Streptomyces coelicolor: DNA methylation and differentiation

DNA cytosine methylation is an epigenetic modification regulating many biological processes in eukaryotes, including chromatin organization, genome maintenance and gene expression. The role of DNA cytosine methylation in prokaryotes has not been deeply investigated. In Escherichia coli it was recently demonstrated that cytosine methylation regulates gene expression during stationary phase [1] and that an induced state of cytosine hypermethylation leads to chromosomal DNA cleavage and cell death [2]. Streptomyces coelicolor is a mycelial soil microorganism, which exhibits a complex life cycle that includes three different cell types: unigenomic spores, a compartmentalized mycelium (MI) and a multinucleated mycelium (substrate and aerial mycelium, MII) [3]. The importance of DNA methylation was already described in Streptomycetes [4], but its biological role remains unknown. The main objectives of this study are to analyze cytosine methylation pattern of Streptomyces coelicolor M145 during growth in liquid and on solid media, and to investigate the relationship between DNA cytosine methylation and morphological/physiological differentiation. Cytosine methylation of total genomic DNA extracted from different developmental stages was investigated by dot-blot experiments using antibody anti-5-methylcytosine. Cytosine methylome was analyzed by BiSulphite sequencing. The biological effect of cytosine methylation was studied adding 5-aza-2\u2019-deoxycytidine (aza-dC), a hypomethylating agent, to the cultures. Dot blot analysis revealed that the level of cytosine methylation changes during development (MI, MII and spores). Specifically, DNA methylation is higher at the MI stage than in the MII or spores. BiSulphite sequencing revealed that 30% of S. coelicolor genes contained a methylated motif in their upstream regions. Genes harbouring these motifs included genes related to differentiation (aerial mycelium formation and sporulation), genes involved in DNA repair/replication/condensation, as well as genes encoding proteins with unknown functions. Phenotypic analyses of cultures treated with aza-dC demonstrated that DNA methylation influences germination, aerial mycelium formation and sporulation on solid medium and antibiotic production both, on solid and in liquid medium. Overall, our preliminary results suggest a role for DNA cytosine methylation in morphological and physiological differentiation of S. coelicolor. Further experiments are ongoing to demonstrate the molecular mechanisms and pathways behind the observed phenotypes

Draft Genome Sequence of Chromate-Resistant and Biofilm-Producing Strain Pseudomonas alcaliphila 34

We report the draft genome sequence of Pseudomonas alcaliphila 34, a Cr(VI)-hyperresistant and biofilm-producing bacterium that might be used for the bioremediation of chromate-polluted soils. The genome sequence might be helpful in exploring the mechanisms involved in chromium resistance and biofilm formation

Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti

The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes

Epigenetic control of Streptomyces coelicolor differentiation

DNA cytosine methylation is one of the most important epigenetic modifications in eukaryotes regulating chromatin organization, genome maintenance and gene expression. The role of DNA cytosine methylation in prokaryotes has not been deeply investigated. In Escherichia coli cytosine methylation regulates gene expression during the stationary phase and cytosine hypermethylation leads to chromosomal DNA cleavage and cell death. Streptomyces coelicolor is a mycelial soil microorganism, which exhibits a complex life cycle that includes three different cell types: unigenomic spores, a compartmentalized mycelium (MI) and a multinucleated mycelium (substrate and aerial mycelium, MII). The importance of DNA methylation was already described in Streptomycetes, but its biological role remained unknown. The main objectives of this study were to analyze the pattern of cytosine methylation in Streptomyces coelicolor and to investigate the relationship between DNA cytosine methylation and morphological/physiological differentiation. Dot-blot analysis of genomic DNA using antibody anti-5-methylcytosine revealed that DNA methylation is modulated during hyphae differentiation. Specifically DNA cytosine methylation is higher at the MI stage than in the MII or spores. Cytosine methylome was investigated by bisulphite DNA sequencing showing that 30% of S. coelicolor genes contain a methylated motif in their upstream region. The biological effect of cytosine methylation was studied using 5-aza-2’-deoxycytidine (aza-dC), a hypomethylating agent. Phenotypic analyses of cultures treated with aza-dC demonstrated that they were impaired in germination, aerial mycelium formation and sporulation. In addition, they showed a strong reduction in antibiotic production. Overall, our results suggest a role for DNA cytosine methylation in morphological and physiological differentiation of S. coelicolor. Further experiments are ongoing to characterize the molecular mechanisms and pathways behind the observed phenotypes

Enly: improving draft genomes through reads recycling

The reconstruction of the complete genome sequence of an organism is an important point for comparative, functional and evolutionary genomics. Nevertheless, overcoming the problems encountered while completing the sequence of an entire genome can still be demanding in terms of time and resources. We have developed Enly, a simple tool based on the iterative mapping of sequence reads at contig edges, capable to extend the genomic contigs deriving from high-throughput sequencing, especially those deriving by Newbler-like assemblies. Testing it on a set of de novo draft genomes led to the closure of up to 20% of the gaps originally present. Enly is cross-platform and most of the steps of its pipeline are parallelizable, making easy and fast to improve a draft genome resulting from a de novo assembly

Mixed nodule infection in Sinorhizobium meliloti-medicago sativa symbiosis suggest the presence of cheating behavior

In the symbiosis between rhizobia and legumes, host plants can forms ymbiotic root nodules with multiple rhizobial strains, potentially showing different symbiotic performances in nitrogen fixation. Here, we investigated the presence of mixed nodules, containing rhizobia with different degrees of mutualisms, and evaluate their relative fitness in the Sinorhizobium meliloti-Medicago sativa model symbiosis. We used three S. meliloti strains, the mutualist strains Rm1021 and BL225C and the non-mutualist AK83. We performed competition experiments involving both in vitro and in vivo symbiotic assays with M. sativa host plants. We show the occurrence of a high number (from 27 to 100%) of mixed nodules with no negative effect on both nitrogen fixation and plant growth. The estimation of the relative fitness as non-mutualist/mutualist ratios in single nodules shows that in some nodules the non-mutualist strain efficiently colonized root nodules along with the mutualist ones. In conclusion, we can support the hypothesis that in S. meliloti-M. sativa symbiosis mixed nodules are formed and allow non-mutualist or less-mutualist bacterial partners to be less or not sanctioned by the host plant, hence allowing a potential form of cheating behavior to be present in the nitrogen fixing symbiosis

Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti

The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes

Advances in Host Plant and Rhizobium Genomics to Enhance Symbiotic Nitrogen Fixation in Grain Legumes

Legumes form symbiotic relationship with root-nodule, rhizobia. The nitrogen (N2) fixed by legumes is a renewable source and of great importance to agriculture. Symbiotic nitrogen fixation (SNF) is constrained by multiple stresses and alleviating them would improve SNF contribution to agroecosystems. Genetic differences in adaptation tolerance to various stresses are known in both host plant and rhizobium. The discovery and use of promiscuous germplasm in soybean led to the release of high-yielding cultivars in Africa. High N2-fixing soybean cultivars are commercially grown in Australia and some countries in Africa and South America and those of pea in Russia. SNF is a complex trait, governed by multigenes with varying effects. Few major quantitative trait loci (QTL) and candidate genes underlying QTL are reported in grain and model legumes. Nodulating genes in model legumes are cloned and orthologs determined in grain legumes. Single nucleotide polymorphism (SNP) markers from nodulation genes are available in common bean and soybean. Genomes of chickpea, pigeonpea, and soybean; and genomes of several rhizobium species are decoded. Expression studies revealed few genes associated with SNF in model and grain legumes. Advances in host plant and rhizobium genomics are helping identify DNA markers to aid breeding of legume cultivars with high symbiotic efficiency. A paradigm shift is needed by breeding programs to simultaneously improve host plant and rhizobium to harness the strength of positive symbiotic interactions in cultivar development. Computation models based on metabolic reconstruction pathways are providing greater insights to explore genotype–phenotype relationships in SNF. Models to simulate the response of N2 fixation to a range of environmental variables and crop growth are assisting researchers to quantify SNF for efficient and sustainable agricultural production systems. Such knowledge helps identifying bottlenecks in specific legume–rhizobia systems that could be overcome by legume breeding to enhance SNF. This review discusses the recent developments to improve SNF and productivity of grain legumes