Andres Mäe

Isolation of an extracellular protease gene of Erwinia carotovora subsp. carotovora strain SCC3193 by transposon mutagenesis and the role of protease in phytopathogenicity.

Using mini-Tn5CmR::gusA, a transposon that allows transcriptional fusions to a promoterless beta-glucuronidase gene, a mutant of Erwinia carotovora subsp. carotovora SCC3193 deficient in extracellular protease production and soft-rot pathogenicity in plants was isolated. The mutant, designated SCC6004, produced normal levels of pectate lyase, polygalacturonase and cellulase. The region of the transposon insertion was partially sequenced to permit the design of specific oligonucleotide primers to amplify a 2.7 kb Clal fragment from E. carotovora subsp. carotovora SCC3193. The DNA sequence of the cloned fragment contained two complete and one partial ORFs. One of the complete ORFs (ORF1) was designated prtW and encodes a secreted protease. The deduced amino acid sequence of PrtW showed a high overall identify of 60-66% to the previously described Erwinia chrysanthemi proteases, but no homology to other proteases isolated from different E. carotovora strains. Downstream from ORF1, a further complete ORF (ORF2) and a partial ORF (ORF3) were found, with deduced peptide sequences that have significant similarity to the Inh and PrtD proteins, respectively, from E. chrysanthemi, which are involved in protease secretion. Gene fusion to the gusA reporter was employed to charaterize the regulation of prtW. The prtW gene was found to be strongly induced in the presence of plant extracts. The mutant exhibited reduced virulence, suggesting that PrtW enhances the ability of strain SCC3193 to macerate plant tissue.

Characterization of a new 2,4-dichlorophenoxyacetic acid degrading plasmid pEST4011: physical map and localization of catabolic genes

SUMMARY: Plasmid pEST4011 enables Pseudomonas putida PaW85 to degrade 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoate (3-CBA). This new 2,4-D degradative plasmid has considerable homology with the regions of pJP4 containing the 2,4-D degradative genes (tfd). Restriction fragment BamHI-B of plasmid pEST4011, which has homology with this region, was cloned into the broad-host-range vector pKT240 and studied in P. putida PaW85. Restriction mapping, hybridization analysis and enzyme assays established the location of the genes for 2,4-D monooxygenase (tfdA), 2,4-dichlorophenol hydroxylase (tfdB), chlorocatechol 1,2-dioxygenase (tfdC) and the tfdR and tfdS regulatory genes on this fragment. Plasmid pEST4012 is a derivative of pEST4011 derived through the spontaneous deletion of a 42 kbp DNA fragment, which results in the loss of the 2,4-D+ and 3-CBA+ phenotype. We present here the physical maps of pEST4011 and pEST4012. In spite of the similarities in functions, the size (70 kbp), order of catabolic genes and restriction pattern of pEST4011 are clearly different from those of pJP4.

Quorum Sensing and Expression of Virulence in Pectobacteria

Quorum sensing (QS) is a population density-dependent regulatory mechanism in which gene expression is coupled to the accumulation of a chemical signaling molecule. QS systems are widespread among the plant soft-rotting bacteria. In Pectobacterium carotovorum, at least two QS systems exist being specified by the nature of chemical signals involved. QS in Pectobacterium carotovorum uses N-acylhomoserine lactone (AHL) based, as well as autoinducer-2 (AI-2) dependent signaling systems. This review will address the importance of the QS in production of virulence factors and interaction of QS with other regulatory systems in Pectobacterium carotovorum.

A role for the Rcs phosphorelay in regulating expression of plant cell wall degrading enzymes in Pectobacterium carotovorum subsp. carotovorum.

The Rcs phosphorelay is a signal transduction system that influences the virulence phenotype of several pathogenic bacteria. In the plant pathogen Pectobacterium carotovorum subsp. carotovorum (Pcc) the response regulator of the Rcs phosphorelay, RcsB, represses expression of plant cell wall degrading enzymes (PCWDE) and motility. The focus of this study was to identify genes directly regulated by the binding of RcsB that also regulate expression of PCWDE genes in Pcc. RcsB-binding sites within the regulatory regions of the flhDC operon and the rprA and rsmB genes were identified using DNase I protection assays, while in vivo studies using flhDC : : gusA, rsmB : : gusA and rprA : : gusA gene fusions revealed gene regulation. These experiments demonstrated that the operon flhDC, a flagellar master regulator, was repressed by RcsB, and transcription of rprA was activated by RcsB. Regulation of the rsmB promoter by RcsB is more complicated. Our results show that RcsB represses rsmB expression mainly through modulating flhDC transcription. Neverthless, direct binding of RcsB on the rsmB promoter region is possible in certain conditions. Using an rprA-negative mutant, it was further demonstrated that RprA RNA is not essential for regulating expression of PCWDE under the conditions tested, whereas overexpression of rprA increased protease expression in wild-type cells. Stationary-phase sigma factor, RpoS, is the only known target gene for RprA RNA in Escherichia coli; however, in Pcc the effect of RprA RNA was found to be rpoS-independent. Overall, our results show that the Rcs phosphorelay negatively affects expression of PCWDE by inhibiting expression of flhDC and rsmB.

Lack of RsmA-mediated control results in constant hypervirulence, cell elongation, and hyperflagellation in Pectobacterium wasabiae.

The posttranscriptional regulator RsmA controls the production of plant cell wall degrading enzymes (PCWDE) and cell motility in the Pectobacterium genus of plant pathogens. In this study the physiological role of gene regulation by RsmA is under investigation. Disruption of rsmA gene of the Pectobacterium wasabiae strain, SCC3193 resulted in 3-fold decrease in growth rate and increased virulence. The comparison of mRNA levels of the rsmA− mutant and wild-type using a genome-wide microarray showed, that genes responsible for successful infection, i.e. virulence factors, motility, butanediol fermentation, various secretion systems etc. were up-regulated in the rsmA− strain. The rsmA− strain exhibited a higher propensity to swarm and produce PCWDE compared to the wild-type strain. Virulence experiments in potato tubers demonstrated that in spite of its more efficient tissue maceration, the rsmA− strains ability to survive within the host is reduced and the infection site is taken over by resident bacteria. Taken together, in the absence of RsmA, cells revert to a constitutively infective phenotype characterized by expression of virulence factors and swarming. We hypothesize that lack of control over these costly energetic processes results in decreased growth rate and fitness. In addition, our findings suggest a relationship between swarming and virulence in plant pathogens.

Role and Regulation of the Flp/Tad Pilus in the Virulence of Pectobacterium atrosepticum SCRI1043 and Pectobacterium wasabiae SCC3193

In this study, we characterized a putative Flp/Tad pilus-encoding gene cluster, and we examined its regulation at the transcriptional level and its role in the virulence of potato pathogenic enterobacteria of the genus Pectobacterium. The Flp/Tad pilus-encoding gene clusters in Pectobacterium atrosepticum, Pectobacterium wasabiae and Pectobacterium aroidearum were compared to previously characterized flp/tad gene clusters, including that of the well-studied Flp/Tad pilus model organism Aggregatibacter actinomycetemcomitans, in which this pilus is a major virulence determinant. Comparative analyses revealed substantial protein sequence similarity and open reading frame synteny between the previously characterized flp/tad gene clusters and the cluster in Pectobacterium, suggesting that the predicted flp/tad gene cluster in Pectobacterium encodes a Flp/Tad pilus-like structure. We detected genes for a novel two-component system adjacent to the flp/tad gene cluster in Pectobacterium, and mutant analysis demonstrated that this system has a positive effect on the transcription of selected Flp/Tad pilus biogenesis genes, suggesting that this response regulator regulate the flp/tad gene cluster. Mutagenesis of either the predicted regulator gene or selected Flp/Tad pilus biogenesis genes had a significant impact on the maceration ability of the bacterial strains in potato tubers, indicating that the Flp/Tad pilus-encoding gene cluster represents a novel virulence determinant in Pectobacterium. Soft-rot enterobacteria in the genera Pectobacterium and Dickeya are of great agricultural importance, and an investigation of the virulence of these pathogens could facilitate improvements in agricultural practices, thus benefiting farmers, the potato industry and consumers.

Microbial population dynamics in response to Pectobacterium atrosepticum infection in potato tubers

Endophytes are microbes and fungi that live inside plant tissues without damaging the host. Herein we examine the dynamic changes in the endophytic bacterial community in potato (Solanum tuberosum) tuber in response to pathogenic infection by Pectobacterium atrosepticum, which causes soft rot in numerous economically important crops. We quantified community changes using both cultivation and next-generation sequencing of the 16S rRNA gene and found that, despite observing significant variability in both the mass of macerated tissue and structure of the endophytic community between individual potato tubers, P. atrosepticum is always taken over by the endophytes during maceration. 16S rDNA sequencing revealed bacteria from the phyla Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, Fusobacteria, Verrucomicrobia, Acidobacteria, TM7, and Deinococcus-Thermus. Prior to infection, Propionibacterium acnes is frequently among the dominant taxa, yet is out competed by relatively few dominant taxa as the infection proceeds. Two days post-infection, the most abundant sequences in macerated potato tissue are Gammaproteobacteria. The most dominant genera are Enterobacter and Pseudomonas. Eight days post-infection, the number of anaerobic pectolytic Clostridia increases, probably due to oxygen depletion. These results demonstrate that the pathogenesis is strictly initiated by the pathogen (sensu stricto) and proceeds with a major contribution from the endophytic community.

AepA of Pectobacterium is not involved in the regulation of extracellular plant cell wall degrading enzymes production

Plant cell wall degrading enzymes (PCWDE) are the major virulence determinants in phytopathogenic Pectobacterium, and their production is controlled by many regulatory factors. In this study, we focus on the role of the AepA protein, which was previously described to be a global regulator of PCWDE production in Pectobacterium carotovorum (Murata et al. in Mol Plant Microbe Interact 4:239–246, 1991). Our results show that neither inactivation nor overexpression of aepA affects PCWDE production in either Pectobacterium atrosepticum SCRI1043 or Pectobacterium carotovorum subsp. carotovorum SCC3193. The previously published observation based on the overexpression of aepA could be explained by the presence of the adjacent regulatory rsmB gene in the constructs used. Our database searches indicated that AepA belongs to the YtcJ subfamily of amidohydrolases. YtcJ-like amidohydrolases are present in bacteria, archaea, plants and some fungi. Although AepA has 28% identity with the formamide deformylase NfdA in Arthrobacter pascens F164, AepA was unable to catalyze the degradation of NdfA-specific N-substituted formamides. We conclude that AepA is a putative aminohydrolase not involved in regulation of PCWDE production.

Transposon-mediated mobilization of chromosomally located catabolic operons of the CAM plasmid by TOL plasmid transposon Tn4652 and CAM plasmid transposon Tn3614

The CAM (camphor degradation) plasmid is integrated into the chromosome of Pseudomonas putida PaW-line strains and is not self-transferable as a plasmid via conjugation. Our results show that the mobilization of chromosomally located CAM and the integration of cam-operons into the chromosome of the new Cam+ transconjugants is a recA-independent process mediated by transposons Tn4652 (17 kbp) and Tn3614 (7.2 kbp). Transposon Tn3614 is apparently identical to the left-hand and the right-hand sequences of the TOL plasmid pWW0 transposon Tn4654. The insertion of Tn401 inside the left-hand terminal IR of Tn4652 completely inhibited the mobilization of CAM. According to our data transposons Tn4652 and Tn3614 together with CAM plasmid catabolic operons are integrated into the chromosome. We propose that in pseudomonads the transposons Tn4652 and Tn3614 play a key role in the evolution and spread of new catabolic plasmids in nature.

Expression of nipP.w of Pectobacterium wasabiae is dependent on functional flgKL flagellar genes.

While flagellum-driven motility is hypothesized to play a role in the virulence of Pectobacterium species, there is no direct evidence that genes involved in flagellum assembly regulate the synthesis of virulence factors. The purpose of this study was to identify genes that affect the production or secretion of necrosis-inducing protein (Nip) in the strain SCC3193. Transposon mutagenesis of an RpoS strain overexpressing NipP.w was performed, and a mutant associated with decreased necrosis of tobacco leaves was detected. The mutant contained a transposon in the regulatory region upstream of the flagellar genes flgK and flgL. Additional mutants were generated related to the flagellar genes fliC and fliA. The mutation in flgKL, but not those in fliC and fliA, inhibited nipP.w transcription. Moreover, the regulatory effect of the flgKL mutation on nipP.w transcription was partially dependent on the Rcs phosphorelay. Secretion of NipP.w was also dependent on a type II secretion mechanism. Overall, the results of this study indicate that the flgKL mutation is responsible for reduced motility and lower levels of nipP.w expression.