Molecular characterization of the nodulation gene, nodT, from two biovars of Rhizobium leguminosarum

DNA sequencing of the nodIJ region from Rhizobium leguminosarum biovar trifolii revealed the nodT gene immediately downstream of nodJ. DNA hybridizations using a nodT-specific probe showed that nodT is present in several R. leguminosarum strains. Interestingly, a flavonoid-inducible nodT gene homologue in R. leguminosarum bv. viciae is not in the nodABCIJ operon but is located downstream of nodMN. The sequence of the nodT gene from bv. viciae was determined and a comparison of the predicted amino-acid sequences of the two nodT genes shows them to be conserved; the predicted protein sequences appear to have a potential transit sequence typical of outer-membrane proteins. Mutations affecting nodT in either biovar had no observed effect on nodulation of the legumes tested.     

The nodI gene product of Rhizobium leguminosarum is closely related to ATP-binding bacterial transport proteins; nucleotide sequence analysis of the nodI and nodJ genes

The nucleotide sequence of a 2-kb fragment immediately downstream of the nodABC genes of the Rhizobium leguminosarum symbiotic plasmid pRL1JI has been determined. Genes corresponding to the two open reading frames identified are named nodI and nodJ. Tn 5 insertions into these genes result in a "nodulation-delayed" phenotype. The predicted amino acid sequence of the nodI gene shows considerable homology to inner-membrane-located gene products involved in active transport systems in Escherichia coli and Salmonella typhimurium. The predicted product of the nodJ gene is very hydrophobic, suggesting that it may be an integral membrane protein.     

Jasmonates induce Nod factor production by Bradyrhizobium japonicum

Jasmonates are signaling molecules involved in induced systemic resistance, wounding and stress responses of plants. We have previously demonstrated that jasmonates can induce nod genes of Bradyrhizobium japonicum when measured by beta-galactosidase activity. In order to test whether jasmonates can effectively induce the production and secretion of Nod factors (lipo-chitooligosaccharides, LCOs) from B. japonicum, we induced two B. japonicum strains, 532C and USDA3, with jasmonic acid (JA), methyl jasmonate (MeJA) and genistein (Ge). As genistein is well characterized as an inducer of nod genes it was used a positive control. The high-performance liquid chromatography (HPLC) profile of LCOs isolated following treatment with jasmonates or genistein showed that both JA and MeJA effectively induced nod genes and caused production of LCOs from bacterial cultures. JA and MeJA are more efficacious inducers of LCO production than genistein. Genistein plus JA or MeJA resulted in greater LCO production than either alone. A soybean root hair deformation assay showed that jasmonate induced LCOs were as effective as those induced by genistein. This is the first report that jasmonates induce Nod factor production by B. japonicum. This report establishes the role of jasmonates as a new class of signaling molecules in the Bradyrhizobium-soybean symbiosis.     

Comparison of characteristics of the nodX genes from various Rhizobium leguminosarum strains

We have analyzed the nucleotide sequences of the nodX genes from two strains of Rhizobium leguminosarum bv. viciae able to nodulate Afghan peas (strains A1 and Himalaya) and from two strains of R. leguminosarum bv. trifolii (ANU843 and CSF). The nodX genes of strains A1 and ANU843 were shown to be functional for the induction of nodules on Afghan peas. To analyze the cause of phenotypic differences of strain A1 and strain TOM we have studied the composition of the lipochitin-oligosaccharides (LCOs) produced by strain A1 after induction by the flavonoid naringenin or various pea root exudates. The structural analysis of the LCOs by mass spectrometry revealed that strain A1 synthesizes a family of at least 23 different LCOs. The use of exudates instead of naringenin resulted only in quantitative differences in the ratios of various LCOs produced.     

Role of nodl and nodj in lipo-chitooligosaccharide secretion in Azorhizobium caulinodans and Escherichia coli

Lipo-chitooligosaccharide (LCO) Nod factors are produced and secreted by rhizobia and trigger nodule development in leguminous host plants. The products of the bacterial nodlJ genes are related to transporters of capsular polysaccharides and were proposed to be involved in LCO transport. We have studied nodlJ of Azorhizobium caulinodans ORS571 by analysis of cell-associated and secreted radioactively labelled Nod factors in wild-type ORS571, a nodJ mutant and a complemented strain. Secretion was strongly reduced in the nodJ mutant, and restored to wild-type levels after complementation. Constructs were made for expression of combinations of different nod genes in Escherichia coli DH5a. The strain DH5α(pUCNABCSU) synthesized LCOs, but they were only secreted when a plasmid containing both nodl and nodJ was supplied in trans, nodi or nodJ alone was not sufficient. In E. coli as well as in Azorhizobium, the nod/J-encoded transporter showed a specificity for more hydrophilic LCOs.     

Growth temperature regulation of host-specific modifications of rhizobial lipo-chitin oligosaccharides: the function of nodX is temperature regulated

Lipo-chitin oligosaccharides (LCOs) are usually produced and isolated for structural analysis from bacteria cultured under laboratory rather than field conditions. We have studied the influence of bacterial growth temperature on the LCO structures produced by different Rhizobium leguminosarum strains, using thin-layer chromatographic, high-performance liquid chromatographic, and mass spectrometric analyses. Wild-type R. leguminosarum bv. viciae A1 was shown to produce larger relative amounts of nodX-mediated, acetylated LCOs at 12 degrees C than at 28 degrees C, indicating that the activity of nodX (a gene encoding an LCO O-acetyl transferase) is temperature dependent. Interestingly, symbiotic resistance genes sym1 and sym2 found in primitive pea cultivars are also temperature sensitive, only being active at low temperatures, at which they block nodulation by R. leguminosarum bv. viciae strains lacking nodX. We therefore propose that the gene-for-gene relationship between plant and bacterium has a temperature-sensitive mechanism as an adaptation to environmental conditions. An R. leguminosarum bv. trifolii strain was also shown to produce larger relative amounts of nodX-mediated, acetylated LCOs at 12 degrees C than at 28 degrees C. The major components synthesized by the two strains are produced at both temperatures but in different relative amounts, while some minor components are only produced at one of the two temperatures.     

Effect of organic N source on bacterial growth, lipo-chitooligosaccharide production, and early soybean nodulation by Bradyrhizobium japonicum

Production of Bradyrhizobium japonicum inoculants is problematic because high inoculation rates are necessary but expensive, while production of rhizobial Nod factors (lipo-chitooligosaccharides (LCOs)), key signal molecules in the establishment of legume-rhizobia symbioses, may be inhibited at high culture cell densities. We conducted experiments to determine the effects of growth medium N source on B. japonicum growth, LCO production, and early nodulation of soybean. We found that 1.57 mmol ammonium nitrate x L(-1) resulted in less rhizobial growth and rhizobial capacity to produce LCOs (on a per cell basis) than did 0.4 g yeast extract x L(-1), which contained the same amount of N as the ammonium nitrate. Increasing yeast extract to 0.8 g x L(-1) increased rhizobial growth and LCO production on a volume basis (per litre of culture) and did not affect cell capacity to produce LCOs; however, at 1.4 g yeast extract x L(-1) per cell, production was reduced. A mixture of 0.8 g yeast extract x L(-1) and 1.6 g casein hydrolysate x L(-1) resulted in the greatest bacterial growth and LCO production on a volume basis but reduced LCO production per cell. Changes in organic N level and source increased production of some of the measured LCOs more than others. LCO production was positively correlated with cell density when expressed on a volume basis; however, it was negatively correlated on a per cell basis. We conclude that although quorum sensing affected Nod factor production, increased levels of organic N, and specific compositions of organic N, increased LCO production on a volume basis. Greenhouse inoculation experiments showed that the medium did not modify nodule number and N fixation in soybean, suggesting that it could have utility in inoculant production.     

NodZ of Bradyrhizobium extends the nodulation host range of Rhizobium by adding a fucosyl residue to nodulation signals

The nodulation genes of rhizobia are involved in the production of the lipo-chitin oligosaccharides (LCO), which are signal molecules required for nodule formation. A mutation in nodZ of Bradyrhizobium japonicum results in the synthesis of nodulation signals lacking the wild-type 2- O -methylfucose residue at the reducing-terminal N -acetylglucosamine. This phenotype is correlated with a defective nodulation of siratro ( Macroptilium atropurpureum ). Here we show that transfer of nodZ to Rhizobium leguminosarum biovar (bv) viciae , which produces LCOs that are not modified at the reducing-terminal N -acetylglucosamine, results in production of LCOs with a fucosyl residue on C-6 of the reducing-terminal N -acetylglucosamine. This finding, together with in vitro enzymatic assays, indicates that the product of nodZ functions as a fucosyltransferase. The transconjugant R. leguminosarum strain producing fucosylated LCOs acquires the capacity to nodulate M. atropurpureum Glycine soja Vigna unguiculata and Leucaena leucocephala . Therefore, nodZ extends the narrow host range of R. leguminosarum bv. viciae to include various tropical legumes. However, microscopic analysis of nodules induced on siratro shows that these nodules do not contain bacteroids, showing that transfer of nodZ does not allow R. leguminosarum to engage in a nitrogen-fixing symbiosis with this plant.     

Lipo-Chitin Oligosaccharides,Plant Symbiosis Signalling Molecules That Modulate Mammalian Angiogenesis In Vitro

Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathways. In animals, oligo-chitins also induce innate immunity and other physiological responses but LCO recognition has not been demonstrated. Here LCO and LCO-like compounds are shown to be biologically active in mammals in a structure dependent way through the modulation of angiogenesis, a tightly-regulated process involving the induction and growth of new blood vessels from existing vessels. The testing of 24 LCO, LCO-like or oligo-chitin compounds resulted in structure-dependent effects on angiogenesis in vitro leading to promotion, or inhibition or nil effects. Like plants, the mammalian LCO biological activity depended upon the presence and type of terminal substitutions. Un-substituted oligo-chitins of similar chain lengths were unable to modulate angiogenesis indicating that mammalian cells, like plant cells, can distinguish between LCOs and un-substituted oligo-chitins. The cellular mode-of-action of the biologically active LCOs in mammals was determined. The stimulation or inhibition of endothelial cell adhesion to vitronectin or fibronectin correlated with their pro- or anti-angiogenic activity. Importantly, novel and more easily synthesised LCO-like disaccharide molecules were also biologically active and de-acetylated chitobiose was shown to be the primary structural basis of recognition. Given this, simpler chitin disaccharides derivatives based on the structure of biologically active LCOs were synthesised and purified and these showed biological activity in mammalian cells. Since important chronic disease states are linked to either insufficient or excessive angiogenesis, LCO and LCO-like molecules may have the potential to be a new, carbohydrate-based class of therapeutics for modulating angiogenesis.     

Aberrant nodulation response of Vigna umbellata to a Bradyrhizobium japonicum NodZ mutant and nodulation signals.

The (Brady)rhizobium nodulation gene products synthesize lipo-chitin oligosaccharide (LCO) signal molecules that induce nodule primordia on legume roots. In spot inoculation assays with roots of Vigna umbellata, Bradyrhizobium elkanii LCO and chemically synthesized LCO induced aberrant nodule structures, similar to the activity of these LCOs on Glycine soja (soybean). LCOs containing a pentameric chitin backbone and a reducing-end 2-O-methyl fucosyl moiety were active on V. umbellata. In contrast, the synthetic LCO-IV(C16:0), which has previously been shown to be active on G. soja, was inactive on V. umbellata. A B. japonicum NodZ mutant, which produces LCO without 2-O-methyl fucose at the reducing end, was able to induce nodule structures on both plants. Surprisingly, the individual, purified, LCO molecules produced by this mutant were incapable of inducing nodule formation on V. umbellata roots. However, when applied in combination, the LCOs produced by the NodZ mutant acted cooperatively to produce nodulelike structures on V. umbellata roots.     

Molecular mechanisms of Nod factor diversity

The rhizobia–legume symbiosis is highly specific. Major host specificity determinants are the bacterial Nod factor signals that trigger the nodulation programme in a compatible host. Nod factors are lipo-chitooligosaccharides (LCOs) varying in the oligosaccharide chain length, the nature of the fatty acids and substitutions on the oligosaccharide. The nod genotype of rhizobia, which forms the genetic basis for this structural variety, includes a set of nodulation genes encoding the enzymes that synthesize LCOs. Allelic and non-allelic variation in these genes ensures the synthesis of different LCO structures by the different rhizobia. The nod genotypes co-evolved with host plant divergence in contrast to the rhizobia, which followed a different evolution. Horizontal gene transfer probably played an important role during evolution of symbiosis. The nod genotypes are particularly well equipped for horizontal gene transfer because of their location on transmissible plasmids and/or on 'symbiosis islands', which are symbiotic regions associated with movable elements.     

Genes in the Salmonella pathogenicity island 2 and the Salmonella virulence plasmid are essential for Salmonella-induced apoptosis in intestinal epithelial cells

Intestinal epithelial cells are an important site of the host's interaction with enteroinvasive bacteria. Genes in the chromosomally encoded Salmonella pathogenicity island 2 (SPI 2) that encodes a type III secretion system and genes on the virulence plasmid pSDL2 of Salmonella enteritica serovar Dublin (spv genes) are thought to be important for Salmonella dublin survival in host cells. We hypothesized that genes in those loci may be important also for prolonged Salmonella growth and the induction of apoptosis induced by Salmonella in human intestinal epithelial cells. HT-29 human intestinal epithelial cells were infected with wild-type S. dublin or isogenic mutants deficient in the expression of spv genes or with SPI 2 locus mutations. Neither the spv nor the SPI 2 mutations affected bacterial entry into epithelial cells or intracellular proliferation of Salmonella during the initial 8 h after infection. However, at later periods, bacteria with mutations in the SPI 2 locus or in the spv locus compared to wild-type bacteria, manifested a marked decrease in intracellular proliferation and a different distribution pattern of bacteria within infected cells. Epithelial cell apoptosis was markedly increased in response to infection with wild-type, but not the mutant Salmonella. However, apoptosis of epithelial cells infected with wild-type S. dublin was delayed for approximately 28 h after bacterial entry. Apoptosis was preceded by caspase 3 activation, which was also delayed for approximately 24 h after infection. Despite its late onset, the cellular commitment to apoptosis was determined in the early period after infection as inhibition of bacterial protein synthesis during the first 6 h after epithelial cell infection with wild-type S. dublin, but not at later times, inhibited the induction of apoptosis. These studies indicate that genes in the SPI 2 and the spv loci are crucial for prolonged bacterial growth in intestinal epithelial cells. In addition to their influence on intracellular proliferation of Salmonella, genes in those loci determine the ultimate fate of infected epithelial cells with respect to caspase 3 activation and undergoing death by apoptosis.     

Characteristics of Secretion of Penicillinase, Alkaline Phosphatase, and Nuclease by Bacillus Species

The distribution of alkaline phosphatase and nuclease activity between cells and medium was examined in one strain of Bacillus licheniformis and four strains of B. subtilis. Over 95% of both activities was found in the medium of the B. licheniformis culture, but in the B. subtilis cultures the amount of enzyme activity found in the medium varied with the strain and the enzyme considered. B. licheniformis 749 and its penicillinase magnoconstitutive mutant 749/C were grown in continuous culture with phosphorous as the growth-limiting factor, and the kinetics of penicillinase formation and secretion were examined. Nutrient arrest halted secretion (usually after a lag of about 30 min) in both the inducible and constitutive strains. Chloramphenicol did not eliminate secretion, but under certain circumstances reduced its rate. In the inducible strain treated with a low level of inducer, the rate of secretion was more affected by the rate of synthesis than by the level of cell-bound enzyme. During induction, the onset of accretion of cell-bound penicillinase and secretion of the exoenzyme were nearly simultaneous. It seems unlikely that a long-lived, membrane- or cell-bound intermediate is mandatory in the secretion of the three enzymes by Bacillus species. In the case of penicillinase secretion, there are at least two different phases. When penicillinase synthesis is proceeding rapidly, the rate of secretion is five to six times greater at equivalent concentrations of membrane-bound penicillinase than it is when penicillinase synthesis is reduced. The data require that any membrane-bound intermediate in the formation of exoenzyme be much shorter-lived in cells with a high rate of synthesis than in cells with a low rate. Either there are two separate routes for the secretion of penicillinase or the characteristics of the process vary substantially between the early stages and the declining phase of induction.     

Further characterization of SOS system induction in recBC mutants of Escherichia coli

Expression of several SOS functions such as induction of lambda prophage, inhibition of cell division and induction of both umuC and recA genes after UV-irradiation, nalidixic acid or mitomycin C addition was studied in an RecBC- mutant. UV-irradiation and mitomycin C induced all SOS functions studied in the RecBC- cells but at a lower level and delayed with respect to the wild-type strain. On the contrary, nalidixic acid was unable to trigger any of these SOS functions. In the RecBC- mutant, adenine only had a stimulating effect on the amplification of RecA protein synthesis following UV-irradiation. Nevertheless, in the wild-type strain the stimulating effect occurred in all SOS functions studied following UV-irradiation as well as in the amplification of RecA protein synthesis by nalidixic acid but not in the other SOS functions triggered by this compound. Furthermore, adenine produced a decrease in the mitomycin C-mediated induction of all SOS functions studied in both RecBC- and wild-type strains.     

In vitro induction of lipo-chitooligosaccharide production in Bradyrhizobium japonicum cultures by root extracts from non-leguminous plants

Bradyrhizobium japonicum can form a N2-fixing symbiosis with compatible leguminous plants. It can also act as a plant-growth promoting rhizobacterium (PGPR) for non-legume plants, possibly through production of lipo-chitooligosaccharides (LCOs), which should have the ability to induce disease resistance responses in plants. The objective of this work was to determine whether non-leguminous crop plants can induce LCO formation by B. japonicum cultures. Cultures treated with root extracts of soybean, corn, cotton or winter wheat were assayed for presence and level of LCO. Root extracts of soybean, corn and winter wheat all induced LCO production, with extracts of corn inducing the greatest amounts. Root washings of corn also induced LCO production, but less than the root extract. These results indicated that the stimulation of non-legume plant growth by B. japonicum could be through the production of LCOs, induced by materials excreted by the roots of non-legume plants.     

Response of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> roots to lipo-chitooligosaccharide from <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and other chitin-like compounds

Lipo-chitooligosaccharides (LCOs) are bacteria-to-plant signals required for the establishment of rhizobia–legume nitrogen fixing symbioses. The ability of LCO [Nod Bj V (C_18:1, MeFuc)] isolated from B. japonicum (strain 532C), and of oligomers of chitosan (tetramer, pentamer) and chitin (pentamer) to affect the developmental morphology of roots in Arabidopsis thaliana (L.) Heynh ecotype Columbia (Col-0) was assessed using an interactive scanner-based image analysis system. LCOs have been shown to play a role in plant organogenesis at nanomolar concentrations. LCO and the chitin pentamer promoted root growth and development in Arabidopsis at concentrations of 10 nM and 100 μM, respectively. The LCO treated Arabidopsis plants had about 35% longer roots than untreated control plants. Similarly, treatment with 100 μM chitin pentamer (CHIT5) resulted in 26% longer roots than the untreated plants; however, chitosan oligomer (CH4 or CH5) treated plants did not differ from the control plants at either concentration (100 or 1 μM). Both LCOs and the chitin pentamer at higher concentrations increased root surface area, mean root diameter and number of root tips. However, leaf area increase was observed only in plants treated with LCO at 10 nM.