Whole-Genome Sequencing Of Mesorhizobium huakuii 7653R Provides Molecular Insights into Host Specificity and Symbiosis Island Dynamics

Background Evidence based on genomic sequences is urgently needed to confirm the phylogenetic relationship between Mesorhizobium strain MAFF303099 and M. huakuii. To define underlying causes for the rather striking difference in host specificity between M. huakuii strain 7653R and MAFF303099, several probable determinants also require comparison at the genomic level. An improved understanding of mobile genetic elements that can be integrated into the main chromosomes of Mesorhizobium to form genomic islands would enrich our knowledge of how genome dynamics may contribute to Mesorhizobium evolution in general. Results In this study, we sequenced the complete genome of 7653R and compared it with five other Mesorhizobium genomes. Genomes of 7653R and MAFF303099 were found to share a large set of orthologs and, most importantly, a conserved chromosomal backbone and even larger perfectly conserved synteny blocks. We also identified candidate molecular differences responsible for the different host specificities of these two strains. Finally, we reconstructed an ancestral Mesorhizobium genomic island that has evolved into diverse forms in different Mesorhizobium species. Conclusions Our ortholog and synteny analyses firmly establish MAFF303099 as a strain of M. huakuii. Differences in nodulation factors and secretion systems T3SS, T4SS, and T6SS may be responsible for the unique host specificities of 7653R and MAFF303099 strains. The plasmids of 7653R may have arisen by excision of the original genomic island from the 7653R chromosome

Molecular mechanisms of adaptation emerging from the physics and evolution of nucleic acids and proteins

DNA, RNA and proteins are major biological macromolecules that coevolve and adapt to environments as components of one highly interconnected system. We explore here sequence/structure determinants of mechanisms of adaptation of these molecules, links between them, and results of their mutual evolution. We complemented statistical analysis of genomic and proteomic sequences with folding simulations of RNA molecules, unraveling causal relations between compositional and sequence biases reflecting molecular adaptation on DNA, RNA and protein levels. We found many compositional peculiarities related to environmental adaptation and the life style. Specifically, thermal adaptation of protein-coding sequences in Archaea is characterized by a stronger codon bias than in Bacteria. Guanine and cytosine load in the third codon position is important for supporting the aerobic life style, and it is highly pronounced in Bacteria. The third codon position also provides a tradeoff between arginine and lysine, which are favorable for thermal adaptation and aerobicity, respectively. Dinucleotide composition provides stability of nucleic acids via strong base-stacking in ApG dinucleotides. In relation to coevolution of nucleic acids and proteins, thermostability-related demands on the amino acid composition affect the nucleotide content in the second codon position in Archaea

RNA-Seq and Microarrays Analyses Reveal Global Differential Transcriptomes of <i>Mesorhizobium huakuii</i> 7653R between Bacteroids and Free-Living Cells

<div><p><i>Mesorhizobium huakuii</i> 7653R occurs either in nitrogen-fixing symbiosis with its host plant, <i>Astragalus sinicus</i>, or free-living in the soil. The <i>M. huakuii</i> 7653R genome has recently been sequenced. To better understand the complex biochemical and developmental changes that occur in 7653R during bacteroid development, RNA-Seq and Microarrays were used to investigate the differential transcriptomes of 7653R bacteroids and free-living cells. The two approaches identified several thousand differentially expressed genes. The most prominent up-regulation occurred in the symbiosis plasmids, meanwhile gene expression is concentrated to a set of genes (clusters) in bacteroids to fulfill corresponding functional requirements. The results suggested that the main energy metabolism is active while fatty acid metabolism is inactive in bacteroid and that most of genes relevant to cell cycle are down-regulated accordingly. For a global analysis, we reconstructed a protein-protein interaction (PPI) network for 7653R and integrated gene expression data into the network using Cytoscape. A highly inter-connected subnetwork, with function enrichment for nitrogen fixation, was found, and a set of hubs and previously uncharacterized genes participating in nitrogen fixation were identified. The results described here provide a broader biological landscape and novel insights that elucidate rhizobial bacteroid differentiation, nitrogen fixation and related novel gene functions.</p></div

Schematic view of data from RNA-Seq and Microarrays.

<p>A: Venn diagram summarizing the overlap between differentially expressed genes from RNA-Seq (<i>left</i> circle) and Microarrays (<i>right</i> circle). The number of genes called by both methods is indicated by the overlap between the two circles. It also contains the numbers of up/down-regulated genes for each set in the Venn-Diagram. B: Comparison and correlations analyses of differentially expressed genes in RNA-Seq and Microarrays. The plot shows log2 ratios (bacteroids/free-living cells) of expressed genes in RNA-Seq (<i>x</i> axis) and Microarrays (<i>y</i> axis) (7043 genes in total). Pearson's correlation coefficient (r) is indicated for each comparison. C: Distribution of differentially expressed genes detected by RNA-Seq and Microarrays. Genes detected by RNA-Seq are shown with blue bars. Genes detected by Microarrays are shown with orange bars.</p

The subnetwork of CtrA with interacting proteins in <i>M. huakuii</i> 7653R.

a<p>The protein also in the nitrogen fixation subnetwork.</p>b<p>The protein interacts directly with CtrA.</p

The set of genes in the <i>M. huakuii</i> 7653R subnetwork of nitrogen fixation.

a<p>: The unknown function genes.</p

The putative CtrA regulatory network in <i>M. huakuii</i> 7653R according to the regulation model of cell cycle in <i>C. crescentus</i>.

<p>Blue arrows indicate positive effects and blunt blue lines indicate negative effects. Genes up-regulated were shown in red and down-regulated shown in green in <i>M. huakuii</i> 7653R bacteroids. Phosphorylated CtrA autoregulated its own transcription and CtrA regulated genes associated with DNA replication, DNA methylation, cell division, flagellar assembly, CtrA degradation, cell division, polar morphogenesis and pili biogenesis.</p

An inter-connected, differentially-expressed PPI subnetwork in bacteroids altered for nitrogen fixation.

<p>The genes with differential expression fold change greater than 5 were shown in red; those greater than 20 were shown in dark red; those down-regulated were shown in green and others were shown in yellow.</p

Visualization of global transcriptional profiles as determined by RNA-Seq and Microarrays.

<p>The plot shows a summary of RNA-Seq and Microarrays expression data at all regions in bacteroids. Across the entire genome, the percentages of up-regulated genes of RNA-Seq and Microarrays are shown in yellow and red, while those of the down-regulated shown in gray and blue, respectively. The G+C content is in green. The percentage of differentially expressed genes and the GC content were computed with a sliding window of every 50 genes. Note the most prominent up-regulation in the symbiosis plasmids and the most prominent down-regulation in regions I and II in the chromosome.</p