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July 7, 2019

Exploring the genomic traits of fungus-feeding bacterial genus Collimonas.

Collimonas is a genus belonging to the class of Betaproteobacteria and consists mostly of soil bacteria with the ability to exploit living fungi as food source (mycophagy). Collimonas strains differ in a range of activities, including swimming motility, quorum sensing, extracellular protease activity, siderophore production, and antimicrobial activities.In order to reveal ecological traits possibly related to Collimonas lifestyle and secondary metabolites production, we performed a comparative genomics analysis based on whole-genome sequencing of six strains representing 3 recognized species. The analysis revealed that the core genome represents 43.1 to 52.7 % of the genomes of the six individual strains. These include genes coding for extracellular enzymes (chitinase, peptidase, phospholipase), iron acquisition and type II secretion systems. In the variable genome, differences were found in genes coding for secondary metabolites (e.g. tripropeptin A and volatile terpenes), several unknown orphan polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), nonribosomal peptide synthetase (NRPS) gene clusters, a new lipopeptide and type III and type VI secretion systems. Potential roles of the latter genes in the interaction with other organisms were investigated. Mutation of a gene involved in tripropeptin A biosynthesis strongly reduced the antibacterial activity against Staphylococcus aureus, while disruption of a gene involved in the biosynthesis of the new lipopeptide had a large effect on the antifungal/oomycetal activities.Overall our results indicated that Collimonas genomes harbour many genes encoding for novel enzymes and secondary metabolites (including terpenes) important for interactions with other organisms and revealed genomic plasticity, which reflect the behaviour, antimicrobial activity and lifestylesof Collimonas spp.

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July 7, 2019

Botrytis, the good, the bad and the ugly

Botrytis spp. are efficient pathogens, causing devastating diseases and significant crop losses in a wide variety of plant species. Here we outline our review of these pathogens, as well as highlight the major advances of the past 10 years in studying Botrytis in interaction with its hosts. Progress in molecular genetics and the development of relevant phylogenetic markers in particular, has resulted in the characterisation of approximately 30 species. The host range of Botrytis spp. includes plant species that are members of 170 families of cultivated plants.

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July 7, 2019

Current overview on the study of bacteria in the rhizosphere by modern molecular techniques: a mini–review

The rhizosphere (soil zone influenced by roots) is a complex environment that harbors diverse bacterial populations, which have an important role in biogeochemical cycling of organic matter and mineral nutrients. Nevertheless, our knowledge of the ecology and role of these bacteria in the rhizosphere is very limited, particularly regarding how indigenous bacteria are able to communicate, colonize root environments, and compete along the rhizosphere microsites. In recent decades, the development and improvement of molecular techniques have provided more accurate knowledge of bacteria in their natural environment, refining microbial ecology and generating new questions about the roles and functions of bacteria in the rhizosphere. Recently, advances in soil post?genomic techniques (metagenomics, metaproteomics and metatranscriptomics) are being applied to improve our understanding of the microbial communities at a higher resolution. Moreover, advantages and limitations of classical and post?genomic techniques must be considered when studying bacteria in the rhizosphere. This review provides an overview of the current knowledge on the study of bacterial community in the rhizosphere by using modern molecular techniques, describing the bias of classical molecular techniques, next generation sequencing platforms and post?genomics techniques.

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July 7, 2019

Leafy spurge genomics: A model perennial weed to investigate development, stress responses, and invasiveness

Leafy spurge is wild flower native to Europe that has become an invasive perennial weed in the northern great plains of the USA and Canada. Leafy spurge primarily infests range and recreation lands and costs US land managers millions dollars annually. In its invaded range, leafy spurge can form vast monocultures that significantly impact native flora and fauna and has been attributed to reduced populations of endangered species such as the prairie fringed orchid. Leafy spurge has remarkable plasticity and can persist under environmental extremes—primarily due to the formation of hundreds of underground adventitious buds that can form on its extensive and deep root system. We have developed genomics-based tools to assist our investigations related to vegetative production from these underground buds, as well as its responses to stress, and the potential mechanisms leading to the invasiveness of leafy spurge. Towards these ends, we have utilized Sanger-based sequencing to develop EST-databases from leafy spurge and cassava (a related species) transcriptomes, and developed textasciitilde23,000 element cDNA microarrays representing all of the unigenes identified in these databases. Additionally, numerous cDNA libraries and genomic libraries have been developed including bacterial artificial chromosome libraries useful for identifying and characterizing promoters of differentially expressed genes. Finally, to enhance our ability to identify promoter sequences and transcription factors involved in vegetative production, stress responses, and invasiveness, we have incorporated next generation sequencing approaches to fully sequence the leafy spurge genome. Using global transcriptome profiles, next generation sequencing, bioinformatics programs has provided insights into molecular mechanisms and regulatory pathways that make leafy spurge a particularly invasive and difficult weed to control.

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July 7, 2019

Quorum sensing activity of Aeromonas caviae strain YL12, a bacterium isolated from compost.

Quorum sensing is a well-studied cell-to-cell communication method that involves a cell-density dependent regulation of genes expression mediated by signalling molecules. In this study, a bacterium isolated from a plant material compost pile was found to possess quorum sensing activity based on bioassay screening. Isolate YL12 was identified using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and molecular typing using rpoD gene which identified the isolate as Aeromonas caviae. High resolution tandem mass spectrometry was subsequently employed to identify the N-acyl homoserine lactone profile of Aeromonas caviae YL12 and confirmed that this isolate produced two short chain N-acyl homoserine lactones, namely C4-HSL and C6, and the production was observed to be cell density-dependent. Using the thin layer chromatography (TLC) bioassay, both AHLs were found to activate C. violaceum CV026, whereas only C6-HSL was revealed to induce bioluminescence expression of E. coli [pSB401]. The data presented in this study will be the leading steps in understanding the role of quorum sensing in Aeromonas caviae strain YL12.

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July 7, 2019

Stenotrophomonas comparative genomics reveals genes and functions that differentiate beneficial and pathogenic bacteria.

In recent years, the number of human infections caused by opportunistic pathogens has increased dramatically. Plant rhizospheres are one of the most typical natural reservoirs for these pathogens but they also represent a great source for beneficial microbes with potential for biotechnological applications. However, understanding the natural variation and possible differences between pathogens and beneficials is the main challenge in furthering these possibilities. The genus Stenotrophomonas contains representatives found to be associated with human and plant host.We used comparative genomics as well as transcriptomic and physiological approaches to detect significant borders between the Stenotrophomonas strains: the multi-drug resistant pathogenic S. maltophilia and the plant-associated strains S. maltophilia R551-3 and S. rhizophila DSM14405T (both are biocontrol agents). We found an overall high degree of sequence similarity between the genomes of all three strains. Despite the notable similarity in potential factors responsible for host invasion and antibiotic resistance, other factors including several crucial virulence factors and heat shock proteins were absent in the plant-associated DSM14405T. Instead, S. rhizophila DSM14405T possessed unique genes for the synthesis and transport of the plant-protective spermidine, plant cell-wall degrading enzymes, and high salinity tolerance. Moreover, the presence or absence of bacterial growth at 37°C was identified as a very simple method in differentiating between pathogenic and non-pathogenic isolates. DSM14405T is not able to grow at this human-relevant temperature, most likely in great part due to the absence of heat shock genes and perhaps also because of the up-regulation at increased temperatures of several genes involved in a suicide mechanism.While this study is important for understanding the mechanisms behind the emerging pattern of infectious diseases, it is, to our knowledge, the first of its kind to assess the risk of beneficial strains for biotechnological applications. We identified certain traits typical of pathogens such as growth at the human body temperature together with the production of heat shock proteins as opposed to a temperature-regulated suicide system that is harnessed by beneficials.

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July 7, 2019

Draft genome sequence of Kluyveromyces marxianus strain DMB1, isolated from sugarcane bagasse hydrolysate.

We determined the genome sequence of a thermotolerant yeast, Kluyveromyces marxianus strain DMB1, isolated from sugarcane bagasse hydrolysate, and the sequence provides further insights into the genomic differences between this strain and other reported K. marxianus strains. The genome described here is composed of 11,165,408 bases and has 4,943 protein-coding genes. Copyright © 2014 Suzuki et al.

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July 7, 2019

Draft genome sequence of Kitasatospora cheerisanensis KCTC 2395, which produces plecomacrolide against phytopathogenic fungi.

Kitasatospora cheerisanensis KCTC 2395, which produces antifungal metabolites with bafilomycin derivatives, including bafilomycin C1-amide, was isolated from a soil sample at Mt. Jiri, South Korea. Here, we report its draft genome sequence, which contains 8.04 Mb with 73.6% G+C content and 7,810 protein-coding genes. Copyright © 2014 Hwang et al.

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July 7, 2019

The oxygen-independent metabolism of cyclic monoterpenes in Castellaniella defragrans 65Phen.

The facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic ß-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway.Castellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95 Mb contained a 70 kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58EuT, than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate.The genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen.

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July 7, 2019

Evaluation and validation of de novo and hybrid assembly techniques to derive high-quality genome sequences.

To assess the potential of different types of sequence data combined with de novo and hybrid assembly approaches to improve existing draft genome sequences.Illumina, 454 and PacBio sequencing technologies were used to generate de novo and hybrid genome assemblies for four different bacteria, which were assessed for quality using summary statistics (e.g. number of contigs, N50) and in silico evaluation tools. Differences in predictions of multiple copies of rDNA operons for each respective bacterium were evaluated by PCR and Sanger sequencing, and then the validated results were applied as an additional criterion to rank assemblies. In general, assemblies using longer PacBio reads were better able to resolve repetitive regions. In this study, the combination of Illumina and PacBio sequence data assembled through the ALLPATHS-LG algorithm gave the best summary statistics and most accurate rDNA operon number predictions. This study will aid others looking to improve existing draft genome assemblies.All assembly tools except CLC Genomics Workbench are freely available under GNU General Public License.brownsd@ornl.govSupplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

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July 7, 2019

Genome sequence of Pseudomonas chlororaphis strain PA23.

Pseudomonas chlororaphis strain PA23 is a plant-beneficial bacterium that is able to suppress disease caused by the fungal pathogen Sclerotinia sclerotiorum through a process known as biological control. Here we present a 7.1-Mb assembly of the PA23 genome. Copyright © 2014 Loewen et al.

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July 7, 2019

Dissecting a hidden gene duplication: the Arabidopsis thaliana SEC10 locus.

Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both SEC10 genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover, we show that the use of the existing A. thaliana reference genome sequence as a guide for sequence assembly of new Arabidopsis accessions or related species has at least in some cases led to error propagation.

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July 7, 2019

FGAP: an automated gap closing tool.

The fast reduction of prices of DNA sequencing allowed rapid accumulation of genome data. However, the process of obtaining complete genome sequences is still very time consuming and labor demanding. In addition, data produced from various sequencing technologies or alternative assemblies remain underexplored to improve assembly of incomplete genome sequences.We have developed FGAP, a tool for closing gaps of draft genome sequences that takes advantage of different datasets. FGAP uses BLAST to align multiple contigs against a draft genome assembly aiming to find sequences that overlap gaps. The algorithm selects the best sequence to fill and eliminate the gap.FGAP reduced the number of gaps by 78% in an E. coli draft genome assembly using two different sequencing technologies, Illumina and 454. Using PacBio long reads, 98% of gaps were solved. In human chromosome 14 assemblies, FGAP reduced the number of gaps by 35%. All the inserted sequences were validated with a reference genome using QUAST. The source code and a web tool are available at http://www.bioinfo.ufpr.br/fgap/.

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July 7, 2019

Genome sequencing of two Neorhizobium galegae strains reveals a noeT gene responsible for the unusual acetylation of the nodulation factors.

The species Neorhizobium galegae comprises two symbiovars that induce nodules on Galega plants. Strains of both symbiovars, orientalis and officinalis, induce nodules on the same plant species, but fix nitrogen only in their own host species. The mechanism behind this strict host specificity is not yet known. In this study, genome sequences of representatives of the two symbiovars were produced, providing new material for studying properties of N. galegae, with a special interest in genomic differences that may play a role in host specificity.The genome sequences confirmed that the two representative strains are much alike at a whole-genome level. Analysis of orthologous genes showed that N. galegae has a higher number of orthologs shared with Rhizobium than with Agrobacterium. The symbiosis plasmid of strain HAMBI 1141 was shown to transfer by conjugation under optimal conditions. In addition, both sequenced strains have an acetyltransferase gene which was shown to modify the Nod factor on the residue adjacent to the non-reducing-terminal residue. The working hypothesis that this gene is of major importance in directing host specificity of N. galegae could not, however, be confirmed.Strains of N. galegae have many genes differentiating them from strains of Agrobacterium, Rhizobium and Sinorhizobium. However, the mechanism behind their ecological difference is not evident. Although the final determinant for the strict host specificity of N. galegae remains to be identified, the gene responsible for the species-specific acetylation of the Nod factors was identified in this study. We propose the name noeT for this gene to reflect its role in symbiosis.

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July 7, 2019

Genome Sequence of Pseudomonas brassicacearum DF41.

Pseudomonas brassicacearum DF41, a Gram-negative soil bacterium, is able to suppress the fungal pathogen Sclerotinia sclerotiorum through a process known as biological control. Here, we present a 6.8-Mb assembly of its genome, which is the second fully assembled genome of a P. brassicacearum strain.

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