Pectobacterium parvum sp. nov., having a Salmonella SPI-1-like Type III secretion system and low virulence.

Pectobacterium strains isolated from potato stems in Finland, Poland and the Netherlands were subjected to polyphasic analyses to characterize their genomic and phenotypic features. Phylogenetic analysis based on 382 core proteins showed that the isolates clustered closest to Pectobacterium polaris but could be divided into two clades. Average nucleotide identity (ANI) analysis revealed that the isolates in one of the clades included the P. polaris type strain, whereas the second clade was at the border of the species P. polaris with a 96 % ANI value. In silico genome-to-genome comparisons between the isolates revealed values below 70%, patristic distances based on 1294 core proteins were at the level observed between closely related Pectobacterium species, and the two groups of bacteria differed in genome size, G+C content and results of amplified fragment length polymorphism and Biolog analyses. Comparisons between the genomes revealed that the isolates of the atypical group contained SPI-1-type Type III secretion island and genes coding for proteins known for toxic effects on nematodes or insects, and lacked many genes coding for previously characterized virulence determinants affecting rotting of plant tissue by soft rot bacteria. Furthermore, the atypical isolates could be differentiated from P. polaris by their low virulence, production of antibacterial metabolites and a citrate-negative phenotype. Based on the results of a polyphasic approach including genome-to-genome comparisons, biochemical and virulence assays, presented in this report, we propose delineation of the atypical isolates as a novel species Pectobacterium parvum, for which the isolate s0421T (CFBP 8630T=LMG 30828T) is suggested as a type strain.

A metatranscriptomics-based assessment of small-scale mixing of sulfidic and oxic waters on redoxcline prokaryotic communities.

Lateral intrusions of oxygen caused by small-scale mixing are thought to shape microbial activity in marine redoxclines. To examine the response of prokaryotes to such mixing events we employed a shipboard mixing experiment in the euxinic central Baltic Sea: oxic, nitrate containing and sulfidic water samples without detectable oxygenized substances were incubated directly or after mixing. While nitrate, nitrite and ammonium concentrations stayed approximately constant in all incubations, we observed a decrease of sulfide after the contact with oxygen in the sulfide containing incubations. The transcription of marker genes from chemolithoauthotrophic key players including archaeal nitrifiers as well as gammaproteobacterial and campylobacterial autotrophic organisms that couple denitrification with sulfur-oxidation were followed at four time points within 8.5 h. The temporally contrasting transcriptional profiles of gammaproteobacterial and campylobacterial denitrifiers that depend on the same inorganic substrates pointed to a niche separation. Particular archaeal and campylobacterial marker genes involved in nitrification, denitrification and sulfur oxidation, which depend on oxidized substrates, were highly upregulated in the anaerobic sulfidic samples. We suggest that, despite the absence of measurable oxygenated compounds in the sulfidic water, frequent intermittent small-scale intrusions stimulate the permanent upregulation of genes involved in nitrification, denitrification and sulfur oxidation.

Metatranscriptomic data reveal the effect of different community properties on multifunctional redundancy.

The assessment of functional redundancy (FR) is essential to understand community structure-function relationships because FR buffers the functional performance of communities against changes in community composition. We introduce a novel metatranscriptome-based approach to quantify FR, which permits multifunctional aspects to be addressed. FR among prokaryotes was ranked in water samples after exposure to changing salinity. FR was higher for functional categories with mostly broad functions shared among many taxa than for functional categories containing many narrow functions. Furthermore, community characteristics had a higher impact on FR than environmental conditions. The metric also allows FR to be estimated between selected groups of taxa, and FR was high between more closely related organisms if communities were grown in similar environmental conditions. Overall, our data revealed a pronounced influence of functional diversity on the one hand but also the characteristics of individual community members on FR, which was specifically high in those communities whose members were more sensitive to salinity changes.

Conserved features and major differences in the outer membrane protein composition of chlamydiae.

Chlamydiae are a highly successful group of obligate intracellular bacteria infecting a variety of eukaryotic hosts. Outer membrane proteins involved in attachment to and uptake into host cells, and cross-linking of these proteins via disulfide bonds are key features of the biphasic chlamydial developmental cycle. In this study, we used a consensus approach to predict outer membrane proteins in the genomes of members of three chlamydial families. By analysing outer membrane protein fractions of purified chlamydiae with highly sensitive mass spectrometry, we show that the protein composition differs strongly between these organisms. Large numbers of major outer membrane protein-like proteins are present at high abundance in the outer membrane of Simkania negevensis and Waddlia chondrophila, whereas yet uncharacterized putative porins dominate in Parachlamydia acanthamoebae. Simkania represents the first case of a chlamydia completely lacking stabilizing cysteine-rich proteins in its outer membrane. In agreement with this, and in contrast to Parachlamydia and Waddlia, the cellular integrity of Simkania is not impaired by conditions that reduce disulfide bonds of these proteins. The observed differences in the protein composition of the outer membrane among members of divergent chlamydial families suggest different stabilities of these organisms in the environment, probably due to adaption to different niches or transmission routes.

Genome and physiology of a model Epsilonproteobacterium responsible for sulfide detoxification in marine oxygen depletion zones.

Eutrophication and global climate change lead to expansion of hypoxia in the ocean, often accompanied by the production of hydrogen sulfide, which is toxic to higher organisms. Chemoautotrophic bacteria are thought to buffer against increased sulfide concentrations by oxidizing hydrogen sulfide before its diffusion to oxygenated surface waters. Model organisms from such environments have not been readily available, which has contributed to a poor understanding of these microbes. We present here a detailed study of "Sulfurimonas gotlandica" str. GD1, an Epsilonproteobacterium isolated from the Baltic Sea oxic-anoxic interface, where it plays a key role in nitrogen and sulfur cycling. Whole-genome analysis and laboratory experiments revealed a high metabolic flexibility, suggesting a considerable capacity for adaptation to variable redox conditions. S. gotlandica str. GD1 was shown to grow chemolithoautotrophically by coupling denitrification with oxidation of reduced sulfur compounds and dark CO(2) fixation. Metabolic versatility was further suggested by the use of a range of different electron donors and acceptors and organic carbon sources. The number of genes involved in signal transduction and metabolic pathways exceeds those of other Epsilonproteobacteria. Oxygen tolerance and environmental-sensing systems combined with chemotactic responses enable this organism to thrive successfully in marine oxygen-depletion zones. We propose that S. gotlandica str. GD1 will serve as a model organism in investigations that will lead to a better understanding how members of the Epsilonproteobacteria are able to cope with water column anoxia and the role these microorganisms play in the detoxification of sulfidic waters.

Comparative genomics of unintrogressed Campylobacter coli clades 2 and 3.

BACKGROUND: Campylobacter jejuni and C. coli share a multitude of risk factors associated with human gastrointestinal disease, yet their phylogeny differs significantly. C. jejuni is scattered into several lineages, with no apparent linkage, whereas C. coli clusters into three distinct phylogenetic groups (clades) of which clade 1 has shown extensive genome-wide introgression with C. jejuni, yet the other two clades (2 and 3) have less than 2% of C. jejuni ancestry. We characterized a C. coli strain (76339) with four novel multilocus sequence type alleles (ST-5088) and having the capability to express gamma-glutamyltranspeptidase (GGT); an accessory feature in C. jejuni. Our aim was to further characterize unintrogressed C. coli clades 2 and 3, using comparative genomics and with additional genome sequences available, to investigate the impact of horizontal gene transfer in shaping the accessory and core gene pools in unintrogressed C. coli. RESULTS: Here, we present the first fully closed C. coli clade 3 genome (76339). The phylogenomic analysis of strain 76339, revealed that it belonged to clade 3 of unintrogressed C. coli. A more extensive respiratory metabolism among unintrogressed C. coli strains was found compared to introgressed C. coli (clade 1). We also identified other genes, such as serine proteases and an active sialyltransferase in the lipooligosaccharide locus, not present in C. coli clade 1 and we further propose a unique scenario for the evolution of Campylobacter ggt. CONCLUSIONS: We propose new insights into the evolution of the accessory genome of C. coli clade 3 and C. jejuni. Also, in silico analysis of the gene content revealed that C. coli clades 2 and 3 have genes associated with infection, suggesting they are a potent human pathogen, and may currently be underreported in human infections due to niche separation.

Genome analysis of Campylobacter jejuni strains isolated from a waterborne outbreak.

BACKGROUND: Waterborne Campylobacter jejuni outbreaks are common in the Nordic countries, and PFGE (pulsed field gel electrophoresis) remains the genotyping method of choice in outbreak investigations. However, PFGE cannot assess the clonal relationship between isolates, leading to difficulties in molecular epidemiological investigations. Here, we explored the applicability of whole genome sequencing to outbreak investigation by re-analysing three C. jejuni strains (one isolated from water and two from patients) from an earlier resolved Finnish waterborne outbreak from the year 2000. RESULTS: One of the patient strains had the same PFGE profile, as well as an identical overall gene synteny and three polymorphisms in comparison with the water strain. However, the other patient isolate, which showed only minor differences in the PFGE pattern relative to the water strain, harboured several polymorphisms as well as rearrangements in the integrated element CJIE2. We reconstructed the genealogy of these strains with ClonalFrame including in the analysis four C. jejuni isolated from chicken in 2012 having the same PFGE profile and sequence type as the outbreak strains. The three outbreak strains exhibited a paraphyletic relationship, implying that the drinking water from 2000 was probably contaminated with at least two different, but related, C. jejuni strains. CONCLUSIONS: Our results emphasize the capability of whole genome sequencing to unambiguously resolve the clonal relationship between isolates of C. jejuni in an outbreak situation and evaluate the diversity of the C. jejuni population.

Genomic variation between Campylobacter jejuni isolates associated with milk-borne-disease outbreaks.

Bacterial genome sequencing has led to the development of new approaches for the analysis of food-borne epidemics and the exploration of the relatedness of outbreak-associated isolates and their separation from nonassociated isolates. Using Illumina technology, we sequenced a total of six isolates (two from patients, two from raw bulk milk, and two from dairy cattle) associated with a milk-borne Campylobacter jejuni outbreak in a farming family and compared their genomes. These isolates had identical pulsed-field gel electrophoresis (PFGE) types, and their multilocus sequence typing (MLST) type was ST-50. We used the Ma_1 isolate (milk) as the reference, and its genome was assembled and tentatively ordered using the C. jejuni NCTC 11168 genome as the scaffold. Using whole-genome MLST (wgMLST), we identified a total of three single-nucleotide polymorphisms (SNPs) and differences in poly(G or C) or poly(A or T) tracts in 12 loci among the isolates. Several new alleles not present in the database were detected. In contrast, the sequences of the unassociated C. jejuni strains P14 and 1-12S (both ST-50) differed by 420 to 454 alleles from the epidemic-associated isolates. We found that the fecal contamination of bulk tank milk occurred by highly related sequence variants of C. jejuni, which are reflected as SNPs and differences in the length of the poly(A or T) tracts. Poly(G or C) tracts are reversibly variable and are thus unstable markers for comparison. Further, unrelated strains of ST-50 were clearly separated from the outbreak-associated isolates, indicating that wgMLST is an excellent tool for analysis. In addition, other useful data related to the genes and genetic systems of the isolates were obtained.

Multilocus sequence typing (MLST) and whole-genome MLST of Campylobacter jejuni isolates from human infections in three districts during a seasonal peak in Finland.

A total of 95 human Campylobacter jejuni isolates acquired from domestic infections and collected from three districts in Finland during the seasonal peak (June to September) in 2012 were analyzed by PCR-based multilocus sequence typing (MLST) and by whole-genome sequencing (WGS). Four predominant sequence types (STs) were detected among the isolates: ST-45 (21%) and ST-230 (14%, ST-45 clonal complex [CC]), ST-267 (21%, ST-283 CC), and ST-677 (19%, ST-677 CC). In districts 1 and 3, most of the infections occurred from early July to the middle of August, with a peak at weeks 29 to 31, but in district 2, the infections were dispersed more evenly throughout 3 months (June to August). WGS data were used for further whole-genome MLST (wgMLST) analyses of the isolates representing the four common STs. Shared loci of the isolates within each ST were analyzed as distance matrices of allelic profiles by the neighbor-net algorithm. The highest allelic variations (>400 different alleles) were detected between different clusters of ST-45 isolates (1,121 shared loci), while ST-230 (1,264 shared loci), ST-677 (1,169 shared loci), and ST-267 isolates (1,217 shared loci) were less diverse with the clusters differing by <40 alleles. Closely related isolates showing no allelic variation (subclusters) were detected among all four major STs. In some cases, they originated from different districts, suggesting that isolates can be epidemiologically connected and may have the same infection source despite being originally identified as sporadic infections.

Notch3 regulates Mybl2 via HeyL to limit proliferation and tumor initiation in breast cancer.

Notch signaling is a conserved signaling pathway that participates in many aspects of mammary gland development and homeostasis, and has extensively been associated with breast tumorigenesis. Here, to unravel the as yet debated role of Notch3 in breast cancer development, we investigated its expression in human breast cancer samples and effects of its loss in mice. Notch3 expression was very weak in breast cancer cells and was associated with good patient prognosis. Interestingly, its expression was very strong in stromal cells of these patients, though this had no prognostic value. Mechanistically, we demonstrated that Notch3 prevents tumor initiation via HeyL-mediated inhibition of Mybl2, an important regulator of cell cycle. In the mammary glands of Notch3-deficient mice, we observed accelerated tumor initiation and proliferation in a MMTV-Neu model. Notch3-null tumors were enriched in Mybl2 mRNA signature and protein expression. Hence, our study reinforces the anti-tumoral role of Notch3 in breast tumorigenesis.

Complete genome sequence of a variant of Campylobacter jejuni NCTC 11168.

Campylobacter jejuni NCTC 11168 is widely used in research, but at least two variants have been reported. The available genome was sequenced from a variant which later showed a different phenotype and gene expression profile. Here we present the complete genome sequence of a second variant of C. jejuni NCTC 11168.

Identification and characterization of a lipopolysaccharide α,2,3-sialyltransferase from the human pathogen Helicobacter bizzozeronii.

Terminal sialic acid in the lipopolysaccharides (LPSs) of mucosal pathogens is an important virulence factor. Here we report the characterization of a Helicobacter sialyltransferase involved in the biosynthesis of sialylated LPS in Helicobacter bizzozeronii, the only non-pylori gastric Helicobacter species isolated from humans thus far. Starting from the genome sequences of canine and human strains, we identified potential sialyltransferases downstream of three genes involved in the biosynthesis of N-acetylneuraminic acid. One of these candidates showed monofunctional α,2,3-sialyltransferase activity with a preference for N-acetyllactosamine as a substrate. The LPSs from different strains were shown by SDS-PAGE and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) to contain sialic acid after neuraminidase treatment. The expression of this sialyltransferase and sialyl-LPS appeared to be a phase-variable characteristic common to both human and canine H. bizzozeronii strains. The sialylation site of the LPSs of two H. bizzozeronii strains was determined to be NeuAc-Hex-HexNAc, suggesting terminal 3'-sialyl-LacNAc. Moreover, serological typing revealed the possible presence of sialyl-Lewis X in two additional strains, indicating that H. bizzozeronii could also mimic the surface glycans of mammalian cells. The expression of sialyl-glycans may influence the adaptation process of H. bizzozeronii during the host jump from dogs to humans.

Genome sequence of Helicobacter bizzozeronii strain CIII-1, an isolate from human gastric mucosa.

The canine-adapted Helicobacter bizzozeronii is the only nonpylori Helicobacter species isolated from human gastric biopsy tissue. Here we present the genome sequence of strain CIII-1, isolated from a 45-year-old female patient with severe gastric symptoms. This is the first genome sequence of nonpylori gastric Helicobacter isolated from human gastritis.

Comparative genomics of Helicobacter pylori and the human-derived Helicobacter bizzozeronii CIII-1 strain reveal the molecular basis of the zoonotic nature of non-pylori gastric Helicobacter infections in humans.

BACKGROUND: The canine Gram-negative Helicobacter bizzozeronii is one of seven species in Helicobacter heilmannii sensu lato that are detected in 0.17-2.3% of the gastric biopsies of human patients with gastric symptoms. At the present, H. bizzozeronii is the only non-pylori gastric Helicobacter sp. cultivated from human patients and is therefore a good alternative model of human gastric Helicobacter disease. We recently sequenced the genome of the H. bizzozeronii human strain CIII-1, isolated in 2008 from a 47-year old Finnish woman suffering from severe dyspeptic symptoms. In this study, we performed a detailed comparative genome analysis with H. pylori, providing new insights into non-pylori Helicobacter infections and the mechanisms of transmission between the primary animal host and humans. RESULTS: H. bizzozeronii possesses all the genes necessary for its specialised life in the stomach. However, H. bizzozeronii differs from H. pylori by having a wider metabolic flexibility in terms of its energy sources and electron transport chain. Moreover, H. bizzozeronii harbours a higher number of methyl-accepting chemotaxis proteins, allowing it to respond to a wider spectrum of environmental signals. In this study, H. bizzozeronii has been shown to have high level of genome plasticity. We were able to identify a total of 43 contingency genes, 5 insertion sequences (ISs), 22 mini-IS elements, 1 genomic island and a putative prophage. Although H. bizzozeronii lacks homologues of some of the major H. pylori virulence genes, other candidate virulence factors are present. In particular, we identified a polysaccharide lyase (HBZC1_15820) as a potential new virulence factor of H. bizzozeronii. CONCLUSIONS: The comparative genome analysis performed in this study increased the knowledge of the biology of gastric Helicobacter species. In particular, we propose the hypothesis that the high metabolic versatility and the ability to react to a range of environmental signals, factors which differentiate H. bizzozeronii as well as H. felis and H. suis from H. pylori, are the molecular basis of the of the zoonotic nature of H. heilmannii sensu lato infection in humans.

Mitochondrial Genome of Fagus sylvatica L. as a Source for Taxonomic Marker Development in the Fagales.

European beech, Fagus sylvatica L., is one of the most important and widespread deciduous tree species in Central Europe and is widely managed for its hard wood. The complete DNA sequence of the mitochondrial genome of Fagus sylvatica L. was assembled and annotated based on Illumina MiSeq reads and validated using long reads from nanopore MinION sequencing. The genome assembled into a single DNA sequence of 504,715 bp in length containing 58 genes with predicted function, including 35 protein-coding, 20 tRNA and three rRNA genes. Additionally, 23 putative protein-coding genes were predicted supported by RNA-Seq data. Aiming at the development of taxon-specific mitochondrial genetic markers, the tool SNPtax was developed and applied to select genic SNPs potentially specific for different taxa within the Fagales. Further validation of a small SNP set resulted in the development of four CAPS markers specific for Fagus, Fagaceae, or Fagales, respectively, when considering over 100 individuals from a total of 69 species of deciduous trees and conifers from up to 15 families included in the marker validation. The CAPS marker set is suitable to identify the genus Fagus in DNA samples from tree tissues or wood products, including wood composite products.

The complete chloroplast genome sequence of Pinus cembra L. (Pinaceae).

The Swiss pine (Pinus cembra) is a montane tree in Central Europe and, therefore, known for its hardiness against severe winter colds. The seeds are harvested and eaten as pine nuts. We assembled and characterized the complete chloroplast genome of P. cembra to serve as a valuable resource in future genetic studies. The complete plastome sequence is 116,609 bp in length and contains 113 genes including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. A phylogenetic analysis of 34 Pinus plastome sequences shows that Pinus sibirica is the nearest relative to P. cembra and that there is a distinct clustering together with the other members of the section Quinquefoliae.

Effect of large magnetotactic bacteria with polyphosphate inclusions on the phosphate profile of the suboxic zone in the Black Sea.

The Black Sea is the world's largest anoxic basin and a model system for studying processes across redox gradients. In between the oxic surface and the deeper sulfidic waters there is an unusually broad layer of 10-40 m, where neither oxygen nor sulfide are detectable. In this suboxic zone, dissolved phosphate profiles display a pronounced minimum at the upper and a maximum at the lower boundary, with a peak of particulate phosphorus in between, which was suggested to be caused by the sorption of phosphate on sinking particles of metal oxides. Here we show that bacterial polyphosphate inclusions within large magnetotactic bacteria related to the genus Magnetococcus contribute substantially to the observed phosphorus peak, as they contain 26-34% phosphorus compared to only 1-5% in metal-rich particles. Furthermore, we found increased gene expression for polyphosphate kinases by several groups of bacteria including Magnetococcaceae at the phosphate maximum, indicating active bacterial polyphosphate degradation. We propose that large magnetotactic bacteria shuttle up and down within the suboxic zone, scavenging phosphate at the upper and releasing it at the lower boundary. In contrast to a passive transport via metal oxides, this bacterial transport can quantitatively explain the observed phosphate profiles.

Structural and functional self-organization of Photosystem II in grana thylakoids.

The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.

Genomic sequence of 'Candidatus Liberibacter solanacearum' haplotype C and its comparison with haplotype A and B genomes.

Haplotypes A and B of 'Candidatus Liberibacter solanacearum' (CLso) are associated with diseases of solanaceous plants, especially Zebra chip disease of potato, and haplotypes C, D and E are associated with symptoms on apiaceous plants. To date, one complete genome of haplotype B and two high quality draft genomes of haplotype A have been obtained for these unculturable bacteria using metagenomics from the psyllid vector Bactericera cockerelli. Here, we present the first genomic sequences obtained for the carrot-associated CLso. These two genomic sequences of haplotype C, FIN114 (1.24 Mbp) and FIN111 (1.20 Mbp), were obtained from carrot psyllids (Trioza apicalis) harboring CLso. Genomic comparisons between the haplotypes A, B and C revealed that the genome organization differs between these haplotypes, due to large inversions and other recombinations. Comparison of protein-coding genes indicated that the core genome of CLso consists of 885 ortholog groups, with the pan-genome consisting of 1327 ortholog groups. Twenty-seven ortholog groups are unique to CLso haplotype C, whilst 11 ortholog groups shared by the haplotypes A and B, are not found in the haplotype C. Some of these ortholog groups that are not part of the core genome may encode functions related to interactions with the different host plant and psyllid species.