APX2 Is an Ascorbate Peroxidase-Related Protein that Regulates the Levels of Plastocyanin in Chlamydomonas.

The function of ascorbate peroxidase-related (APX-R) proteins, present in all green photosynthetic eukaryotes, remains unclear. This study focuses on APX-R from Chlamydomonas reinhardtii, namely, ascorbate peroxidase 2 (APX2). We showed that apx2 mutants exhibited a faster oxidation of the photosystem I primary electron donor, P700, upon sudden light increase and a slower re-reduction rate compared to the wild type, pointing to a limitation of plastocyanin. Spectroscopic, proteomic and immunoblot analyses confirmed that the phenotype was a result of lower levels of plastocyanin in the apx2 mutants. The redox state of P700 did not differ between wild type and apx2 mutants when the loss of function in plastocyanin was nutritionally complemented by growing apx2 mutants under copper deficiency. In this case, cytochrome c6 functionally replaces plastocyanin, confirming that lower levels of plastocyanin were the primary defect caused by the absence of APX2. Overall, the results presented here shed light on an unexpected regulation of plastocyanin level under copper-replete conditions, induced by APX2 in Chlamydomonas.

Comparative genomics and pangenome-oriented studies reveal high homogeneity of the agronomically relevant enterobacterial plant pathogen Dickeya solani.

BACKGROUND: Dickeya solani is an important plant pathogenic bacterium causing severe losses in European potato production. This species draws a lot of attention due to its remarkable virulence, great devastating potential and easier spread in contrast to other Dickeya spp. In view of a high need for extensive studies on economically important soft rot Pectobacteriaceae, we performed a comparative genomics analysis on D. solani strains to search for genetic foundations that would explain the differences in the observed virulence levels within the D. solani population. RESULTS: High quality assemblies of 8 de novo sequenced D. solani genomes have been obtained. Whole-sequence comparison, ANIb, ANIm, Tetra and pangenome-oriented analyses performed on these genomes and the sequences of 14 additional strains revealed an exceptionally high level of homogeneity among the studied genetic material of D. solani strains. With the use of 22 genomes, the pangenome of D. solani, comprising 84.7% core, 7.2% accessory and 8.1% unique genes, has been almost completely determined, suggesting the presence of a nearly closed pangenome structure. Attribution of the genes included in the D. solani pangenome fractions to functional COG categories showed that higher percentages of accessory and unique pangenome parts in contrast to the core section are encountered in phage/mobile elements- and transcription- associated groups with the genome of RNS 05.1.2A strain having the most significant impact. Also, the first D. solani large-scale genome-wide phylogeny computed on concatenated core gene alignments is herein reported. CONCLUSIONS: The almost closed status of D. solani pangenome achieved in this work points to the fact that the unique gene pool of this species should no longer expand. Such a feature is characteristic of taxa whose representatives either occupy isolated ecological niches or lack efficient mechanisms for gene exchange and recombination, which seems rational concerning a strictly pathogenic species with clonal population structure. Finally, no obvious correlations between the geographical origin of D. solani strains and their phylogeny were found, which might reflect the specificity of the international seed potato market.

Isoforms of Acyl-CoA:Diacylglycerol Acyltransferase2 Differ Substantially in Their Specificities toward Erucic Acid.

In most oilseeds, two evolutionarily unrelated acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, are the main contributors to the acylation of diacylglycerols in the synthesis of triacylglycerol. DGAT1 and DGAT2 are both present in the important crop oilseed rape (Brassica napus), with each type having four isoforms. We studied the activities of DGAT isoforms during seed development in microsomal fractions from two oilseed rape cultivars: edible, low-erucic acid (22:1) MONOLIT and nonedible high-erucic acid MAPLUS. Whereas the specific activities of DGATs were similar with most of the tested acyl-CoA substrates in both cultivars, MAPLUS had 6- to 14-fold higher activity with 22:1-CoA than did MONOLIT. Thus, DGAT isoforms with different acyl-CoA specificities are differentially active in the two cultivars. We characterized the acyl-CoA specificities of all DGAT isoforms in oilseed rape in the microsomal fractions of yeast cells heterologously expressing these enzymes. All four DGAT1 isoforms showed similar and broad acyl-CoA specificities. However, DGAT2 isoforms had much narrower acyl-CoA specificities: two DGAT2 isoforms were highly active with 22:1-CoA, while the ability of the other two isoforms to use this substrate was impaired. These findings elucidate the importance, which a DGAT isoform with suitable acyl-CoA specificity may have, when aiming for high content of a particular fatty acid in plant triacylglycerol reservoirs.

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.

Pectobacterium polonicum sp. nov. isolated from vegetable fields.

Gram-stain-negative, rod-shaped pectinolytic bacteria strains designated as DPMP315T, DPMP316, DPMP317 and DPMP318 isolated from groundwater sampled from a vegetable field in the North of Poland, were subjected to the polyphasic analyses. Multilocus sequence analyses based on five housekeeping genes (gyrA, recA, recN, rpoA and rpoS) revealed their distinctiveness from the other species of the genus, simultaneously indicating that the newly described species, Pectobacterium punjabense, as well as Pectobacterium parmentieri and P. wasabiae, to be the closest relatives. In silico DNA-DNA hybridization (<43.1 %) and average nucleotide identity (<92.5 %) values of strain DPMP315T with other type strains of species of the genus Pectobacterium supported the delineation of the novel strain as representing a novel species. The phenotypic comparisons, fatty acid methyl esters compositions, genetic rep PCR fingerprint and detailed whole-cell MALDI-TOF mass spectrometry proteomic profiles permitted the differentiation of Polish strains from the type strains of all other known species of the genus Pectobacterium. The results of polyphasic analyses performed for four Polish strains are the basis for the distinction of the novel species. Here, we propose to establish DPMP315T as a type strain (=PCM3006T=LMG 31077T) with the name Pectobacterium polonicum sp. nov.

Differences in the constituents of bacterial microbiota of soils collected from two fields of diverse potato blackleg and soft rot diseases incidences, a case study.

The presence of bacteria from the Dickeya spp. and Pectobacterium spp. in farmlands leads to global crop losses of over $420 million annually. Since 1982, the scientists have started to suspect that the development of disease symptoms in crops might be inhibited by bacteria present in the soil. Here, we characterized in terms of physicochemical properties and the composition of bacterial soil microbiota two fields differing, on the basis of long-term studies, in the occurrence of Dickeya spp.- and Pectobacterium spp.-triggered infections. Majority, i.e. 17 of the investigated physicochemical features of the soils collected from two fields of either low or high potato blackleg and soft rot diseases incidences turned out to be similar, in contrast to the observed 4 deviations in relation to Mg, Mn, organic C and organic substance contents. By performing microbial cultures and molecular diagnostics-based identification, 20 Pectobacterium spp. strains were acquired from the field showing high blackleg and soft rot incidences. In addition, 16S rRNA gene amplicon sequencing followed by bioinformatic analysis revealed differences at various taxonomic levels in the soil bacterial microbiota of the studied fields. We observed that bacteria from the genera Bacillus, Rumeliibacillus, Acidobacterium and Gaiella turned out to be more abundant in the soil samples originating from the field of low comparing to high frequency of pectinolytic bacterial infections. In the herein presented case study, it is shown for the first time that the composition of bacterial soil microbiota varies between two fields differing in the incidences of soft rot and blackleg infections.

Comparative Genomics, from the Annotated Genome to Valuable Biological Information: A Case Study.

High availability of fast, cheap, and high-throughput next generation sequencing techniques resulted in acquisition of numerous de novo sequenced and assembled bacterial genomes. It rapidly became clear that digging out useful biological information from such a huge amount of data presents a considerable challenge. In this chapter we share our experience with utilization of several handy open source comparative genomic tools. All of them were applied in the studies focused on revealing inter- and intraspecies variation in pectinolytic plant pathogenic bacteria classified to Dickeya solani and Pectobacterium parmentieri. As the described software performed well on the species within the Pectobacteriaceae family, it presumably may be readily utilized on some closely related taxa from the Enterobacteriaceae family. First of all, implementation of various annotation software is discussed and compared. Then, tools computing whole genome comparisons including generation of circular juxtapositions of multiple sequences, revealing the order of synteny blocks or calculation of ANI or Tetra values are presented. Besides, web servers intended either for functional annotation of the genes of interest or for detection of genomic islands, plasmids, prophages, CRISPR/Cas are described. Last but not least, utilization of the software designed for pangenome studies and the further downstream analyses is explained. The presented work not only summarizes broad possibilities assured by the comparative genomic approach but also provides a user-friendly guide that might be easily followed by nonbioinformaticians interested in undertaking similar studies.

Comparative Genomics and Physiological Investigation of a New Arthrospira/Limnospira Strain O9.13F Isolated from an Alkaline, Winter Freezing, Siberian Lake.

Cyanobacteria from the genus Arthrospira/Limnospira are considered haloalkalotolerant organisms with optimal growth temperatures around 35 °C. They are most abundant in soda lakes in tropical and subtropical regions. Here, we report the comprehensive genome-based characterisation and physiological investigation of the new strain O9.13F that was isolated in a temperate climate zone from the winter freezing Solenoye Lake in Western Siberia. Based on genomic analyses, the Siberian strain belongs to the Arthrospira/Limnospira genus. The described strain O9.13F showed the highest relative growth index upon cultivation at 20 °C, lower than the temperature 35 °C reported as optimal for the Arthrospira/Limnospira strains. We assessed the composition of fatty acids, proteins and photosynthetic pigments in the biomass of strain O9.13F grown at different temperatures, showing its potential suitability for cultivation in a temperate climate zone. We observed a decrease of gamma-linolenic acid favouring palmitic acid in the case of strain O9.13F compared to tropical strains. Comparative genomics showed no unique genes had been found for the Siberian strain related to its tolerance to low temperatures. In addition, this strain does not possess a different set of genes associated with the salinity stress response from those typically found in tropical strains. We confirmed the absence of plasmids and functional prophage sequences. The genome consists of a 4.94 Mbp with a GC% of 44.47% and 5355 encoded proteins. The Arthrospira/Limnospira strain O9.13F presented in this work is the first representative of a new clade III based on the 16S rRNA gene, for which a genomic sequence is available in public databases (PKGD00000000).

Arthrospiribacter ruber gen. nov., sp. nov., a novel bacterium isolated from Arthrospira cultures.

Polyphasic analysis of ten isolates of the red-pigmented bacteria isolated from ten Arthrospira cultures originating from different parts of the world is described. The 16S rRNA analysis showed <95 % identity with the known bacteria on public databases, therefore, additional analyses of fatty acids profiles, MALDI-TOF/MS, genome sequencing of the chosen isolate and following phylogenomic analyses were performed. Gram-stain-negative, strictly aerobic rods were positive for catalase, negative for oxidase, proteolytic and urease activity. Major fatty acids were 15 : 0 iso, 17:0 iso 3 OH and 17:1 iso w9c/16:0 10-methyl. The whole phylogenomic analyses revealed that the genomic sequence of newly isolated strain DPMB0001 was most closely related to members of Cyclobacteriaceae family and clearly indicated distinctiveness of newly isolated bacteria. The average nucleotide identity and in silico DNA-DNA hybridisation values were calculated between representative of the novel strains DPMB0001 and its phylogenetically closest species, Indibacter alkaliphilus CCUG57479 (LW1)T (ANI 69.2 % is DDH 17.2 %) and Mariniradius saccharolyticus AK6T (ANI 80.02 % isDDH 26.1 %), and were significantly below the established cut-off <94 % (ANI) and <70 % (isDDH) for species and genus delineation. The obtained results showed that the analysed isolates represent novel genus and species, for which names Arthrospiribacter gen nov. and Arthrospiribacter ruber sp. nov. (type strain DPMB0001=LMG 31078=PCM 3008) is proposed.

Pectobacterium zantedeschiae sp. nov. a new species of a soft rot pathogen isolated from Calla lily (Zantedeschia spp.).

Four Gram-negative, rod-shaped pectinolytic bacterial strains designated as 2M, 9M, DPMP599 and DPMP600 were subjected to polyphasic analyses that revealed their distinctiveness from the other Pectobacterium species. Strains 2M and 9M were isolated from Calla lily bulbs cultivated in Central Poland. DPMP599 and DPMP600 strains were isolated from Calla lily leaves from plants grown in Serbia. Phylogenetic analyses based on nine housekeeping genes (gapA, gyrA, icdA, pgi, proA, recA, recN, rpoA, and rpoS), as well as phylogeny based on the 381 most conserved universal proteins confirmed that Pectobacterium zantedeschiae strains were distantly related to the other Pectobacterium, and indicated Pectobacterium atrosepticum, Pectobacterium betavasculorum, Pectobacterium parmentieri and Pectobacterium wasabiae as the closest relatives. Moreover, the analysis revealed that Pectobacterium zantedeschiae strains are not akin to Pectobacterium aroidearum strains, which were likewise isolated from Calla lily. The genome sequencing of the strains 2M, 9M and DPMP600 and their comparison with whole genome sequences of other Pectobacterium type strains confirmed their distinctiveness and separate species status within the genus based on parameters of in silico DNA-DNA hybridization and average nucleotide identity (ANI) values. The MALDI-TOF MS proteomic profile supported the proposition of delineation of the P. zantedeschiae and additionally confirmed the individuality of the studied strains. Based on of all of these data, it is proposed that the strains 2M, 9M, DPMP599, and DPMP600 isolated from Calla lily, previously assigned as P. atrosepticum should be reclassified as Pectobacterium zantedeschiae sp. nov. with the strain 9MT (PCM2893=DSM105717=IFB9009) as the type strain.

Transfer of Pectobacterium carotovorum subsp. carotovorum strains isolated from potatoes grown at high altitudes to Pectobacterium peruviense sp. nov.

Seven Gram-negative, rod-shaped pectinolytic bacteria strains designated as IFB5227, IFB5228, IFB5229, IFB5230, IFB5231, IFB5232, IFB5636, isolated from potato tubers cultivated in Peru at high altitude (2400-3800m) were subjected to polyphasic analyses that revealed their distinctiveness from the other Pectobacterium species. Phylogenetic analyses based on five housekeeping genes (gyrA, recA, recN, rpoA and rpoS) clearly showed strains separateness, simultaneously indicating Pectobacterium atrosepticum, Pectobacterium wasabiae, Pectobacterium parmentieri and Pectobacterium betavasculorum as the closest relatives. In silico DNA-DNA hybridization of strain IFB5232T with other Pectobacterium type strains revealed significant drop in DDH value below 70%, which is a prerequisite to distinguish Pectobacterium peruviense. The ANI values supported the proposition of delineation of the P. peruviense. Genetic REP-PCR fingerprint and detailed MALDI-TOF MS proteomic profile sealed the individuality of the studied strains. However, phenotypic assays do not indicate immense differences. Provided results of analyses performed for seven Peruvian strains are the basis for novel species distinction and reclassification of the strains IFB5227-5232 and IFB5636, previously classified as Pectobacterium carotovorum subsp. carotovorum. Here, we propose to establish the IFB5232 isolate as a type strain (=PCM2893T=LMG30269T=SCRI179T) with the name Pectobacterium peruviense sp. nov.

Consensus assessment of the contamination level of publicly available cyanobacterial genomes.

Publicly available genomes are crucial for phylogenetic and metagenomic studies, in which contaminating sequences can be the cause of major problems. This issue is expected to be especially important for Cyanobacteria because axenic strains are notoriously difficult to obtain and keep in culture. Yet, despite their great scientific interest, no data are currently available concerning the quality of publicly available cyanobacterial genomes. As reliably detecting contaminants is a complex task, we designed a pipeline combining six methods in a consensus strategy to assess the contamination level of 440 genome assemblies of Cyanobacteria. Two methods are based on published reference databases of ribosomal genes (SSU rRNA 16S and ribosomal proteins), one is indirectly based on a reference database of marker genes (CheckM), and three are based on complete genome analysis. Among those genome-wide methods, Kraken and DIAMOND blastx share the same reference database that we derived from Ensembl Bacteria, whereas CONCOCT does not require any reference database, instead relying on differences in DNA tetramer frequencies. Given that all the six methods appear to have their own strengths and limitations, we used the consensus of their rankings to infer that >5% of cyanobacterial genome assemblies are highly contaminated by foreign DNA (i.e., contaminants were detected by 5 or 6 methods). Our results will help researchers to check the quality of publicly available genomic data before use in their own analyses. Moreover, we argue that journals should make mandatory the submission of raw read data along with genome assemblies in order to facilitate the detection of contaminants in sequence databases.

Edible Cyanobacterial Genus Arthrospira: Actual State of the Art in Cultivation Methods, Genetics, and Application in Medicine.

The cyanobacterial genus Arthrospira appears very conserved and has been divided into five main genetic clusters on the basis of molecular taxonomy markers. Genetic studies of seven Arthrospira strains, including genome sequencing, have enabled a better understanding of those photosynthetic prokaryotes. Even though genetic manipulations have not yet been performed with success, many genomic and proteomic features such as stress adaptation, nitrogen fixation, or biofuel production have been characterized. Many of above-mentioned studies aimed to optimize the cultivation conditions. Factors like the light intensity and quality, the nitrogen source, or different modes of growth (auto-, hetero-, or mixotrophic) have been studied in detail. The scaling-up of the biomass production using photobioreactors, either closed or open, was also investigated to increase the production of useful compounds. The richness of nutrients contained in the genus Arthrospira can be used for promising applications in the biomedical domain. Ingredients such as the calcium spirulan, immulina, C-phycocyanin, and γ-linolenic acid (GLA) show a strong biological activity. Recently, its use in the fight against cancer cells was documented in many publications. The health-promoting action of "Spirulina" has been demonstrated in the case of cardiovascular diseases and age-related conditions. Some compounds also have potent immunomodulatory properties, promoting the growth of beneficial gut microflora, acting as antimicrobial and antiviral. Products derived from Arthrospira were shown to successfully replace biomaterial scaffolds in regenerative medicine. Supplementation with the cyanobacterium also improves the health of livestock and quality of the products of animal origin. They were also used in cosmetic preparations.

Draft Genome Sequence of the Axenic Strain Phormidesmispriestleyi ULC007, a Cyanobacterium Isolated from Lake Bruehwiler (Larsemann Hills, Antarctica).

Phormidesmis priestleyi ULC007 is an Antarctic freshwater cyanobacterium. Its draft genome is 5,684,389 bp long. It contains a total of 5,604 protein-encoding genes, of which 22.2% have no clear homologues in known genomes. To date, this draft genome is the first one ever determined for an axenic cyanobacterium from Antarctica.