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Abstract: Phage ΦM12 is an important transducing phage of the nitrogen-fixing rhizobial bacterium Sinorhizobium meliloti. Here we report the genome, phylogenetic analysis, and proteome of ΦM12, the first report of the genome and proteome of a rhizobium-infecting T4-superfamily phage. The structural genes of ΦM12 are most similar to T4-superfamily phages of cyanobacteria. ΦM12 is the first reported T4-superfamily phage to lack genes encoding class I ribonucleotide reductase (RNR) and exonuclease dexA, and to possess a class II coenzyme B12-dependent RNR. ΦM12’s novel collection of genes establishes it as the founder of a new group of T4-superfamily phages, fusing features of cyanophages and phages of enteric bacteria. [Copyright &y& Elsevier]

Summary: The formation of nitrogen‐fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen‐fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin‐motif receptor‐like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild‐type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild‐type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan‐defective strains was achieved by in trans rescue with a Nod factor‐deficient S. meliloti mutant. While the Nod factor‐deficient strain was always more abundant inside nodules, the succinoglycan‐deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules. Significance Statement: This work should be of interest across the field of plant–microbe endosymbioses by providing insights into key determinants of both bacteria and plants for successful microbial host invasion. It is timely with much current interest in symbiotic roles of plant LysM‐RLK proteins and the evolutionary origins of nitrogen‐fixing endosymbiosis. MtLYK10 is a Medicago truncatula component that specifically intervenes in rhizobial infection, independently of succinoglycan recognition. [ABSTRACT FROM AUTHOR]

Bacteriophages of nitrogen-fixing rhizobial bacteria are revealing a wealth of novel structures, diverse enzyme combinations and genomic features. Here we report the cryo-EM structure of the phage capsid at 4.9–5.7 Å-resolution, the phage particle proteome, and the genome of the Sinorhizobium meliloti -infecting Podovirus ΦM5. This is the first structure of a phage with a capsid and capsid-associated structural proteins related to those of the LUZ24-like viruses that infect Pseudomonas aeruginosa . Like many other Podoviruses, ΦM5 is a T = 7 icosahedron with a smooth capsid and short, relatively featureless tail. Nonetheless, this group is phylogenetically quite distinct from Podoviruses of the well-characterized T7, P22, and epsilon 15 supergroups. Structurally, a distinct bridge of density that appears unique to ΦM5 reaches down the body of the coat protein to the extended loop that interacts with the next monomer in a hexamer, perhaps stabilizing the mature capsid. Further, the predicted tail fibers of ΦM5 are quite different from those of enteric bacteria phages, but have domains in common with other rhizophages. Genomically, ΦM5 is highly mosaic. The ΦM5 genome is 44,005 bp with 357 bp direct terminal repeats (DTRs) and 58 unique ORFs. Surprisingly, the capsid structural module, the tail module, the DNA-packaging terminase, the DNA replication module and the integrase each appear to be from a different lineage. One of the most unusual features of ΦM5 is its terminase whose large subunit is quite different from previously-described short-DTR-generating packaging machines and does not fit into any of the established phylogenetic groups. [ABSTRACT FROM AUTHOR]

Abstract: ΦM12 is the first example of a T=19l geometry capsid, encapsulating the recently sequenced genome. Here, we present structures determined by cryo-EM of full and empty capsids. The structure reveals the pattern for assembly of 1140 HK97-like capsid proteins, pointing to interactions at the pseudo 3-fold symmetry axes that hold together the asymmetric unit. The particular smooth surface of the capsid, along with a lack of accessory coat proteins encoded by the genome, suggest that this interface is the primary mechanism for capsid assembly. Two-dimensional averages of the tail, including the neck and baseplate, reveal that ΦM12 has a relatively narrow neck that attaches the tail to the capsid, as well as a three-layer baseplate. When free from DNA, the icosahedral edges expand by about 5nm, while the vertices stay at the same position, forming a similarly smooth, but bowed, T=19l icosahedral capsid. [Copyright &y& Elsevier]

Background: We have used the genomic data in the Integrated Microbial Genomes system of the Department of Energy's Joint Genome Institute to make predictions about rhizobial open reading frames that play a role in nodulation of host plants. The genomic data was screened by searching for ORFs conserved in α-proteobacterial rhizobia, but not conserved in closely-related non-nitrogen-fixing α-proteobacteria. Results: Using this approach, we identified many genes known to be involved in nodulation or nitrogen fixation, as well as several new candidate genes. We knocked out selected new genes and assayed for the presence of nodulation phenotypes and/or nodule-specific expression. One of these genes, SMc00911, is strongly expressed by bacterial cells within host plant nodules, but is expressed minimally by free-living bacterial cells. A strain carrying an insertion mutation in SMc00911 is not defective in the symbiosis with host plants, but in contrast to expectations, this mutant strain is able to out-compete the S. meliloti 1021 wild type strain for nodule occupancy in co-inoculation experiments. The SMc00911 ORF is predicted to encode a "SodM-like" (superoxide dismutase-like) protein containing a rhodanese sulfurtransferase domain at the N-terminus and a chromate-resistance superfamily domain at the C-terminus. Several other ORFs (SMb20360, SMc01562, SMc01266, SMc03964, and the SMc01424-22 operon) identified in the screen are expressed at a moderate level by bacteria within nodules, but not by free-living bacteria. Conclusions: Based on the analysis of ORFs identified in this study, we conclude that this comparative genomics approach can identify rhizobial genes involved in the nitrogen-fixing symbiosis with host plants, although none of the newly identified genes were found to be essential for this process. [ABSTRACT FROM AUTHOR]