP, the regulators FleQ and AmrZ bound to adjacent sequences at the wss promoter inhibiting its expression, but only FleQ responded to the presence of c-di-GMP by activating cellulose production. In the present work, we advance in the knowledge of this complex regulation in Pto DC3000 by shedding light over the role of FleN in this process. The distinctive features of this system are that FleN and FleQ are both required for repression and activation of the wss operon under low and high c-di-GMP levels, respectively. We have also identified three putative FleQ binding sites at the wss promoter and show that FleQ/FleN-ATP binds at those sites under low c-di-GMP levels, inducing a distortion of DNA, impairing RNA polymerase binding, and repressing wss transcription. However, binding of c-di-GMP induces a conformational change in the FleQ/FleN-ATP complex, which relieves the DNA distortion, allows promoter access to the RNA polymerase, and leads to activation of wss transcription. On the other hand, AmrZ is always bound at the wss promoter limiting its expression independently of FleQ, FleN and c-di-GMP levels

DNA melting within a binary σ(54)-promoter DNA complex

9 pages, 7 figures, 1 table.-- PMID: 11036081 [PubMed].-- Available online Oct 17, 2000.-- Full-text version available Open Access at the journal site.The σ(54) subunit of the bacterial RNA polymerase requires the action of specialized enhancer-binding activators to initiate transcription. Here we show that σ(54) is able to melt promoter DNA when it is bound to a DNA structure representing the initial nucleation of DNA opening found in closed complexes. Melting occurs in response to activator in a nucleotide-hydrolyzing reaction and appears to spread downstream from the nucleation point toward the transcription start site. We show that σ(54) contains some weak determinants for DNA melting that are masked by the Region I sequences and some strong ones that require Region I. It seems that σ(54) binds to DNA in a self-inhibited state, and one function of the activator is therefore to promote a conformational change in σ(54) to reveal its DNA-melting activity. Results with the holoenzyme bound to early melted DNA suggest an ordered series of events in which changes in core to σ(54) interactions and σ(54)-DNA interactions occur in response to activator to allow σ(54) isomerization and the holoenzyme to progress from the closed complex to the open complex.This work was supported by a Wellcome Trust grant (to M. B.) and by a Biotechnology Marie Curie fellowship (to M.-T. G.). Work was conducted in the Imperial College Center for Structural Biology.Peer reviewe

Caracterización del regulador transcripcional XyIS del plásmido tol de pseudomonas putida

Tesis Universidad de Granada. Facultad de Ciencia

Distinctive features of the Gac-Rsm pathway in plant-associated Pseudomonas

Productive plant–bacteria interactions, either beneficial or pathogenic, require that bacteria successfully sense, integrate and respond to continuously changing environmental and plant stimuli. They use complex signal transduction systems that control a vast array of genes and functions. The Gac-Rsm global regulatory pathway plays a key role in controlling fundamental aspects of the apparently different lifestyles of plant beneficial and phytopathogenic Pseudomonas as it coordinates adaptation and survival while either promoting plant health (biocontrol strains) or causing disease (pathogenic strains). Plant-interacting Pseudomonas stand out for possessing multiple Rsm proteins and Rsm RNAs, but the physiological significance of this redundancy is not yet clear. Strikingly, the components of the Gac-Rsm pathway and the controlled genes/pathways are similar, but the outcome of its regulation may be opposite. Therefore, identifying the target mRNAs bound by the Rsm proteins and their mode of action (repression or activation) is essential to explain the resulting phenotype. Some technical considerations to approach the study of this system are also given. Overall, several important features of the Gac-Rsm cascade are now understood in molecular detail, particularly in Pseudomonas protegens CHA0, but further questions remain to be solved in other plant-interacting Pseudomonas.This research was supported by grants BIO2014-55075-P and BIO2017-83533-P from the ERDF/Spanish Ministry of Science, Innovation and Universities - State Research Agency. M.D.F. was supported by a FPU contract from the Spanish MECD/MEFP (ECD/1619/2013)

Single amino acid substitution mutants of Klebsiella pneumoniae σ(54) defective in transcription

Transcription initiation by the σ(54) RNA polymerase requires specialised activators and their associated nucleoside triphosphate hydrolysis. To explore the roles of σ(54) in initiation we used random mutagenesis of rpoN and an in vivo activity screen to isolate functionally altered σ(54) proteins. Five defective mutants, each with a different single amino acid substitution, were obtained. Three failed in transcription after forming a closed complex. One such mutant mapped to regulatory Region I of σ(54), the other two to Region III. The Region I mutant allowed transcription independently of activator and showed reduced activator-dependent σ(54) isomerisation. The two Region III mutants displayed altered behaviour in a σ(54) isomerisation assay and one failed to stably bind early melted DNA as the holoenzyme; they may contribute to a communication pathway linking changes in σ to open complex formation. Two further Region III mutants showed gross defects in overall DNA binding. For one, sufficient residual DNA binding activity remained to allow us to demonstrate that other activities were largely unaffected. Changes in DNA binding preferences and core polymerase-dependent properties were evident amongst the mutants

Antibacterial actions of flavonoids

Accumulation of flavonoids and isoflavonoids in response to pathogen attack has been observed in many plant species and its importance as antimicrobial phytoalexins is well established. Moreover, the mechanisms of action of several flavonoids on bacteria have been elucidated, proving that they act on multiple cell targets. Flavonoids cause damage to the cytoplasmic membrane perforating it and/or reducing its fluidity; block the synthesis of nucleic acids by inhibiting topoisomerase and/or DNA gyrase, paralyze energy metabolism by inhibiting the respiratory chain and ATP synthase, prevent cell wall synthesis by acting as competitive inhibitors of the D-alanine-D-alanine ligase, and cell membrane synthesis by inhibiting enzymes of the fatty acid synthesis pathway. Furthermore, it has been demonstrated that flavonoids have synergistic activity modulating antibiotic resistance and attenuating bacterial pathogenicity by inhibiting the production of several virulence factors that causes the inhibition of motility, bacterial adhesion to the host, biofilm formation.The authors are grateful for the continued funding by grants from ‘Dirección General de Investigación’ of the Spanish Ministry fof Economy and Competitiveness (MINECO) (BIO2011-23032), grants from ‘Programa de Incentivos, Consejería de Innovación, Ciencia y Empresa’ of the Andalusian Regional Goverment (P08-CVI-3475, P10-CVI-05800), and FEDER funds.Peer reviewe

Pathogenic and mutualistic plant-bacteria interactions: ever increasing similarities

Plant-interacting bacteria can establish either mutualistic or pathogenic interactions that cause beneficial or detrimental effects respectively, to their hosts. In spite of the completely different outcomes, accumulating evidence indicates that similar molecular bases underlie the establishment of these two contrasting plant-bacteria associations. Recent findings observed in the mutualistic nitrogen-fixing Rhizobium-legume symbiosis add new elements to the increasing list of similarities. Amongst these, in this review we describe the role of plant resistance proteins in determining host specificity in the Rhizobium-legume symbiosis that resemble the gene-for-gene resistance of plant-pathogen interactions, and the production of antimicrobial peptides by certain legumes to control rhizobial proliferation within nodules. Amongst common bacterial strategies, cyclic diguanylate (c-di-GMP) appears to be a second messenger used by both pathogenic and mutualistic bacteria to regulate key features for interaction with their plant hosts.Peer Reviewe