Rhizobium leguminosarum 7

 

Genospecies E – Rhizobium leguminosarum sensu stricto

Genospecies E includes the type strain of Rhizobium leguminosarum, so it is, by definition, the true R. leguminosarum in the narrow sense.

The last time I looked (25/07/2020), there were 68 gsE genomes in GenBank. The same set of genomes is found in the group with ANI > 97% to the type strain and in a well-separated clade in the phylogeny. They are, in alphabetical order:

R._gallicum_R602sp

R._indigoferae_CCBAU_71042

R._leguminosarum_128C53

R._leguminosarum_23B

R._leguminosarum_3B

R._leguminosarum_4292

R._leguminosarum_4B

R._leguminosarum_ATCC_14479

R._leguminosarum_BIHB_1217

R._leguminosarum_CB1

R._leguminosarum_CC283b

R._leguminosarum_CZF1F8

R._leguminosarum_CZP1G1

R._leguminosarum_CZP1G4

R._leguminosarum_CZP1G9

R._leguminosarum_CZP1H7

R._leguminosarum_CZP3C9

R._leguminosarum_CZP3G7

R._leguminosarum_CZP3H7

R._leguminosarum_FRF1A1

R._leguminosarum_FRF3E2

R._leguminosarum_FRP2D7

R._leguminosarum_FRP3A12

R._leguminosarum_FRP3E11

R._leguminosarum_FRP3E5

R._leguminosarum_FRP3G5

R._leguminosarum_FRP4D11

R._leguminosarum_FRP4H3

R._leguminosarum_FRP5A4

R._leguminosarum_FRP5C5

R._leguminosarum_FRP5D3

R._leguminosarum_FRP5H7

R._leguminosarum_GB51

R._leguminosarum_GD25

R._leguminosarum_IAUb11

R._leguminosarum_JHI1084

R._leguminosarum_JHI944

R._leguminosarum_L113

R._leguminosarum_L125

R._leguminosarum_L324

R._leguminosarum_LCS0306

R._leguminosarum_OV152

R._leguminosarum_P1.14

R._leguminosarum_P2.59

R._leguminosarum_P221

R._leguminosarum_RSP1E11

R._leguminosarum_Rt24.2

R._leguminosarum_SEP2G2

R._leguminosarum_SEP3F1

R._leguminosarum_SEP4B10

R._leguminosarum_SEP5D11

R._leguminosarum_SEP5D7

R._leguminosarum_SM126A_gsE

R._leguminosarum_SM135A_gsE

R._leguminosarum_SM135B_gsE

R._leguminosarum_SM141B_gsE

R._leguminosarum_SM149A_gsE

R._leguminosarum_SM159_gsE

R._leguminosarum_SM160_gsE

R._leguminosarum_SM168A_gsE

R._leguminosarum_SM168B_gsE

R._leguminosarum_SM45_gsE

R._leguminosarum_SM52_gsE

R._leguminosarum_UPM1137

R._leguminosarum_UPM791

R._leguminosarum_UPM988

R._leguminosarum_USDA_2370_GCF_003058385.1

R._leguminosarum_USDA_2370_GCF_002008365.1

The last two entries are two genomes for the type strain, sequenced by different groups. Fortunately, they are more or less identical (ANI 99.9981). More problematic are the first two entries on the list, which are labelled as different species altogether. R. indigoferae CCBAU 71042 is a well known problem. The type strain of this species, which should be related to R. gallicum according to the original publication, has been lost and replaced by a strain of R. leguminosarum. This has been discussed by the Taxonomy Subcommittee (https://doi.org/10.1099/ijsem.0.002974 minute 18). Now we have a complete genome sequence to prove that the available culture is R. leguminosarum, not R. indigoferae! The R. gallicum genome seems to be a different kind of mistake. This genome (GCF_000373025.1) was published by JGI, who seem to have got hold of the wrong strain. Fortunately, the real R. gallicum type strain R602 has been sequenced by CCG UNAM and is available as GCF_000816845.1.

That leaves genome sequences of 65 different known strains of genospecies E. The majority of these are symbiovar viciae, although the SM strains are sv. trifolii from our Danish project (Cavassim et al. 2020, https://doi.org/10.1099/mgen.0.000351). Since we already know the genospecies of all strains from that study, this has been appended to their names to reassure us that we are looking at the right genospecies.

Genospecies E is shown below in an extract from the phylogeny based on 120 genes. It is clearly defined by a long, well supported branch. There is a very tight group around the type strain (USDA2370), and some looser groups, matching the pattern of the ANI values that we saw in the previous post. I think the strains in the tight group are all symbiovar viciae, except for 4292 (phaseoli), but they are from many different places. There is a clade of trifolii strains at the bottom, mostly Danish but a couple from Russia. The other strains are a mix of viciae, trifolii and phaseoli, and sometimes closely related strains are of different symbiovars, e.g. 23B (trifolii) and P1.14 (viciae), SM45 (trifolii) and UPM1137 (viciae).

 

Despite their diverse geographic origins and various symbiovars, the strains in gsE form a tight group, easily distinguished by phylogeny and ANI from any other strains. Whatever the mechanisms that created and maintained it, genospecies E is a real biological entity that deserves recognition. If the rest of the Rlc is as clear as this, our job will soon be done! Next time, I’ll look at gsD.

Rhizobium leguminosarum 6

 

Rhizobium anhuiense

Before we get stuck into the Rhizobium leguminosarum species complex (Rlc), let us have a quick look at its sister species, R. anhuiense. So far, this is a very compact clade that is on a long branch and clearly forms a single species, with all strains having ANI  > 98 with the type strain. Here is an excerpt from the 120-gene phylogeny:

 

Here is the list of genomes that are included in R. anhuiense, with their ANI to the type strain:

R._anhuiense_CCBAU_23252_GCF_003985145.1.fna	100.00
R._anhuiense_S10_GCF_002531655.1.fna	98.92
R._anhuiense_J3_GCF_002531645.1.fna	98.91
R._sp._BK398_BK398_GCF_004343265.1.fna	98.88
R._anhuiense_Y27_GCF_002531615.1.fna	98.87
R._anhuiense_JX3_GCF_002531635.1.fna	98.87
R._anhuiense_CCBAU43229_GCF_012276505.1.fna	98.82
R._anhuiense_C15_GCF_002531695.1.fna	98.81
R._anhuiense_WYCCWR10015_GCF_001632995.1.fna	98.79
R._tropici_CF286_GCF_000800135.1.fna	98.43
R._leguminosarum_CF307_GCF_000799945.1.fna	98.40

 

This homogeneity is surprising given their diverse origins. The type strain, CCBAU23252, is a symbiont of Vicia faba, as is CCBAU43229, whereas WYCCWR10015 nodulates Trifolium repens, and C15, S10, J3, JX3, Y27 are all Phaseolus vulgaris symbionts. They are all from China, but represent three symbiovars. Strains CF286 and CF307 were isolated by the JGI from Populus root endosphere, presumably in the USA. They were published before R. anhuiense was defined, but it is not clear why they were assigned to different species when they are so similar. The origin of BK398 is unspecified, but it is probably also from Populus. There are also three assemblies from a JGI study of single cells from Arabidopsis roots that fall in the R. anhuiense clade, but the genomes are very incomplete and error-prone, so they are not shown in the phylogeny.

R. anhuiense seems to be widespread as a legume symbiont across China, with various symbiovars, but I am not aware of any reports of symbionts from outside China. Has anyone else seen it? The fact that it keeps cropping up in non-legume rhizospheres in the USA suggests that it might have a much wider distribution.

From now on, we will turn our attention to the Rlc itself.

Rhizobium leguminosarum 5

 

Average Nucleotide Identity

In recent years, genome-based ANI has taken over from the old laboratory technique of DNA-DNA hybridisation (DDH) as the main criterion for deciding whether two strains belong to the same species. It was designed to mimic certain features of DDH (an approach that was developed in the 1960s, before DNA sequencing had been invented). The principle is to identify all stretches of DNA in one genome that are deemed to ‘match’ sequences in a second genome, and to calculate the percentage of identical nucleotides in these stretches. Like DDH, the set of sequences used is different for every pairwise comparison, because it is a mixture of universal core genes and those accessory genes that happen to be in common between the two strains. Like DDH, there is a threshold value above which two strains are considered to be conspecific – this is somewhere around 95-96%. Like DDH, there is also another arbitrary threshold that is hidden and seldom discussed, namely the threshold similarity that is used to decide whether to compare two stretches of sequence in the first place. Like DDH, there is no standard method for calculating ANI, and different methods may give slightly different results (Palmer et al. 2020,  https://doi.org/10.1099/ijsem.0.004124). Like DDH, the results are not expected to be exactly symmetric (the ANI of A to B is not the same as the ANI of B to A). 

In my view, a phylogeny based on a sufficiently large number of core genes is a safer basis for taxonomy than ANI, particularly in bacteria such as rhizobia that have large and variable accessory genomes, but it is important to look at ANI because it provides a different perspective and is widely used in bacterial taxonomy. I chose to use FastANI (Jain et al. 2018, https://doi.org/10.1038/s41467-018-07641-9) because it really is fast and gives comparable results to other methods in the critical 90-100% range (Palmer et al. 2020).

 

 

 

Here is a plot of ANI, ranked from highest to lowest, for 765 genomes labelled “Rhizobium” in GenBank, compared to USDA2370, the type strain of R. leguminosarum (which, in turn, is the type species of the genus Rhizobium). ANI does a good job of separating different levels of relatedness within Rhizobium, reflecting exactly the nested clades we observed in the phylogeny in post 3. USDA2370 belongs to genospecies E, and all other gsE genomes have ANI > 97.40. Then there is a clear gap before the gsD genomes (ANI 94.54 – 95.00). We know that gsD and gsE are closely related (Cavassim et al. 2020, https://doi.org/10.1099/mgen.0.000351). After another gap, the rest of the genospecies of the R. leguminosarum species cluster (Rlc) form a continuous group (ANI 92.15 – 93.52). There is another gap followed by 11 genomes of R. anhuiense (ANI 91.33 – 91.55). We saw in the phylogeny that this species is the sister group of the Rlc. There is then a large gap punctuated by a single spot. This is Rhizobium sp. L43 (ANI 90.18), a strain with no close relatives that was also the sister taxon of the Rlc-anhuiense clade in the phylogeny.

Tong et al. (2018, https://doi.org/10.1016/j.syapm.2018.03.001) have previously shown that R. anhuiense is the sister taxon of the Rlc, based on a phylogeny of 1458 shared genes, and their ANI and dDDH values support this. Their highest ANI and dDDH values for strain L43 were with R. anhuiense and the Rlc, but in their phylogeny it is not closest neighbour of these taxa, so the affinities of this strain need more investigation (but not now, since it is clearly outside the Rlc). Incidentally, the phylogeny that Tong et al. obtained from concatenated atpD-glnII-recA (their Fig. 3) shows L43 within the Rlc, illustrating the hazards of basing taxonomy on just a few genes.

Returning to our ANI plot, the rest of the leguminosarum/etli clade (with multiple species) has ANI 86.68 – 89.32. After that is a large drop to R. alamii at ANI 83.16, the sister taxon of the leguminosarum-etli clade, reflecting a long branch in the tree. After that, there are no more breaks in ANI, even at the boundary of the genus Rhizobium. Indeed, there is some overlap in ANI values between strains within Rhizobium and those in related genera. This seems a little surprising, considering that the phylogeny shows a long, well supported branch at the base of the genus, but ANI (and especially FastANI) is not very sensitive at these lower values. ANI also suffers from the general problem with pairwise distance measures, that slowly evolving lineages (with short branches) can give high similarity scores even if they are phylogenetically distant.

In summary, ANI fully corroborates the phylogeny and can identify clear breakpoints that define genospecies, the Rlc, and higher groupings within the genus Rhizobium. FastANI is not, however, useful for defining the genus boundary. In the following posts, we will look at each of the genospecies of the Rlc and the strains that are included in them.

Rhizobium leguminosarum 4

 

What’s in a name?

Last time, we saw that Rhizobium leguminosarum, in the traditional sense, is a very clearly defined clade in the genomic phylogeny. We could just go on treating this as a single species, with the reassurance that it is clearly defined. This would save a lot of trouble, because people with new isolates would just have to check that they were in this clade. If they were, they could safely call them R. leguminosarum, as we always did in the past, and need not enquire further. Job done!

The problem is that this broadly-defined group has too much diversity to fit comfortably within one species. As we showed in our first genome-based survey, ANI values between strains in this group can be as low as 93%, and the strains clustered in a number of tighter clades that corresponded more closely to the species that people were defining in other bacteria – we called them genospecies A to E (Kumar et al. 2015, http://dx.doi.org/10.1098/rsob.140133).

In response to these low ANI values (or the antiquated DNA-DNA hybridisation that ANI has replaced), a number of crews have already moved in and carved off chunks of the R. leguminosarum species complex (Rlc), setting them up as separate species. First we had R. laguerreae (Saïdi et al. 2014, https://doi.org/10.1099/ijs.0.052191-0), then R. sophorae (Jiao et al. 2015, https://doi.org/10.1099/ijs.0.068916-0), and this year we have already had R. ruizarguesonis (Jorrin et al. 2020, https://doi.org/10.1016/j.syapm.2020.126090) and R. indicum (Rahi et al. 2020, https://doi.org/10.1016/j.syapm.2020.126127). All these new species are perfectly justifiable and were well described in accordance with the conventions of bacterial taxonomy. In each case, there were several (3 to 6) strains assigned to the new species. The remnants of R. leguminosarum are not really a viable taxonomic unit without these chunks, though, so there is now a strong motivation to sort the situation out.

The standard approach to bacterial taxonomy is what I call “flag sticking”. Back in the bad old days of European imperialism, sea captains ranged over the oceans without proper maps and when they found a piece of coastline that hadn’t been claimed (by other Europeans), they stuck their national flag on it and gave it a name. Often, they didn’t really know how big the territory was that they were claiming – was it Brazil, or was it just Bermuda? Similarly, bacterial taxonomists happen upon a strain that hasn’t been “claimed”, because its similarity to all published type strains is less than the magic number, so they make it the type strain of a new species. There is no obligation, or even expectation, that they will attempt to define the boundaries of the territory that they are claiming. The better studies include several strains, but the rules allow a species to be based on a single isolate. Typical bacterial taxonomy is, in fact, the taxonomy of bacterial type strains. The result is a map on which the capital cities are clearly marked, but the international borders are very hazy.

What we really need is a satellite image of the entire landscape, so that we can see where the landmasses are, and whether there are clear natural boundaries between them. Now that we have hundreds of genomes, we are beginning to see such an image. Of course, it has some serious limitations because the genomes that have been sequenced were selected from a limited range of sources. There are undoubtedly parts of the landscape that are blank because no satellite has yet passed over them. However, as we will see in the following posts, there are some clearly defined territories (genospecies) within the Rlc, and some are much larger (represented by more genomes) than others. We will discover that R. ruizarguesonis is Brazil, while R. sophorae is Bermuda and R. laguerreae is Indonesia.

In the next post, I’ll have a look at what ANI (Average Nucleotide Identity) can tell us about the Rlc.

Rhizobium leguminosarum 3

A preliminary genome-based phylogeny

The most usual way to make a genome-based phylogeny is to select a set of representative genes, concatenate their sequences, and apply standard phylogenetic methods. The genes used should, if possible, be (a) present in all the genomes, (b) present in a single copy per genome, (c) unlikely to have a history of horizontal gene transfer. There need to be enough genes so that the consensus view will outvote any anomalous behaviour of individual genes. Many different sets of genes have been proposed for this purpose and used in the literature. Here are a few.

(1)   In our paper that defined chromids, Peter Harrison found 305 genes that were shared by all chromid-bearing bacteria (Harrison et al. 2010, http://dx.doi.org/10.1016/j.tim.2009.12.010), and we used that set in our first study of Rlc genome diversity (Kumar et al. 2015, http://dx.doi.org/10.1098/rsob.140133) and for a phylogeny of the 196 genomes in our recent work (Cavassim et al. 2020, https://doi.org/10.1099/mgen.0.000351).

(2)   Parks et al. (2018, http://dx.doi.org/10.1038/nbt.4229) used 120 ubiquitous single-copy proteins for a taxonomy covering 94,759 genomes across the whole domain of Bacteria. They set up the Genome Taxonomy Database (GTDB http://gtdb.ecogenomic.org/, described in a recent article by Parks et al., 2020, https://www.nature.com/articles/s41587-020-0501-8), which is interesting because it proposes taxon boundaries based on normalised phylogenetic depth, and recognises a number of “species” within the Rlc. At some point, we will need to check how their classification maps onto the genospecies structure of the Rlc. Their “species” boundaries may prove useful in deciding on ambiguous cases.

(3)   Na et al. (2018, http://dx.doi.org/10.1007/s12275-018-8014-6) defined a set of 92 genes as their up-to-date bacterial core gene set (UBCG) and provide tools for using them (https://www.ezbiocloud.net/tools/ubcg).

Undoubtedly there is considerable overlap among these sets, and undoubtedly there are many other comparable sets proposed in the literature. Any reasonably large set of core genes will probably give a similar phylogeny, so the choice is probably not critical, but it would be good to try more than one.

For a first try, I have used the 120 Parks proteins. However, I used the DNA sequences rather than amino acid sequences, because we want to resolve differences within a single genus, not to construct a tree for all bacteria. I first checked for recent uploads and found that there were now 834 Rhizobium genomes (on 25/07/2020). There were 797 genome assemblies that had all 120 genes, and this is the phylogeny I got:

 

The clade on a yellow background is the genus Rhizobium, as currently defined. I have indicated three major clades within it: tropici, gallicum and leguminosarum/etli. On a green background is the Rhizobium leguminosarum complex (Rlc). The great news is that this is very clearly defined, on a long branch (arrowed), so there is no ambiguity about which strains belong to it and which do not.

From now on, our main focus is going to be on the Rlc, but it is worth spending a little time looking at the rest of this tree. The Rlc has a very clear sister taxon, R. anhuiense, with 11 genomes in a tight group. There are many species and a great deal of complexity in the rest of the Rhizobium genus that needs to be sorted out at some future date. At the moment, the assemblies have the taxon names that are assigned to them in GenBank, and many of these are clearly incorrect. There are many genomes labelled “R. leguminosarum” that are outside the Rlc in other parts of the leguminosarum/etli clade. For example, CF307 is R. anhuiense, WSM2012 is R. hidalgonense, WSM2304 is R. acidisoli, CCGM1 is R. phaseoli. The outgroups in black and grey are other genera in the Rhizobium/Agrobacterium group, but they are very incomplete because these are only the genomes that are mislabelled “Rhizobium” in GenBank! There are many more genomes available under their proper genus names, apart from Allorhizobium, which NCBI has decided does not exist so calls them all Rhizobium. The genus names are as assigned in GTDB. The temporary label g__Rhizobium_A is for a genus that does not currently have a name, but includes straminoryzae, rhizoryzae and pseudoryzae (though not oryzae, which is in Neorhizobium). All these genera need some sorting out now that many genomes are available, but we must not get distracted from the Rlc. In the next post, I’ll consider whether Rhizobium leguminosarum is a species or a species complex.

Rhizobium leguminosarum 2

 

Finding the genome sequences

I downloaded all the genome sequences for Rhizobium (taxid 379) from NCBI. I used a very handy script called genbank_get_genomes_by_taxon.py that is distributed as part of the pyani package (http://widdowquinn.github.io/pyani/). When I first tried this on 12 February 2020 there were 682 genomes. By 24 April there were 799. Since then, there have been a few more, but my first analyses use the 24 April set.

Why did I search for Rhizobium and not just Rhizobium leguminosarum? Because the taxonomic labels on database entries cannot be trusted. If we want to be sure of finding all the genomes in the R. leguminosarum cluster (Rlc), we don’t want to miss those labelled (quite legitimately) R. laguerreae or just Rhizobium sp. On the other hand, there are many genomes that are labelled R. leguminosarum although they are way outside the Rlc. These may reflect changes in taxonomy, or simply be misidentified. I assumed that authors would at least get the genus right, although this turned out not to be completely true. The   download included an outlier labelled Rhizobium tropici NFR14 (GCA_00300175) that is actually a Bradyrhizobium!

Of the other 798 downloaded genomes, 388 were described as R. leguminosarum, 231 as Rhizobium sp., and the remainder were assigned to 66 other named Rhizobium species, except for one strain that was mysteriously labelled “Pseudomonas sp. SLBN-2”, although the GenBank taxonomy correctly placed it in Rhizobium.

This is an extraordinary wealth of information, particularly for R. leguminosarum, so we should be able to do some serious genome-based taxonomy. First, we need to check whether the genome sequences are consistent with the taxonomic names they have been given. I’ll tackle that in the next post, and we’ll get a first hint as to whether R. leguminosarum is a definable group within the genus.

Towards an understanding of Rhizobium leguminosarum

I have been trying to understand the diversity of Rhizobium leguminosarum  for most of my career. When I first started work on R. leguminosarum in the 1980s, our concept of this species was in the middle of a radical change. Until then, Rhizobium species had been defined by their host range, so strains nodulating peas and related legumes were R. leguminosarum, those nodulating clover were R. trifolii, and those nodulating Phaseolus beans were R. phaseoli. However, the taxonomic evidence indicated that they were not really distinct apart from their host range, so D. C. Jordan proposed (in Bergey’s Manual, 1984) that they be amalgamated as three biovars within a single species, R. leguminosarum. My colleagues at the John Innes Institute had provided genetic evidence to support this, since they had converted “R. phaseoli” and “R. trifolii” into “R. leguminosarum” by transferring a symbiosis plasmid (Johnston et al. 1978. Nature 276, 634–636). My own contribution was to provide evidence from population genetics: I showed that isolates from pea, clover and bean shared the same pool of genetic variation (Young 1985, https://doi.org/10.1099/00221287-131-9-2399). The concept of biovars as symbiosis phenotypes that can be transferred horizontally between strains has become firmly established and we see that it applies to rhizobia in general, not just R. leguminosarum. They are now called ‘symbiovars’ because they are specifically concerned with symbiosis, and it is likely that other ‘adaptive packages’ exist.

Much has changed since then. In 1993, R. etli was split off from R. leguminosarum (Segovia et al. 193, https://doi.org/10.1099/00207713-43-2-374), and many other species in the leguminosarum-etli clade have been defined since. In 2006, we published the first genome sequence of an R. leguminosarum strain (Young et al. 2006, https://doi.org/10.1186/gb-2006-7-4-r34). The sequence was determined by an expert team at the Sanger Institute, using Sanger sequencing (of course). It took three years and cost £350 000. We never really considered sequencing the type strain because it was geneticists who needed the genome sequence, not taxonomists, and nobody had ever used the type strain for any genetic study. We discussed whether to sequence the strain 8401pRL1 (now called A34), because this had been used most extensively for genetic studies, but it was one of the “artificial” strains created by substituting the symbiosis plasmid, and I argued that we needed something closer to “natural” as the representative of the species, so we settled on strain 3841, which is derived from the field isolate 300 with just a spontaneous mutation to streptomycin resistance.

That genome sequence has been a very important reference point, but at that high cost we were clearly not going to get a lot more genomes. The next game-changer was the development of next-generation sequencing. In late 2005, when the genome sequence had been submitted for pubication, I wrote a grant proposal to explore the genes shared by different rhizobia that were living together, using microarrays. By the time this work was eventually funded, our university had acquired a 454 sequencer, and my postdoc Xavier Bailly was  smart enough to suggest that we diverted the project from microarrays to genome sequencing. We published the Sinorhizobium medicae part of the study in 2011 (Bailly et al. 2011, http://dx.doi.org/10.1038/ismej.2011.55), but the R. leguminosarum results were more complex and did not appear until 2015 (Kumar et al. 2015, http://dx.doi.org/10.1098/rsob.140133). Despite very low genome coverage (all we could afford!), they delivered a new view of R. leguminosarum as a complex of related but distinct genospecies. Later work has shown that the five genospecies that we found in a single square metre are, in fact, widespread in Europe (Cavassim et al. 2020, https://doi.org/10.1099/mgen.0.000351) and that, unsurprisingly, there are more than five genospecies in the complex (Boivin et al. 2020, https://doi.org/10.1111/nph.16392).

As a result of recent hard work by many different people, there are now more than 800 Rhizobium genomes in the public databases, of which about half are in the R. leguminosarum complex. We are in the midst of another revolution in our understanding of this species, and I think the time is right to develop a new definition of the R. leguminosarum complex (Rlc) and the genospecies within it, based on genome sequences.

I have already started some analyses of the available genome data, and I aim to share some of these in future posts to this blog. Eventually, I will assemble the more convincing analyses into a formal publication, but I see this as a joint enterprise by the R. leguminosarum research community, and I expect the publication to have many authors. This is, if you like, an experiment in “open research”, and I welcome comments, criticisms and suggestions, as well as offers of help, from anyone who is interested in joining in.

In my next post, I will look at the set of genomes that is available.

These posts are also available on my rhizobium blog at https://rhizobium.wordpress.com/