Rhizobium meliloti competitiveness and the alfalfa agglutinin

We have isolated two types of isolates having identical colony morphologies from stock cultures of two different Rhizobium meliloti strains. One isolate was agglutinated at a high-dilution titer (HA, highly agglutinable) of the alfalfa agglutinin and was sensitive to phage F20, and the other was agglutinated at a lower agglutinin titer (LA) and was sensitive to phage 16B. All LA isolates from the original slant produced nodules on alfalfa earlier than did HA strains from the original slant. When these HA and LA strains were mixed and used as the inoculum in both vermiculite and field soil in the laboratory, LA strains were always the predominant strains recovered from the nodules. LA strains were obtained from HA cells by selection for resistance to phage F20, and HA strains were obtained from LA cells by selection for resistance to phage 16B. All of the strains with the HA phenotype that were derived from LA strains by phage selection had the nodulation properties of the HA strains from the original slant. Two classes of strains with the LA phenotype were obtained from HA cells by phage selection. One was identical to the original LA strains from the slant, and the other had the nodulation properties of the HA strains. Thus, we have shown that some cell surface properties change the nodulation abilities of R. meliloti strains and, furthermore, that specific phages can be used to enrich for more competitive rhizobia.     

The NodA proteins of Rhizobium meliloti and Rhizobium tropici specify the N-acylation of Nod factors by different fatty acids

Rhizobia synthesize mono- N -acylated chitooligosaccharide signals, called Nod factors, that are required for the specific infection and nodulation of their legume hosts. The biosynthesis of Nod factors is under the control of nodulation ( nod ) genes, including the nodABC genes present in all rhizobial species. The N -acyl substitution can vary between species and can play a role in host specificity. In Rhizobium meliloti , an alfalfa symbiont, the acyl chain is a C16 unsaturated or a (ω-1) hydroxylated fatty acid, whereas in Rhizobium tropici , a bean symbiont, it is vaccenic acid (C18:1). We constructed R. meliloti derivatives having a non-polar deletion of nodA , and carrying a plasmid with either the R. meliloti or the R. tropici nodA gene. The strain with the R. tropici nodA gene produced Nod factors acylated by vaccenic acid, instead of the C16 unsaturated or hydroxylated fatty acids characteristic of R. meliloti Nod factors, and infected and nodulated alfalfa with a significant delay. These results show that NodA proteins of R. meliloti and R. tropici specify the N -acylation of Nod factors by different fatty acids, and that allelic variation of the common nodA gene can contribute to the determination of host range.     

Rhizobium meliloti distribution in the soil following alfalfa inoculation

Summary Alfalfa seeds, inoculated with an antibiotic-resistantRhizobium meliloti strain, were planted in three replicated field plots at Clayton, N.C. Core samples were taken three times in the next year at 0, 10, and 20 cm from the edge of each plot. Soil subsamples were taken from within each core sample at 0, 6, 12, and 18 cm depths. The numbers of the inoculum Rhizobium strain in each soil subsample were determined by inoculation of alfalfa plants with diluted soil samples. In general the distribution of rhizobia showed some movement outward and downward in the soil. Lower counts were obtained at the surface during summer. The Rhizobium persistence pattern in the soil differed in the three plots which is consistent with the variability in Rhizobium numbers often observed in established alfalfa stands. Cooperative investigation of the United States Department of Agriculture, Science and Education Administration, Agricultural Research and the North Carolina Agricultural Research Service, Raleigh, North Carolina. Paper No. 6818 of the Journal Series of the North Carolina Agricultural Research Service at Raleigh.     

Structural modifications in Rhizobium meliloti Nod factors influence their stability against hydrolysis by root chitinases

Acylated chitooligosaccharide signals (Nod factors) trigger the development of root nodules on leguminous plants and play an important role in determining host specificity in the Rhizobium-plant symbiosis. Here, the ability of plant chitinases to hydrolyze different Nod factors and the potential significance of the structural modifications of Nod factors in stabilizing them against enzymatic inactivation were investigated. Incubation of the sulfated Nod factors of Rhizobium meliloti, NodRm-IV(S) and NodRm-V(S), as well as their desulfated derivatives NodRm-IV and NodRm-V, with purified chitinases from the roots of the host plant Medicago and the nonhost plant Vicia resulted in the release of the acylated lipotrisaccharide NodRm-III from NodRm-V, NodRm-IV and NodRm-V(S), whereas NodRm-IV(S) was completely resistant to digestion by both chitinases. Kinetic analysis showed that the structural parameters determining host specificity, the length of the oligosaccharide chain, the acylation at the nonreducing end and the sulfatation at the reducing end of the lipooligosaccharide, influence the stability of the molecule against degradation by chitinases. When the Nod factors were incubated in the presence of intact roots of Medicago, as well as of Vicia, the acylated lipotrisaccharide was similarly released in vivo from all Nod factors except NodRm-IV(S). In addition, a dimer-forming activity was observed in intact roots which also cleaved NodRm-IV(S). This activity was much greater in Medicago than in Vicia and increased upon incubation. The initial overall degradation rate of the Nod factors on Medicago was inversely correlated with their biological activities on Medicago roots. These results open the possibility that the activity of Nod factors on Medicago may partly be determined by the action of chitinases.     

Developmental fate of Rhizobium meliloti bacteroids in alfalfa nodules.

Nitrogen-fixing bacteroids are degraded during nodule senescence. This is in contrast to recent implications that viable bacteroids can be released into soil from legume nodules. Rhizobia originating from persistent infection threads in senescing nodule plant cells seem to be the source of viable cells required for perpetuation of the Rhizobium spp. population in the soil. Our conclusions were derived from electron microscopic examination of stages of development and senescence of alfalfa root nodules.     

Interaction of nod and exo Rhizobium meliloti in alfalfa nodulation

Among the genes of Rhizobium meliloti SU47 that affect nitrogen-fixing symbiosis with alfalfa are nod genes, in which mutations block nodule induction, and exo genes, in which mutations allow nodule formation but block rhizobial exopolysaccharide production as well as nodule invasion and nitrogen fixation. To investigate whether an exo+ bacterium can "help" (that is, reverse the symbiotic defect of) an exo mutant in trans, we have coinoculated alfalfa with pairs of rhizobia of different genotypes. Coinoculant genotypes were chosen so that the exo+ helper strain was nif while the exo "indicator" strain was nif+, and thus any fixation observed was carried out by the exo coinoculant. We find that a nod exo+ coinoculant can help an exo mutant both to invade nodules and to fix nitrogen. However, a nod+ exo+ coinoculant cannot help an exo mutant: Few exo bacteria are recovered from nodules, some bacteroids differentiate into bizarre aberrant forms, and the nodules fail to fix nitrogen. In a triple coinoculation, the effect of nod+ helper supersedes that of nod helper. Implications of these results for interaction of nod and exo gene products are discussed.     

根瘤菌结瘤因子的结构和功能

结瘤因子是根瘤菌分泌的寡糖,它作为外在信号,诱发宿主植物根部各种生理反应。引起根毛变形,诱导皮层细胞分裂,形成根瘤原基,作者主要就这一早期结瘤过程中结瘤因子的结构和功能作一综述。     

The exoD gene of Rhizobium meliloti encodes a novel function needed for alfalfa nodule invasion

During the symbiotic interaction between alfalfa and the nitrogen-fixing bacterium Rhizobium meliloti, the bacterium induces the formation of nodules on the plant roots and then invades these nodules. Among the bacterial genes required for nodule invasion are the exo genes, involved in production of an extracellular polysaccharide, and the ndv genes, needed for production of a periplasmic cyclic glucan. Mutations in the exoD gene result in altered exopolysaccharide production and in a nodule invasion defect. In this work we show that the stage of symbiotic arrest of exoD mutants is similar to that of other exo and ndv mutants. However, the effects of exoD mutations on exopolysaccharide production and growth on various media are different from the effects of other exo and ndv mutations. Finally, exoD mutations behave differently from other exo mutations in their ability to be suppressed or complemented extracellularly. The results suggest that exoD represents a new class of Rhizobium genes required for nodule invasion, distinct from the other exo genes and the ndv genes. We discuss models for the function of exoD.     

NoIR controls expression of the Rhizobium meliloti nodulation genes involved in the core Nod factor synthesis

The synthesis of Rhizobium meliloti Nod signal molecules, encoded by the nod gene products, is finely regulated. A negative control of plasmid-borne nod gene expression is provided by the NoIR repressor encoded by the chromosomal noIR gene. NoIR was previously shown to downregulate the expression of the activator nodD1 gene and the common nodABC operon by binding to an overlapping region of the two promoters adjacent to the n1 nod-box (Kondorosi et al., 1989). We demonstrate here that NoIR also controls the expression of two additional genes, nodD2 and nodM, but does not directly regulate the expression of the host-specific nod genes located downstream of the n2, n3 and n5 nod-boxes. Thus, the nod genes are differentially regulated by NoIR and only those providing common nodulation functions, by determining the synthesis of the core Nod factor structure, are subjected to this negative regulation. Furthermore, NoIR has a strong negative effect on the production of Nod metabolites, the level of which may serve as a fine-tuning mechanism for optimal nodulation, specific to host-plant genotypes. In addition, it elicits preferential synthesis of Nod factors carrying unsaturated C₁₆ fatty acids. Expression of noIR was high both in the free-living bacterium and in the bacteroid and it was downregulated by its own product and by the nod gene inducer luteolin.     

Microscopic studies of cell divisions induced in alfalfa roots by Rhizobium meliloti

We have used spot-inoculation and new cytological procedures to observe the earliest events stimulated in alfalfa (Medicago sativa L.) roots by Rhizobium meliloti. Roots were inoculated with 1–10 nl of concentrated bacteria, fixed in paraformaldehyde, and after embedding and sectioning stained with a combination of acridine orange and DAPI (4-6-diamidino-2-phenylindole hydrochloride). Normal R. meliloti provoke cell dedifferentiation and mitosis in the inner cortex of the root within 21–24 h after inoculation. This activation of root cells spreads progressively, leading to nodule formation. In contrast, the R. meliloti nodA and nodC mutants do not stimulate any activation or mitosis. Thus the primary and earliest effect of Rhizobium nod gene action is plant cellular activation. A rapid, whole-mount visualization by lactic acid shows that the pattern of nodule form varies widely. Some R. meliloti strains were found to be capable of stimulating on alfalfa roots both normal nodules and a hybrid structure intermediate between a nodule and a lateral root.     

Vitamin B12-dependent methionine synthesis in Rhizobium meliloti

Methionine, among the various additions to the medium, could only replace cobalt ion or vitamin B₁₂ required for the growth of $\text{Rhizobium meliloti}$. It was demonstrated that there exists a vitamin B₁₂-dependent terminal step in the methionine synthesis, that is, N⁵CH₃-tetrahydrofolate-homocysteine transmethylase, which can also catalyze the methyl transfer from CH₃B₁₂ to homocysteine, in the cell-free extracts of Rhizobium meliloti. These facts seem to indicate that the vitamin B₁₂-dependent pathway to methionine functions mainly among the B₁₂-dependent enzymatic systems in the wild-type symbionts and this is the chief nutritional significance of cobalt.     

Erwinia herbicola isolates from alfalfa plants may play a role in nodulation of alfalfa by Rhizobium meliloti

Erwinia herbicola was isolated from roots of plants derived from surface-sterilized seeds of all alfalfa varieties that were tested. Some of these E. herbicola strains affected nodulation by certain strains of Rhizobium meliloti. In previously published work we presented the isolation of slow-and fast-nodulating variants from a single culture of R. meliloti 102F51. In the absence of E. herbicola, the slow-nodulating variant induced the formation of nodules on alfalfa as rapidly as the faster-nodulating strain. The rates of nodulation by the faster-nodulating variant were the same in the presence and absence of E. herbicola. All of the previously reported slower-nodulating strains derived from R. meliloti 102F51 nodulated more rapidly on sterilized plants than in the presence of certain E. herbicola isolates.     

A Rhizobium meliloti lipopolysaccharide mutant altered in competitiveness for nodulation of alfalfa.

A transposon Tn5-induced mutant of Rhizobium meliloti Rm2011, designated Rm6963, showed a rough colony morphology on rich and minimal media and an altered lipopolysaccharide (LPS). Major differences from the wild-type LPS were observed in (i) hexose and 2-keto-3-deoxyoctonate elution profiles of crude phenol extracts chromatographed in Sepharose CL-4B, (ii) silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis patterns of crude and purified LPS fractions, and (iii) immunoreactivities otherwise present in purified LPS of the parental strain Rm2011. In addition, Rm6963 lost the ability to grow in Luria-Bertani medium containing the hydrophobic compounds sodium deoxycholate or SDS and showed a decrease in survival in TY medium supplemented with high calcium concentrations. The mutant also had altered symbiotic properties. Rm6963 formed nodules that fixed nitrogen but showed a delayed or even reduced ability to nodulate the primary root of alfalfa without showing changes in the position of nodule distribution profiles along the roots. Furthermore, 2 to 3 weeks after inoculation, plants nodulated by Rm6963 were smaller than control plants inoculated with wild-type bacteria in correlation with a transient decrease in nitrogen fixation. In most experiments, the plants recovered later by expressing a full nitrogen-fixing phenotype and developing an abnormally high number of small nodules in lateral roots after 1 month. Rm6963 was also deficient in the ability to compete for nodulation. In coinoculation experiments with equal bacterial numbers of both mutant and wild-type rhizobia, only the parent was recovered from the uppermost root nodules. A strain ratio of approximately 100 to 1 favoring the mutant was necessary to obtain an equal ratio (1:1) of nodule occupancy. These results show that alterations in Rm6963 which include LPS changes lead to an altered symbiotic phenotype during the association with alfalfa that affects the timing of nodule emergence, the progress of nitrogen fixation, and the strain competitiveness for nodulation.