The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

Data from multilocus enzyme electrophoresis of bacterial populations were analyzed using a statistical test designed to detect associations between genes at different loci. Some species (e.g., Salmonella) were found to be clonal at all levels of analysis. At the other extreme, Neisseria gonorrhoeae is panmictic, with random association between loci. Two intermediate types of population structure were also found. Neisseria meningitidis displays what we have called an "epidemic" structure. There is significant association between loci, but this arises only because of the recent, explosive, increase in particular electrophoretic types; when this effect is eliminated the population is found to be effectively panmictic. In contrast, linkage disequilibrium in a population of Rhizobium meliloti exists because the sample consisted of two genetically isolated divisions, often fixed for different alleles: within each division association between loci was almost random. The method of analysis is appropriate whenever there is doubt about the extent of genetic recombination between members of a population. To illustrate this we analyzed data on protozoan parasites and again found panmictic, epidemic, and clonal population structures.

https://www.pnas.org/content/pnas/90/10/4384.full.pdf

How clonal are bacteria

Common bean (Phaseolus vulgaris) has become a cosmopolitan crop, but was originally domesticated in the Americas and has been grown in Latin America for several thousand years. Consequently an enormous diversity of bean nodulating bacteria have developed and in the centers of origin the predominant species in bean nodules is R. etli. In some areas of Latin America, inoculation, which normally promotes nodulation and nitrogen fixation is hampered by the prevalence of native strains. Many other species in addition to R. etli have been found in bean nodules in regions where bean has been introduced. Some of these species such as R. leguminosarum bv. phaseoli, R. gallicum bv. phaseoli and R. giardinii bv. phaseoli might have arisen by acquiring the phaseoli plasmid from R. etli. Others, like R. tropici, are well adapted to acid soils and high temperatures and are good inoculants for bean under these conditions. The large number of rhizobia species capable of nodulating bean supports that bean is a promiscuous host and a diversity of bean-rhizobia interactions exists. Large ranges of dinitrogen fixing capabilities have been documented among bean cultivars and commercial beans have the lowest values among legume crops. Knowledge on bean symbiosis is still incipient but could help to improve bean biological nitrogen fixation.

Diversity of Rhizobium-Phaseolus vulgaris symbiosis: overview and perspectives

The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

http://science.sciencemag.org/content/sci/293/5530/668.full.pdf

The composite genome of the legume symbiont Sinorhizobium meliloti.

This study examines the potential, magnitude, and causes of enhanced biological N2 fixation (BNF) by common beans (Phaseolus vulgaris L.) through bio-char additions (charcoal, biomass-derived black carbon). Bio-char was added at 0, 30, 60, and 90 g kg−1 soil, and BNF was determined using the isotope dilution method after adding 15N-enriched ammonium sulfate to a Typic Haplustox cropped to a potentially nodulating bean variety (CIAT BAT 477) in comparison to its non-nodulating isoline (BAT 477NN), both inoculated with effective Rhizobium strains. The proportion of fixed N increased from 50% without bio-char additions to 72% with 90 g kg−1 bio-char added. While total N derived from the atmosphere (NdfA) significantly increased by 49 and 78% with 30 and 60 g kg−1 bio-char added to soil, respectively, NdfA decreased to 30% above the control with 90 g kg−1 due to low total biomass production and N uptake. The primary reason for the higher BNF with bio-char additions was the greater B and Mo availability, whereas greater K, Ca, and P availability, as well as higher pH and lower N availability and Al saturation, may have contributed to a lesser extent. Enhanced mycorrhizal infections of roots were not found to contribute to better nutrient uptake and BNF. Bean yield increased by 46% and biomass production by 39% over the control at 90 and 60 g kg−1 bio-char, respectively. However, biomass production and total N uptake decreased when bio-char applications were increased to 90 g kg−1. Soil N uptake by N-fixing beans decreased by 14, 17, and 50% when 30, 60, and 90 g kg−1 bio-char were added to soil, whereas the C/N ratios increased from 16 to 23.7, 28, and 35, respectively. Results demonstrate the potential of bio-char applications to improve N input into agroecosystems while pointing out the needs for long-term field studies to better understand the effects of bio-char on BNF.

http://www.css.cornell.edu/faculty/lehmann/publ/BiolFertSoils%2043,%20699-708,%202007%20Rondon.pdf

Biological nitrogen fixation by common beans (Phaseolus vulgaris L.) increases with bio-char additions

The Rhizobium Genome

Nitrogen fixation gene sequences are reiterated in R. phaseoli. Three different DNA regions containing such sequences were isolated fxom a genome library of strain CFN-42 (C.Quinto et al., 1982). Here we report on the organization of nitrogenase reductase gene (H) sequences.

Reiteration of nitrogen fixation gene sequences in Rhizobium phaseoli

Fifty-one isolates of Rhizobium leguminosarum biovar phaseoli from various geographic and ecological sources, largely in Mexico, were characterized by the electrophoretic mobilities of 15 metabolic enzymes, and 46 distinctive multilocus genotypes (electrophoretic types [ETs]) were distinguished on the basis of allele profiles at the enzyme loci. Mean genetic diversity per enzyme locus among the 46 ETs was 0.691, the highest value yet recorded for any species of bacterium. The occurrence of strong nonrandom associations of alleles over loci suggested a basically clonal population structure, reflecting infrequent recombination of chromosomal genes. Multilocus genotypic diversity was unusually high, with the most strongly differentiated pairs of ETs having distinctive alleles at all 15 loci and major clusters of ETs diverging at genetic distances as large as 0.89. This great diversity in the chromosomal genome raises the possibility that R. leguminosarum biovar phaseoli is a polyphyletic assemblage of strains. As other workers have suggested, the inclusion of all strains capable of nodulating beans in a single biovar or species is genetically unrealistic and taxonomically misleading. A biologically meaningful classification of Rhizobium spp. should be based on assessment of variation in the chromosomal genome rather than on phenotypic characters, especially those mediated for the most part or wholly by plasmid-borne genes, such as host relationships.

https://aem.asm.org/content/aem/54/11/2825.full.pdf

Genetic diversity and relationships among isolates of Rhizobium leguminosarum biovar phaseoli.

Summary: We have previously reported that Rhizobium phaseoli has multiple copies of nitrogen-fixation gene sequences. In this work we extend our analysis to cover a broader range of R. phaseoli strains and other rhizobia isolated from different legumes. Our results indicate that most R. phaseoli strains have reiterated nifH sequences. Reiterations are also found in rhizobia from nodules of other Phaseolus species and from the close relative of Phaseolus, Pachyrhizus. However, nifH gene reiteration is not always found in the rhizobia able to nodulate and fix nitrogen in Phaseolus, since some strains isolated from nodules of Phaseolus vulgaris as well as from other legumes are able to establish an effective symbiosis with Phaseolus vulgaris and do not show reiterations. We propose that there are different evolutionary lines of R. phaseoli. The reiteration of nif genes may be considered a marker for the most abundant and specialized of these evolutionary lines.

Reiteration of Nitrogen Fixation Gene Sequences and Specificity of Rhizobium in Nodulation and Nitrogen Fixation in Phaseolus vulgaris

Rhizobium meliloti nodulation (nod) genes required for specific infection and nodulation of alfalfa have been cloned. Transposon Tn5 mutagenesis defined three nod regions spanning 16 kilobases of the pSym megaplasmid. Genetic and cytological studies of 62 nodulation-defective mutants allowed the assignment of symbiotic developmental phenotypes to common and specific nod loci. Root hair curling was determined by both common (region I) and specific (region III) nod transcription units; locus IIIb (nodH gene) positively controlled curling on the homologous host alfalfa, whereas loci IIIa (nodFE) and IIIb (nodH) negatively controlled curling on heterologous hosts. Region I (nodABC) was required for bacterial penetration and infection thread initiation in shepherd's crooks, and the nodFE transcription unit controlled infection thread development within the alfalfa root hair. In contrast, induction of nodule organogenesis, which can be triggered from a distance, seemed to be controlled by common nodABC genes and not to require specific nod genes nodFE and nodH. Region II affected the efficiency of hair curling and infection thread formation.

Esperanza Martinez

Papers

The Rhizobium Genome

Reiteration of nitrogen fixation gene sequences in Rhizobium phaseoli

Genetic diversity and relationships among isolates of Rhizobium leguminosarum biovar phaseoli.

Reiteration of Nitrogen Fixation Gene Sequences and Specificity of Rhizobium in Nodulation and Nitrogen Fixation in Phaseolus vulgaris

Assignment of symbiotic developmental phenotypes to common and specific nodulation (nod) genetic loci of Rhizobium meliloti.