May Bente Brurberg

Functional analysis of promoters involved in quorum sensing‐based regulation of bacteriocin production in Lactobacillus

Bacteriocin production in Lactobacillus sake LTH673 involves at least four operons: a regulatory operon (sppIPKR); two operons encoding bacteriocins and their immunity proteins (sppAiA and orfX); and an operon needed for secretion (sppTE). We show here that the response regulator encoded by sppR in L. sake LTH673, as well as the homologous response regulators encoded by plnC and plnD in Lactobacillus plantarum C11, bind to characteristic repeats found in the −80 to −40 regions of spp operons. The promoters controlling bacteriocin operons are strictly regulated, and their activity is increased more than 1000‐fold upon activation. Constitutive expression for the regulatory and transport operons is driven, at least in part, by promoters upstream of the −80 to −40 regions. Peak promoter activity of the regulatory and transporter operons precedes that of the two bacteriocin operons. The results reveal how promoters involved in quorum sensing‐based regulation of bacteriocin production in Lactobacillus differ in strength, leakiness and timing of their activity.

Genetic analysis of Phytophthora infestans populations in the Nordic European countries reveals high genetic variability

Late blight, caused by the oomycete Phytophthora infestans, is the most important disease of potato (Solanum tuberosum). The pathogen is highly adaptable and to get an overview of the genetic variation in the Nordic countries, Denmark, Finland, Norway and Sweden we have analyzed 200 isolates from different fields using nine simple-sequence repeat (SSR) markers. Forty-nine alleles were detected among the nine SSR loci and isolates from all four Nordic countries shared the most common alleles across the loci. In total 169 multilocus genotypes (based on seven loci) were identified among 191 isolates. The genotypic diversities, quantified by a normalized Shannons diversity index (H(s)), were 0.95 for the four Nordic countries. The low F(ST) value of 0.04 indicates that the majority of variation is found within the four Nordic countries. The large number of genotypes and the frequency distribution of mating types (60% A1) support the hypothesis that sexual reproduction is contributing notably to the genetic variation of P. infestans in the Nordic countries.

Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1

BackgroundLactobacillus plantarum is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. The bacterium has great potential as food-grade cell factory and for in situ delivery of biomolecules. Since protein secretion is important both for probiotic activity and in biotechnological applications, we have carried out a genome-wide experimental study of signal peptide (SP) functionality.ResultsWe have constructed a library of 76 Sec-type signal peptides from L. plantarum WCFS1 that were predicted to be cleaved by signal peptidase I. SP functionality was studied using staphylococcal nuclease (NucA) as a reporter protein. 82% of the SPs gave significant extracellular NucA activity. Levels of secreted NucA varied by a dramatic 1800-fold and this variation was shown not to be the result of different mRNA levels. For the best-performing SPs all produced NucA was detected in the culture supernatant, but the secretion efficiency decreased for the less well performing SPs. Sequence analyses of the SPs and their cognate proteins revealed four properties that correlated positively with SP performance for NucA: high hydrophobicity, the presence of a transmembrane helix predicted by TMHMM, the absence of an anchoring motif in the cognate protein, and the length of the H+C domain. Analysis of a subset of SPs with a lactobacillal amylase (AmyA) showed large variation in production levels and secretion efficiencies. Importantly, there was no correlation between SP performance with NucA and the performance with AmyA.ConclusionThis is the first comprehensive experimental study showing that predicted SPs in the L. plantarum genome actually are capable of driving protein secretion. The results reveal considerable variation between the SPs that is at least in part dependent on the protein that is secreted. Several SPs stand out as promising candidates for efficient secretion of heterologous proteins in L. plantarum. The results for NucA provide some hints as to the sequence-based prediction of SP functionality, but the general conclusion is that such prediction is difficult. The vector library generated in this study is based on exchangeable cassettes and provides a powerful tool for rapid experimental screening of SPs.

Purification, Characterization, and Gene Sequence of Michiganin A, an Actagardine-Like Lantibiotic Produced by the Tomato Pathogen Clavibacter michiganensis subsp. michiganensis

ABSTRACT Members of the actinomycete genus Clavibacter are known to produce antimicrobial compounds, but so far none of these compounds has been purified and characterized. We have isolated an antimicrobial peptide, michiganin A, from the tomato pathogen Clavibacter michiganensis subsp. michiganensis, using ammonium sulfate precipitation followed by cation-exchange and reversed-phase chromatography steps. Upon chemical derivatization of putative dehydrated amino acids and lanthionine bridges by alkaline ethanethiol, Edman degradation yielded sequence information that proved to be sufficient for cloning of the gene by a genome-walking strategy. The mature unmodified peptide consists of 21 amino acids, SSSGWLCTLTIECGTIICACR. All of the threonine residues undergo dehydration, and three of them interact with cysteines via thioether bonds to form methyllanthionine bridges. Michiganin A resembles actagardine, a type B lantibiotic with a known three-dimensional structure, produced by Actinoplanes liguriae, which is a filamentous actinomycete. The DNA sequence of the gene showed that the michiganin A precursor contains an unusual putative signal peptide with no similarity to well-known secretion signals and only very limited similarity to the (only two) available leader peptides of other type B lantibiotics. Michiganin A inhibits the growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of ring rot of potatoes, with MICs in the low nanomolar range. Thus, michiganin A may have some potential in biological control of potato ring rot.

Chalara fraxinea Isolated from Diseased Ash in Norway

European ash (Fraxinus excelsior), also known as common ash, occurs naturally inland in lower areas of southeastern Norway and along the southern coast of the country. It is important both as a forest and ornamental tree. During the last decade, dieback has become a disastrous disease on F. excelsior in many European countries. The anamorphic fungus Chalara fraxinea T. Kowalski (1), described for the first time from dying ash trees in Poland, is now considered the cause of ash dieback (2). In May of 2008, C. fraxinea was isolated from 1.5 m high diseased F. excelsior in a nursery in Østfold County in southeastern Norway. Symptoms included wilting, necrotic lesions around leaf scars and side branches, and discoloration of the wood. From symptomatic branches, small pieces (approximately 1 cm3) were excised in the transition area between healthy and discolored wood. After surface sterilization (10 s in 70% ethanol + 90 s in NaOCl), the pieces were air dried for 1 min in a safety cabinet, cut into smaller pieces, and placed on media. The fungus was isolated on potato dextrose agar (PDA) and water agar (WA). On PDA, the cultures were tomentose, light orange, and grew slowly (21 mm mean colony diameter after 2 weeks at room temperature). Typical morphological features of C. fraxinea developed in culture. Brownish phialides (14.8 to 30.0 [19.5] × 2.5 to 5.0 [4.1] μm, n = 50) first appeared in the center of the colonies on the agar plugs that had been transferred. The agar plugs were 21 days old when phialides were observed. Abundant sporulation occurred 3 days later. Conidia (phialospores) extruded apically from the phialides and formed droplets. Conidia measured 2.1 to 4.0 (3.0) × 1.4 to 1.9 (1.7) μm (n = 50). The first-formed conidia from each phialide were different in size and shape from the rest by being longer (6 μm, n = 10) and more narrow in the end that first appeared at the opening of the phialide. Internal transcribed spacer sequencing confirmed that the morphological identification was correct (Accession No. EU848544 in GenBank). A pathogenicity test was carried out in June of 2008 by carefully removing one leaf per plant on 10 to 25 cm high F. excelsior trees (18 trees) and placing agar plugs from a 31-day-old C. fraxinea culture (isolate number 10636) on the leaf scars and covering with Parafilm. After 46 days, isolations were carried out as described above from discolored wood that had developed underneath necrotic lesions in the bark and subsequently caused wilting of leaves. All the inoculated plants showed symptoms, and C. fraxinea was successfully reisolated. No symptoms were seen on uninoculated control plants (eight trees) that had received the same treatment except that sterile PDA agar plugs had been used. References: (1) T. Kowalski. For. Pathol. 36:264, 2006. (2) T. Kowalski and O. Holdenrieder, For. Pathol. Online publication, doi: 10.1111/j.1439-0329.2008.00565.x, 2008.

A ddRAD based linkage map of the cultivated Strawberry, Fragaria xananassa

The cultivated strawberry (Fragaria ×ananassa Duch.) is an allo-octoploid considered difficult to disentangle genetically due to its four relatively similar sub-genomic chromosome sets. This has been alleviated by the recent release of the strawberry IStraw90 whole genome genotyping array. However, array resolution relies on the genotypes used in the array construction and may be of limited general use. SNP detection based on reduced genomic sequencing approaches has the potential of providing better coverage in cases where the studied genotypes are only distantly related from the SNP array’s construction foundation. Here we have used double digest restriction-associated DNA sequencing (ddRAD) to identify SNPs in a 145 seedling F1 hybrid population raised from the cross between the cultivars Sonata (♀) and Babette (♂). A linkage map containing 907 markers which spanned 1,581.5 cM across 31 linkage groups representing the 28 chromosomes of the species. Comparing the physical span of the SNP markers with the F. vesca genome sequence, the linkage groups resolved covered 79% of the estimated 830 Mb of the F. ×ananassa genome. Here, we have developed the first linkage map for F. ×ananassa using ddRAD and show that this technique and other related techniques are useful tools for linkage map development and downstream genetic studies in the octoploid strawberry.

Expression of putative virulence factors in the potato pathogen Clavibacter michiganensis subsp. sepedonicus during infection

The Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus is the causal agent of bacterial wilt and ring rot of potato. So far, only two proteins have been shown to be essential for virulence, namely a plasmid-encoded cellulase CelA and a hypersensitive response-inducing protein. We have examined the relative expression of CelA and eight putative virulence factors during infection of potato and in liquid culture, using quantitative real-time PCR. The examined putative virulence genes were celB, a cellulase-encoding gene and genes encoding a pectate lyase, a xylanase and five homologues of the Clavibacter michiganensis subsp. michiganensis pathogenicity factor Pat-1 thought to encode a serine protease. Six of the nine assayed genes were up-regulated during infection of potato, including celA, celB, the xylanase gene, and two of the pat genes. The pectate lyase gene showed only slightly elevated expression, whereas three of the five examined pat genes were down-regulated during infection in potato. Interestingly, the two up-regulated pat genes showed a noticeable sequence difference compared to the three down-regulated pat genes. These results reveal several new proteins that are likely to be involved in Clavibacter michiganensis subsp. sepedonicus pathogenicity.

Characterization of Dickeya and Pectobacterium strains obtained from diseased potato plants in different climatic conditions of Norway and Poland

Soft rot and blackleg of potato caused by pectinolytic bacteria lead to severe economic losses in potato production worldwide. To investigate the species composition of bacteria causing soft rot and black leg of potato in Norway and Poland, bacteria were isolated from potato tubers and stems. Forty-one Norwegian strains and 42 Polish strains that formed cavities on pectate medium were selected for potato tuber maceration assays and sequencing of three housekeeping genes (dnaX, icdA and mdh) for species identification and phylogenetic analysis. The distribution of the species causing soft rot and blackleg in Norway and Poland differed: we have demonstrated that mainly P. atrosepticum and P. c. subsp. carotovorum are the causal agents of soft rot and blackleg of potatoes in Norway, while P. wasabiae was identified as one of the most important soft rot pathogens in Poland. In contrast to the other European countries, D. solani seem not to be a major pathogen of potato in Norway and Poland. The Norwegian and Polish P. c. subsp. carotovorum and P. wasabiae strains did not cluster with type strains of the respective species in the phylogenetic analysis, which underlines the taxonomic complexity of the genus Pectobacterium. No correlation between the country of origin and clustering of the strains was observed. All strains tested in this study were able to macerate potato tissue. The ability to macerate potato tissue was significantly greater for the P. c. subsp. carotovorum and Dickeya spp., compared to P. atrosepticum and P. wasabiae.

Expression of resistance gene analogs in woodland strawberry (Fragaria vesca) during infection with Phytophthora cactorum

Important losses in strawberry production are often caused by the oomycete Phytophthora cactorum, the causal agent of crown rot. However, very limited studies at molecular levels exist of the mechanisms related to strawberry resistance against this pathogen. To begin to rectify this situation, a PCR-based approach (NBS profiling) was used to isolate strawberry resistance gene analogs (RGAs) with altered expression in response to P. cactorum during a time course (2, 4, 6, 24, 48, 96 and 192 h post-infection). Twenty-three distinct RGA fragments of the NB-LRR type were identified from a resistance genotype (Bukammen) of the wild species Fragaria vesca. The gene transcriptional profiles after infection showed that the response of most RGAs was quicker and stronger in the resistance genotype (Bukammen) than in the susceptible one (FDP821) during the early infection stage. The transcriptional patterns of one RGA (RGA109) were further monitored and compared during the P. cactorum infection of two pairs of resistant and susceptible genotype combinations (Bukammen/FDP821 and FDR1218/1603). The 5′ end sequence was cloned, and its putative protein was characteristic of NBS-LRR R protein. Our results yielded a first insight into the strawberry RGAs responding to P. cactorum infection at molecular level.

First report of Phytophthora ramorum causing shoot dieback on bilberry (Vaccinium myrtillus) in Norway.

In the annual Norwegian Phytophthora ramorum survey in 2009, wild bilberry samples, collected during September and October in a semimanaged park (arboretum) in the southwest coast of Norway, tested positive in a P. ramorum-specific real-time PCR test (1). Necrotic lesions were observed in shoot tips, branching points, and around leaf abscission scars. The lesions were of variable dimensions. In the samples collected in October, some lesions were confluent and completely covered some shoots. After direct detection on plant material, P. ramorum was isolated from necrotic lesions of the stems on semiselective media PARP (corn meal agar amended with pimaricin, ampicillin, rifampicin, and pentacloronitrobenzene) (2). The isolates were identified by the production of abundant chlamydospores on agar and deciduous, semipapillate sporangia that is characteristic of P. ramorum (3). Sexual structures were not observed. Three pure cultures obtained from different plant samples also tested positive for P. ramorum by the specific real-time PCR test (1). All positive samples were found in close vicinity of infected rhododendron plants. In this location, P. ramorum had already been detected on rhododendron in 2005. A pathogenicity test was performed with two isolates from bilberry and one from rhododendron. Wild asymptomatic bilberry plants were collected at the end of June in the forest around Oslo. Two shoot tips with 6 to 10 leaves each and one small part of a branch with several shoots and immature berries were used for testing each isolate. The inoculations were made by dipping the shoots in a zoospore suspension (2 to 3 × 104 zoospores ml-1) for 1 min. Inoculated material was placed in moist incubation chambers and incubated at room temperature (19 to 24°C). Controls were obtained by dipping shoots in sterile water. After 2 days, lesions were observed on leaf laminae from all the shoots inoculated with the three different isolates. After 4 days, severe petiole necroses were observed and leaves abscised easily from the stems. Symptoms on the stems were observed in the apical part or areas around the nodes. Some shoots were almost completely necrotic. Heavy sporulation was observed on the berries. P. ramorum was reisolated from leaves and stems of inoculated shoots for all the isolates. P. ramorum was not recovered from control plants. To our knowledge, this is the first report of P. ramorum on bilberry in Norway. References: (1) K. L. Hughes et al. Phytopathology 96:975, 2006. (2) M. E. Kannwischer and D. J. Mitchell. Phytopathology 68:1760, 1978. (3) S. Werres et al. Mycol. Res.105:1155, 2001.