Plastic and terrestrial organic matter degradation by the humic lake microbiome continues throughout the seasons.

Boreal freshwaters go through four seasons, however, studies about the decomposition of terrestrial and plastic compounds often focus only on summer. We compared microbial decomposition of 13C-polyethylene, 13C-polystyrene, and 13C-plant litter (Typha latifolia) by determining the biochemical fate of the substrate carbon and identified the microbial decomposer taxa in humic lake waters in four seasons. For the first time, the annual decomposition rate including separated seasonal variation was calculated for microplastics and plant litter in the freshwater system. Polyethylene decomposition was not detected, whereas polystyrene and plant litter were degraded in all seasons. In winter, decomposition rates of polystyrene and plant litter were fivefold and fourfold slower than in summer, respectively. Carbon from each substrate was mainly respired in all seasons. Plant litter was utilized efficiently by various microbial groups, whereas polystyrene decomposition was limited to Alpha- and Gammaproteobacteria. The decomposition was not restricted only to the growth season, highlighting that the decomposition of both labile organic matter and extremely recalcitrant microplastics continues throughout the seasons.

NIRis: A low-cost, versatile imaging system for near-infrared fluorescence detection of phototrophic cell colonies used in research and education.

A variety of costly research-grade imaging devices are available for the detection of spectroscopic features. Here we present an affordable, open-source and versatile device, suitable for a range of applications. We provide the files to print the imaging chamber with commonly available 3D printers and instructions to assemble it with easily available hardware. The imager is suitable for rapid sample screening in research, as well as for educational purposes. We provide details and results for an already proven set-up which suits the needs of a research group and students interested in UV-induced near-infrared fluorescence detection of microbial colonies grown on Petri dishes. The fluorescence signal confirms the presence of bacteriochlorophyll a in aerobic anoxygenic phototrophic bacteria (AAPB). The imager allows for the rapid detection and subsequent isolation of AAPB colonies on Petri dishes with diverse environmental samples. To this date, 15 devices have been build and more than 7000 Petri dishes have been analyzed for AAPB, leading to over 1000 new AAPB isolates. Parts can be modified depending on needs and budget. The latest version with automated switches and double band pass filters costs around 350€ in materials and resolves bacterial colonies with diameters of 0.5 mm and larger. The low cost and modular build allow for the integration in high school classes to educate students on light properties, fluorescence and microbiology. Computer-aided design of 3D-printed parts and programming of the employed Raspberry Pi computer could be incorporated in computer sciences classes. Students have been also inspired to do agar art with microbes. The device is currently used in seven different high schools in Finland. Additionally, a science education network of Finnish universities has incorporated it in its program for high school students. Video guides have been produced to facilitate easy operation and accessibility of the device.

Multifaceted photoreceptor compositions in dual phototrophic systems - A genomic analysis.

For microbes and their hosts, sensing of external cues is essential for their survival. For example, in the case of plant associated microbes, the light absorbing pigment composition of the plant as well as the ambient light conditions determine the well-being of the microbe. In addition to light sensing, some microbes can utilize xanthorhodopsin based proton pumps and bacterial photosynthetic complexes that work in parallel for energy production. They are called dual phototrophic systems. Light sensing requirements in these type of systems are obviously demanding. In nature, the photosensing machinery follows mainly the same composition in all organisms. However, the specific role of each photosensor in specific light conditions is elusive. In this study, we provide an overall picture of photosensors present in dual phototrophic systems. We compare the genomes of the photosensor proteins from dual phototrophs to those from similar microbes with "single" phototrophicity or microbes without phototrophicity. We find that the dual phototrophic bacteria obtain a larger variety of photosensors than their light inactive counterparts. Their rich domain composition and functional repertoire remains similar across all microbial photosensors. Our study calls further investigations of this particular group of bacteria. This includes protein specific biophysical characterization in vitro, microbiological studies, as well as clarification of the ecological meaning of their host microbial interactions.

Glyphosate-based herbicide use affects individual microbial taxa in strawberry endosphere but not the microbial community composition.

AIMS: In a field study, the effects of treatments of glyphosate-based herbicides (GBHs) in soil, alone and in combination with phosphate fertilizer, were examined on the performance and endophytic microbiota of garden strawberry. METHODS AND RESULTS: The root and leaf endophytic microbiota of garden strawberries grown in GBH-treated and untreated soil, with and without phosphate fertilizer, were analyzed. Next, bioinformatics analysis on the type of 5-enolpyruvylshikimate-3-phosphate synthase enzyme was conducted to assess the potential sensitivity of strawberry-associated bacteria and fungi to glyphosate, and to compare the results with field observations. GBH treatments altered the abundance and/or frequency of several operational taxonomic units (OTUs), especially those of root-associated fungi and bacteria. These changes were partly related to their sensitivity to glyphosate. Still, GBH treatments did not shape the overall community structure of strawberry microbiota or affect plant performance. Phosphate fertilizer increased the abundance of both glyphosate-resistant and glyphosate-sensitive bacterial OTUs, regardless of the GBH treatments. CONCLUSIONS: These findings demonstrate that although the overall community structure of strawberry endophytic microbes is not affected by GBH use, some individual taxa are.

Ecosystem consequences of herbicides: the role of microbiome.

Non-target organisms are globally exposed to herbicides. While many herbicides - for example, glyphosate - were initially considered safe, increasing evidence demonstrates that they have profound effects on ecosystem functions via altered microbial communities. We provide a comprehensive framework on how herbicide residues may modulate ecosystem-level outcomes via alteration of microbiomes. The changes in soil microbiome are likely to influence key nutrient cycling and plant-soil processes. Herbicide-altered microbiome affects plant and animal performance and can influence trophic interactions such as herbivory and pollination. These changes are expected to lead to ecosystem and even evolutionary consequences for both microbes and hosts. Tackling the threats caused by agrochemicals to ecosystem functions and services requires tools and solutions based on a comprehensive understanding of microbe-mediated risks.

Decomposition rate and biochemical fate of carbon from natural polymers and microplastics in boreal lakes.

Microbial mineralization of organic compounds is essential for carbon recycling in food webs. Microbes can decompose terrestrial recalcitrant and semi-recalcitrant polymers such as lignin and cellulose, which are precursors for humus formation. In addition to naturally occurring recalcitrant substrates, microplastics have been found in various aquatic environments. However, microbial utilization of lignin, hemicellulose, and microplastics as carbon sources in freshwaters and their biochemical fate and mineralization rate in freshwaters is poorly understood. To fill this knowledge gap, we investigated the biochemical fate and mineralization rates of several natural and synthetic polymer-derived carbon in clear and humic lake waters. We used stable isotope analysis to unravel the decomposition processes of different 13C-labeled substrates [polyethylene, polypropylene, polystyrene, lignin/hemicellulose, and leaves (Fagus sylvatica)]. We also used compound-specific isotope analysis and molecular biology to identify microbes associated with used substrates. Leaves and hemicellulose were rapidly decomposed compared to microplastics which were degraded slowly or below detection level. Furthermore, aromatic polystyrene was decomposed faster than aliphatic polyethylene and polypropylene. The major biochemical fate of decomposed substrate carbon was in microbial biomass. Bacteria were the main decomposers of all studied substrates, whereas fungal contribution was poor. Bacteria from the family Burkholderiaceae were identified as potential leaf and polystyrene decomposers, whereas polypropylene and polyethylene were not decomposed.

Biodegradation of microplastic in freshwaters: A long-lasting process affected by the lake microbiome.

Plastics have been produced for over a century, but definitive evidence of complete plastic biodegradation in different habitats, particularly freshwater ecosystems, is still missing. Using 13 C-labelled polyethylene microplastics (PE-MP) and stable isotope analysis of produced gas and microbial membrane lipids, we determined the biodegradation rate and fate of carbon in PE-MP in different freshwater types. The biodegradation rate in the humic-lake waters was much higher (0.45% ± 0.21% per year) than in the clear-lake waters (0.07% ± 0.06% per year) or the artificial freshwater medium (0.02% ± 0.02% per year). Complete biodegradation of PE-MP was calculated to last 100-200 years in humic-lake waters, 300-4000 years in clear-lake waters, and 2000-20,000 years in the artificial freshwater medium. The concentration of 18:1ω7, characteristic phospholipid fatty acid in Alpha- and Gammaproteobacteria, was a predictor of faster biodegradation of PE. Uncultured Acetobacteraceae and Comamonadaceae among Alpha- and Gammaproteobacteria, respectively, were major bacteria related to the biodegradation of PE-MP. Overall, it appears that microorganisms in humic lakes with naturally occurring refractory polymers are more adept at decomposing PE than those in other waters.

Tissue-Specific Dynamics in the Endophytic Bacterial Communities in Arctic Pioneer Plant Oxyria digyna.

The rapid developments in the next-generation sequencing methods in the recent years have provided a wealth of information on the community structures and functions of endophytic bacteria. However, the assembly processes of these communities in different plant tissues are still currently poorly understood, especially in wild plants in natural settings. The aim of this study was to compare the composition of endophytic bacterial communities in leaves and roots of arcto-alpine pioneer plant Oxyria digyna, and investigate, how plant tissue (leaf or root) or plant origin affect the community assembly. To address this, we planted micropropagated O. digyna plants with low bacterial load (bait plants) in experimental site with native O. digyna population, in the Low Arctic. The endophytic bacterial community structures in the leaves and roots of the bait plants were analyzed after one growing season and one year in the field, and compared to those of the wild plants growing at the same site. 16S rRNA gene targeted sequencing revealed that endophytic communities in the roots were more diverse than in the leaves, and the diversity in the bait plants increased in the field, and was highest in the wild plants. Both tissue type and plant group had strong impact on the endophytic bacterial community structures. Firmicutes were highly abundant in the leaf communities of both plant types. Proteobacteria and Bacteroidetes were more abundant in the roots, albeit with different relative abundances in different plant groups. The community structures in the bait plants changed in the field over time, and increasingly resembled the wild plant endophytic communities. This was due to the changes in the relative abundances of several bacterial taxa, as well as species acquisition in the field, but with no species turnover. Several OTUs that were acquired by the bait plants in the field and represent phosphate solubilizing and diazotrophic bacterial taxa, suggesting major role in nutrient acquisition of these bacteria for this nonmycorrhizal plant, thriving in the nutrient poor arctic soils.

Heritable Epichloë symbiosis shapes fungal but not bacterial communities of plant leaves.

Keystone microbial species have driven eco-evolutionary processes since the origin of life. However, due to our inability to detect the majority of microbiota, members of diverse microbial communities of fungi, bacteria and viruses have largely been ignored as keystone species in past literature. Here we tested whether heritable Epichloë species of pooidae grasses modulate microbiota of their shared host plant.

Strong Regionality and Dominance of Anaerobic Bacterial Taxa Characterize Diazotrophic Bacterial Communities of the Arcto-Alpine Plant Species Oxyria digyna and Saxifraga oppositifolia.

Arctic and alpine biomes are most often strongly nitrogen-limited, and hence biological nitrogen fixation is a strong driver of these ecosystems. Both biomes are characterized by low temperatures and short growing seasons, but they differ in seasonality of solar radiation and in soil water balance due to underlying permafrost in the Arctic. Arcto-alpine plant species are well-adapted to the low temperatures that prevail in their habitats, and plant growth is mainly limited by the availability of nutrients, in particular nitrogen, due to slow mineralization. Nitrogen fixing bacteria are likely important for plant growth in these habitats, but very little is known of these bacteria or forces shaping their communities. In this study, we characterized the potential nitrogen fixing bacterial (PNFB) communities associated with two arcto-alpine pioneer plant species, Oxyria digyna (mountain sorrel) and Saxifraga oppositifolia (blue saxifrage), in three climate regions. Both of these plants readily colonize low nutrient mineral soils. Our goal was to investigate how climate (region) and, on the other hand, host plant and plant species shape these communities. To our knowledge, this is the first comprehensive study describing PNFB communities associated with pioneer plants in different arcto-alpine biomes. Replicate samples were taken from two arctic regions, Kilpisjärvi and Ny-Ålesund, and one alpine region, Mayrhofen. In these, the PNFB communities in the bulk and rhizosphere soils and the plant endospheres were characterized by nifH-targeted PCR and massive parallel sequencing. The data revealed strong effects of climatic region on the dominating nitrogen fixers. Specifically, nifH sequences related to Geobacter (δ-Proteobacteria) were present in high relative abundances in the nitrogen-fixing communities in the Mayrhofen and Kilpisjärvi regions, while members of the Clostridiales prevailed in the Kilpisjärvi and Ny-Ålesund regions. The bulk and rhizosphere soil as well as the endosphere communities in the Mayrhofen region were all characterized by high relative abundances of nifH sequences related to Geobacter. In contrast, the endosphere and soil (bulk or rhizosphere soil) communities in the High Arctic were highly divergent: endosphere communities in the arctic regions were shaped by Clostridium spp., while nifH sequences representing δ-Proteobacteria, ß-Proteobacteria, Cyanobacteria (in Ny-Ålesund), and Verrucomicrobia (in Kilpisjärvi) dominated the soil communities. Interestingly, the major PNFB genera identified in this study have been previously identified as members of conserved core microbiomes in the endospheres and seeds of these plants by 16S rRNA gene based analyses combined with bacterial isolation, suggesting a very tight interaction between diazotrophic bacteria and these arctic pioneer plants. Overall, anaerobic bacterial taxa dominated the PNFB communities of the endospheres and rhizospheres of the two plant species in all study sites. This could indicate anoxic conditions in and around plant roots at the time of sampling (early growth season), created by melting snow and underlying permafrost.

Native arbuscular mycorrhizal symbiosis alters foliar bacterial community composition.

The effects of arbuscular mycorrhizal (AM) fungi on plant-associated microbes are poorly known. We tested the hypothesis that colonization by an AM fungus affects microbial species richness and microbial community composition of host plant tissues. We grew the grass, Deschampsia flexuosa in a greenhouse with or without the native AM fungus, Claroideoglomus etunicatum. We divided clonally produced tillers into two parts: one inoculated with AM fungus spores and one without AM fungus inoculation (non-mycorrhizal, NM). We characterized bacterial (16S rRNA gene) and fungal communities (internal transcribed spacer region) in surface-sterilized leaf and root plant compartments. AM fungus inoculation did not affect microbial species richness or diversity indices in leaves or roots, but the AM fungus inoculation significantly affected bacterial community composition in leaves. A total of three OTUs in leaves belonging to the phylum Firmicutes positively responded to the presence of the AM fungus in roots. Another six OTUs belonging to the Proteobacteria (Alpha, Beta, and Gamma) and Bacteroidetes were significantly more abundant in NM plants when compared to AM fungus-inoculated plants. Further, there was a significant correlation between plant dry weight and leaf microbial community compositional shift. Also, there was a significant correlation between leaf bacterial community compositional shift and foliar nitrogen content changes due to AM fungus inoculation. The results suggest that AM fungus colonization in roots has a profound effect on plant physiology that is reflected in leaf bacterial community composition.

Plants Assemble Species Specific Bacterial Communities from Common Core Taxa in Three Arcto-Alpine Climate Zones.

Evidence for the pivotal role of plant-associated bacteria to plant health and productivity has accumulated rapidly in the last years. However, key questions related to what drives plant bacteriomes remain unanswered, among which is the impact of climate zones on plant-associated microbiota. This is particularly true for wild plants in arcto-alpine biomes. Here, we hypothesized that the bacterial communities associated with pioneer plants in these regions have major roles in plant health support, and this is reflected in the formation of climate and host plant specific endophytic communities. We thus compared the bacteriomes associated with the native perennial plants Oxyria digyna and Saxifraga oppositifolia in three arcto-alpine regions (alpine, low Arctic and high Arctic) with those in the corresponding bulk soils. As expected, the bulk soil bacterial communities in the three regions were significantly different. The relative abundances of Proteobacteria decreased progressively from the alpine to the high-arctic soils, whereas those of Actinobacteria increased. The candidate division AD3 and Acidobacteria abounded in the low Arctic soils. Furthermore, plant species and geographic region were the major determinants of the structures of the endophere communities. The plants in the alpine region had higher relative abundances of Proteobacteria, while plants from the low- and high-arctic regions were dominated by Firmicutes. A highly-conserved shared set of ubiquitous bacterial taxa (core bacteriome) was found to occur in the two plant species. Burkholderiales, Actinomycetales and Rhizobiales were the main taxa in this core, and they were also the main contributors to the differences in the endosphere bacterial community structures across compartments as well as regions. We postulate that the composition of this core is driven by selection by the two plants.

Microbial community composition but not diversity changes along succession in arctic sand dunes.

The generality of increasing diversity of fungi and bacteria across arctic sand dune succession was tested. Microbial communities were examined by high-throughput sequencing of 16S rRNA genes (bacteria) and internal transcribed spacer (ITS) regions (fungi). We studied four microbial compartments (inside leaf, inside root, rhizosphere and bulk soil) and characterized microbes associated with a single plant species (Deschampsia flexuosa) across two sand dune successional stages (early and late). Bacterial richness increased across succession in bulk soil and leaf endosphere. In contrast, soil fungal richness remained constant while root endosphere fungal richness increased across succession. There was, however, no significant difference in Shannon diversity indices between early and late successional stage in any compartment. There was a significant difference in the composition of microbial communities between early and late successional stage in all compartments, although the major microbial OTUs were shared between early and late successional stage. Co-occurrence network analysis revealed successional stage-specific microbial groups. There were more co-occurring modules in early successional stage than in late stage. Altogether, these results emphasize that succession strongly affects distribution of microbial species, but not microbial diversity in arctic sand dune ecosystem and that fungi and bacteria may not follow the same successional trajectories.

Culturable endophytic microbial communities in the circumpolar grass, Deschampsia flexuosa in a sub-Arctic inland primary succession are habitat and growth stage specific.

Little is known about endophytic microbes in cold climate plants and how their communities are formed.We compared culturable putative endophytic bacteria and fungi in the ecologically important circumpolargrass, Deschampsia flexuosa growing in two successional stages of subarctic sand dune (68°29'N).Sequence analyses of partial 16S rRNA and internal transcribed spacer (ITS) sequences of culturable endophytes showed that diverse bacteria and fungi inhabit different tissues of D. flexuosa. A total of 178 bacterial isolates representing seven taxonomic divisions, Alpha, Beta and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Acidobacteria, and 30 fungal isolates representing the phylum Ascomycota were identified. Several endophytes were affiliated with specific plant tissues or successional stages. This first report of bacterial endophytes in D. flexuosa revealed that the genus Pseudomonas is tightly associated with D. flexuosa, and encompassed 39% of the bacterial isolates, and 58% of seed isolates. Based on 16S rRNA and ITS sequence data, most of the D. flexuosa endophytes were closely related to microbes from other cold environments. The majority of seed endophytic bacterial isolates were able to solubilize organic form of phosphate suggesting that these endophytes could play a role in resource mobilization in germinating seeds in nutrient-poor habitat.

Endophytic bacterial communities in three arctic plants from low arctic fell tundra are cold-adapted and host-plant specific.

Endophytic bacteria inhabit internal plant tissues, and have been isolated from a large diversity of plants, where they form nonpathogenic relationships with their hosts. This study combines molecular and culture-dependent approaches to characterize endophytic bacterial communities of three arcto-alpine plant species (Oxyria digyna, Diapensia lapponica and Juncus trifidus) sampled in the low Arctic (69°03'N). Analyses of a 325 bacterial endophyte isolates, as well as seven clone libraries, revealed a high diversity. In particular, members of the Actinobacteria, Bacteroidetes, Firmicutes, Acidobacteria, and Proteobacteria were found. The compositions of the endophytic bacterial communities were dependent on host-plant species as well as on snow cover at sampling sites. Several bacterial genera were found to be associated tightly with specific host-plant species. In particular, Sphingomonas spp. were characteristic for D. lapponica and O. digyna, and their phylogenetic grouping corresponded to the host plant. Most of the endophyte isolates grew well and retained activity at +4 °C, and isolate as well as clone library sequences were often highly similar to sequences from bacteria from cold environments. Taken together, this study shows that arctic plants harbour a diverse community of bacterial endophytes, a portion of which seems to be tightly associated with specific plant species.

Hcp2, a secreted protein of the phytopathogen Pseudomonas syringae pv. tomato DC3000, is required for fitness for competition against bacteria and yeasts.

When analyzing the secretome of the plant pathogen Pseudomonas syringae pv. tomato DC3000, we identified hemolysin-coregulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the P. syringae pv. tomato DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of the hcp genes and tested the fitness of hcp knockout mutants in host plant colonization and in intermicrobial competition. We found that the hcp2 gene is expressed most actively at the stationary growth phase and that the Hcp2 protein is secreted via the T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and does not contribute to virulence in or colonization of tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with the suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition with yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive under conditions where it has to compete with other microorganisms for resources.

The putative secreted serine protease Chp-7 is required for full virulence and induction of a nonhost hypersensitive response by Clavibacter michiganensis subsp. sepedonicus.

Molecular biological studies on Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato, have gained greater feasibility due to the recent availability of whole genomic sequences and genetic tools for related taxa. Here, we describe the first report of construction and characterization of a transposon (Tn) mutant library of C. michiganensis subsp. sepedonicus sp. strain R10. Since virulence of R10 in potato has been shown previously to be associated with elicitation of a nonhost hypersensitive response (HR), the mutant library was screened initially for loss of HR in tobacco. The screen identified two HR-negative mutants containing Tn insertions within the same gene, CMS2989 (chp-7), although at distinct locations. chp-7 is one of 11 pat-1 homologs in C. michiganensis subsp. sepedonicus. HR-negative mutants of R10 multiplied to the same extent as wild type in planta but were less virulent in potato. Complementation with chp-7 restored virulence as well as the HR phenotype. Together, these findings demonstrate a role for chp-7 in C. michiganensis subsp. sepedonicus-plant interactions.

Microarray profiling of host-extract-induced genes and characterization of the type VI secretion cluster in the potato pathogen Pectobacterium atrosepticum.

Pectobacterium atrosepticum is a Gram-negative plant-pathogenic bacterium that rots potato stems and tubers. Microarray analysis was used to identify genes that were differentially expressed when host extracts were added to the growth medium. Potato extracts downregulated the expression of ribosomal genes and genes related to uptake and metabolism of nutrients, and upregulated genes needed for nitrate or phosphonate use. Some of the observed changes in gene expression in host-extract-induced cultures are similar to those during attachment of the bacterium to host tissues. Other responses indicated defence against toxic metabolites in the extract. Tuber extract induced a large gene cluster having homology to type VI secretion genes shown to be virulence determinants in many, but not all, animal and human pathogens. Two of the genes in the type VI cluster were found to be expressed during infection in potato tubers and stems, and mutants with knockouts of the corresponding genes had increased virulence on potato. One of the type VI secretion mutants was further characterized and found to grow to higher cell density in culture in the presence of host extract and to produce slightly more extracellular tissue-macerating enzymes than the wild-type strain. Analysis of secreted proteins showed that this type VI mutant was affected in the production of haemolysin-coregulated proteins (Hcps), which have been suggested to be secreted by the type VI pathway in other bacteria. The results suggest that the type VI secretion system of P. atrosepticum is needed for secretion of Hcps but not for virulence on its host plant, potato.

HrpN of Erwinia amylovora functions in the translocation of DspA/E into plant cells.

The type III secretion system (T3SS) is required by plant pathogenic bacteria for the translocation of certain bacterial proteins to the cytoplasm of plant cells or secretion of some proteins to the apoplast. The T3SS of Erwinia amylovora, which causes fire blight of pear, apple and other rosaceous plants, secretes DspA/E, which is an indispensable pathogenicity factor. Several other proteins, including HrpN, a critical virulence factor, are also secreted by the T3SS. Using a CyaA reporter system, we demonstrated that DspA/E is translocated into the cells of Nicotiana tabacum'Xanthi'. To determine if other T3-secreted proteins are needed for translocation of DspA/E, we examined its translocation in several mutants of E. amylovora strain Ea321. DspA/E was translocated by both hrpW and hrpK mutants, although with some delay, indicating that these two proteins are dispensable in the translocation of DspA/E. Remarkably, translocation of DspA/E was essentially abolished in both hrpN and hrpJ mutants; however, secretion of DspA/E into medium was not affected in any of the mentioned mutants. In contrast to the more virulent strain Ea273, secretion of HrpN was abolished in a hrpJ mutant of strain Ea321. In addition, HrpN was weakly translocated into plant cytoplasm. These results suggest that HrpN plays a significant role in the translocation of DspA/E, and HrpJ affects the translocation of DspA/E by affecting secretion or stability of HrpN. Taken together, these results explain the critical importance of HrpN and HrpJ to the development of fire blight.

Host-extract induced changes in the secretome of the plant pathogenic bacterium Pectobacterium atrosepticum.

Pectobacterium atrosepticum is a Gram-negative plant pathogenic bacterium that causes rotting in potato stems and tubers. The secreted proteins of this pathogen were analyzed with proteomics from culture supernatant of cells grown in minimal medium supplemented with host extracts. More than 40 proteins were identified, among them known virulence determinants, such as pectic enzymes, metalloprotease, and virulence protein Svx, along with flagella proteins, GroEL and cyclophilin-type chaperones and several hypothetical proteins or proteins with unknown function. Some of the identified proteins may be involved in utilization of nutrients or transport of minerals. Northern and real-time RT-PCR analyses suggested that most of the proteins upregulated by plant extract were transcriptionally regulated. Among the identified proteins were VgrG and four homologs of hemolysin-coregulated proteins (Hcps). A mutant strain lacking one of the hcp genes was not affected in virulence, while a bacterial strain overexpressing the same gene was shown to have increased virulence, which suggests that these proteins may be new virulence determinants of P. atrosepticum. Comparison of the secretomes of wild type cells and hrcC mutant defective in Type III secretion suggested that the production of the identified proteins was independent of functional Type III secretion system.