Conclusions

The phylogenetic analysis carried out using the genome information collected for various SRE confirmed ICMP19477 as a strain of P. carotovorum subsp. brasiliensis (as described in Chapter 2). This strain had previously been identified as belonging to P.

carotovorum subsp. carotovorum (Pitman et al., 2008). It is possible that other

Pectobacterium isolates from potato have been mistakenly mis-classified and that P.

carotovorum subsp. brasiliensis is more common than previously thought. Certainly, recent research has described the widespread distribution of this pathogen on potato in other countries (De Boer et al., 2012; Duarte et al., 2004; Onkendi et al., 2014; Van der Merwe et al., 2010).

Comparative genomic analysis using ICMP19477 and a number of other SRE also enabled the identification of genetic factors that might contribute to the pathogenicity or aggressiveness of P. carotovorum subsp. brasiliensis as well as the GIs and gene islets whose horizontal transfer may have lead to their acquisition. A total of 69 GIs and 10 gene islets were identified that comprised 38.2% of the total CDSs (1694 out of 4435 CDSs) and 37.4% of the assembled genome for ICMP19477. The size of the GIs ranged from 5,415 bps to 165,181 bps. There were 27 GIs larger than 10 Kb, eight GIs > 50 Kb and two GIs > 100 Kb. At least 17 GIs harboured known virulence genes. For example, PbN1_GI12, PbN1_GI22 and PbN1_GI23 encode cfa,

virB and T6SS clusters, respectively. These gene clusters have already been shown to be involved in virulence of P. atrosepticum. Other islands or gene islets encoded clusters with no association with virulence of SRE. One such example was the gene islet carrying an auxin efflux carrier protein along with its transcriptional regulator. These genes were present only in the P. carotovorum subsp. brasiliensis and P.

carotovorum subsp. carotovorum strains. It would be interesting to inactivate these genes and compare the epiphytic fitness of the resulting mutants to the wild type strains to determine whether these genes are responsible for the aggressive nature of these subspecies on tubers, when compared to P. atrosepticum.

176 Although many putative GIs and possible virulence factors were identified, a common set of genes that could be related to the lifestyle of a particular species of SRE or to specific disease symptoms in the host was not determined. For example, the cfa

biosynthetic cluster involved in virulence of P. atrosepticum was found in ICMP19477, but was absent from the other P. carotovorum subsp. brasiliensis strain. Both cause blackleg. Another such example was the presence of an NRPS cluster in blackleg causing Pectobacterium strains (P. atrosepticum and P. carotovorum subsp.

brasiliensis) isolated from potatoes. This cluster was also present in WPP14 but was absent from other P. carotovorum subsp. carotovorum strains isolated from potatoes such as ICM5702 as well as UGC32 and strains of P. wasabiae.

Additional genes involved in the environmental fitness and adaptation of the bacterium in planta were also identified such as the novel bacteriocins of P.

carotovorum subsp. brasiliensis. P. carotovorum subsp. brasiliensis is known to have activity against P. atrosepticum (del Pilar Marquez-Villavicencio et al., 2011). The presence of novel bacteriocin, colicin D, in both the strains of P. cartovorum subsp.

brasiliensis and its absence from the remaining SRE suggests that this bacteriocin may be involved in providing a competitive advantage to this subspecies. Further characterization of this toxin by construction of NRPS mutants, and their use in competition assays, would shed some light on the competitive nature of P.

carotovorum subsp. brasiliensis.

In summary, comparative genomics provided a selection of GIs and gene islets that harbour genes that may be involved in the virulence or ecological fitness of P.

cartovorum subsp. brasiliensis and/or other blackleg causing SRE. In the future, however, key questions that need to be addressed include: i) are the putative virulence genes identified in this study functionally active and what roles do they play in host pathogen interactions or fitness, ii) which in vitro and in planta conditions activate these genes and, iii) what are the events leading to the transfer of these genes?

177

4.6 Appendices

178

Chapter 5

Construction of knockout mutants of P. carotovorum subspecies brasiliensis

ICMP19477

5.1 Abstract

Pectobacterium carotovorum subsp. brasiliensis ICMP19477 harbours various virulence genes encoded on putative GIs and small gene islets. Five genes of interest,

sim (KCO_20372), pad (KCO_03377), nrps1 (KCO_06050), abc (KCO_06055) and cfa7

(KCO_08405) were selected for functional studies on the basis of their presence in blackleg causing Pectobacterium (P. atrosepticum and P. carotovorum subsp.

brasiliensis) and evidence that they are involved in virulence of other bacterial plant pathogens. Non-functional copies of the five genes were cloned into either the suicide vector pKNG101 or the counter-selectable plasmid pK18mobsacB. Allelic exchange mutagenesisof P.carotovorum subsp. brasiliensis ICMP19477 was then performed to create mutants in which these genes were inactivated. Allelic exchange mutagenesis initially involved selection of ‘single crossover’ events, in which plasmid marker genes were incorporated along with the non-functional copy of the target gene. ‘Double crossover’ events were then induced to produce mutants that had lost the plasmid markers and were consequently sucrose resistant. The virulence of resulting mutants was compared to that of wild type P.carotovorum subsp. brasiliensis ICMP19477 in virulence assays. The assays were carried out on stems and tubers of potato plants. No effect on virulence was observed when the NRPS and CFA clusters were disrupted. Single crossover mutants in the islands encoding sim and pad significantly reduced both lesion length on stems of potato plants and maceration of potato tubers. This data suggested that the sim and pad loci might be important for virulence of P. carotovorum subsp. brasiliensis ICMP19477.

179

5.2 Introduction

In the previous chapter, comparative genome analysis led to the identification of candidate virulence factors in the genome of ICMP19477. Many of the candidate virulence factors identified were encoded on GIs and others were present on gene islets. To understand the roles of these genes in virulence of ICMP19477, attempts were made to generate knockout mutants of ICMP19477 in five genes of interest (cfa7,

nrps1, abc, sim and pad). The cfa7, nrps1 and abc genes are encoded on GIs, PbN1_GI15, PbN1_GI24 and PbN1_GI24, respectively. The sim and pad genes are encoded on gene islets. Allelic exchange was used to create knockout mutants for ICMP19477. The virulence of these mutants was then compared to the virulence of wild type ICMP19477.

Reverse genetic analysis through allelic exchange has been extensively used to introduce recombinant or mutated alleles into genomes of both Gram-negative and Gram-positive prokaryotes to decipher the unknown function of genes (Prentki & Krisch, 1984). In general, gene inactivation by allelic exchange mutagenesis is carried out by conjugation. Conjugation is usually performed by triparental matings using a strain transformed with a mutagenic plasmid (carrying the inactivated gene construct) as a donor, a helper strain containing a plasmid with transfer (tra) genes, and the recipient strain.

Suicide vectors carrying the inactivated gene construct are generally desirable for this type of gene inactivation process. However, the suicide vector must have several criteria: it must i) be conditional for replication to allow selection for integration into the chromosome, ii) carry a selectable marker (i.e. a counter-selectable gene) for subsequent selection and, iii) be transferable to a variety of other bacteria for efficient gene inactivation (Zhou et al., 2002). The most commonly used counter-selectable markers include genes that confer sucrose (sacB), streptomycin (rpsL), or fusaric acid (tetAR) sensitivity (Dean, 1981; Gay et al., 1985; Maloy & Nunn, 1981).

180 The first step in allelic exchange mutagenesis involves the integration of a plasmid carrying an inactivated gene construct within the reciprocal target sequence by homologous recombination, producing a chromosomal duplication. Following integration, the integrated plasmid can be excised via a second crossover event, resulting in allelic exchange (Appendix A5.1). Allelic exchange ultimately leaves one copy of the gene on the chromosome, either the wild type copy or the mutant copy. If the plasmid is still integrated in the chromosome, allelic exchange is initiated with the use of the counter-selectable markers as the cell will die in the presence of the counter-selective compound (Reyrat et al., 1998).

5.3 Methods

5.3.1 Bacterial strains

Pectobacterium and E. coli strains were cultured in LB medium at 28°C for 24 h and at 37°C for 16 h, respectively. When appropriate, cultures were grown with antibiotics at the following concentrations: 50 µg/mL kanamycin (Kn), 170 µg/mL chloramphenicol (Chl), 100 µg/mL ampicillin (Amp) and 50 µg/mL streptomycin (Str). Bacterial cells were harvested by centrifugation at 7,500 rpm for 10 min, the supernatant discarded and the pellet used for subsequent DNA preparation. Alternatively, for long term storage, equal volumes of an overnight culture were mixed with 40% glycerol and stored at -80°C. The bacterial strains and plasmid vectors used in this study are listed in Table 5.1.

181 Table 5.1 Bacterial strains and plasmid vectors used in this study

Bacterial strain/plasmid Description/Genotype Source/Reference Antibiotic resistance