Occurrence of choline and glycine betaine uptake and metabolism in the family rhizobiaceae and their roles in osmoprotection

The role of glycine betaine and choline in osmoprotection of various Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, and Bradyrhizobium reference strains which display a large variation in salt tolerance was investigated. When externally provided, both compounds enhanced the growth of Rhizobium tropici, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium galegae, Agrobacterium tumefaciens, Mesorhizobium loti, and Mesorhizobium huakuii, demonstrating their utilization as osmoprotectants. However, both compounds were inefficient for the most salt-sensitive strains, such as Rhizobium leguminosarum (all biovars), Agrobacterium rhizogenes, Rhizobium etli, and Bradyrhizobium japonicum. Except for B. japonicum, all strains exhibit transport activity for glycine betaine and choline. When the medium osmolarity was raised, choline uptake activity was inhibited, whereas glycine betaine uptake was either increased in R. leguminosarum and S. meliloti or, more surprisingly, reduced in R. tropici, S. fredii, and M. loti. The transport of glycine betaine was increased by growing the cells in the presence of the substrate. With the exception of B. japonicum, all strains were able to use glycine betaine and choline as sole carbon and nitrogen sources. This catabolic function, reported for only a few soil bacteria, could increase competitiveness of rhizobial species in the rhizosphere. Choline dehydrogenase and betaine-aldehyde dehydrogenase activities were present in the cells of all strains with the exception of M. huakuii and B. japonicum. The main physiological role of glycine betaine in the family Rhizobiaceae seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains.     

Fluorescent-Antibody Approach to Study of Rhizobia in Soil

Application of fluorescent-antibody (FA) techniques to the study of rhizobia as free-living soil bacteria was explored. Antiserum to a particular strain of Rhizobium japonicum proved specific in both agglutination and FA tests. Within the R. japonicum group, 2 of 12 strains were stained by the conjugate and these fluoresced brightly; all others were entirely negative. FA tests were negative for 7 strains of R. meliloti, 9 strains of R. leguminosarum, 9 strains of R. trifolii, 6 strains of R. phaseoli, and 65 unidentified bacteria isolated from 12 soils. R. japonicum grew in autoclaved soil and was readily detectable by FA examination of contact slides. The FA technique also detected antibody-reacting bacteria in a field soil whose rhizobial content was unknown. Fluorescent cells were probably R. japonicum, since nodules developed on soybean plants grown in the same soil sample and FA preparations of the crushed nodules proved positive. Autofluorescence was not a problem, but nonspecific adsorption of conjugate restricted observations to microscopic fields free from soil particles. Nonspecific adsorption was substantial, irrespective of the soil used.     

Transfer of R factors to and between genetically marked sublines of Rhizobium japonicum.

Plasmids R1822 and pRD1 of the P-1 incompatibility group, for which Rhizobium japonicum had not previously been shown to serve as host, were introduced into a strain of R. japonicum. Acquisition of R68 and R68.45 plasmids by this Rhizobium was equivocal. Transfer of R1822 from Pseudomonas aeruginosa and of pRD1 from Escherichia coli to R. japonicum was unambiguous, because the exconjugants subsequently cotransferred the three R-factor resistance determinants (kanamycin, tetracycline, and penicillin) between genetically marked sublines of strain I-110. Under optimal conditions the transfer of R1822 and pRD1 occurred at frequencies of approximately 10(-3) in plate matings of strains bearing as many as five dissimilar genetic markers. In matings with R1822 on membrane filters, recombinants were formed at incidences as high as 4%.     

Transfer of R factors to and between genetically marked sublines of Rhizobium japonicum.

Plasmids R1822 and pRD1 of the P-1 incompatibility group, for which Rhizobium japonicum had not previously been shown to serve as host, were introduced into a strain of R. japonicum. Acquisition of R68 and R68.45 plasmids by this Rhizobium was equivocal. Transfer of R1822 from Pseudomonas aeruginosa and of pRD1 from Escherichia coli to R. japonicum was unambiguous, because the exconjugants subsequently cotransferred the three R-factor resistance determinants (kanamycin, tetracycline, and penicillin) between genetically marked sublines of strain I-110. Under optimal conditions the transfer of R1822 and pRD1 occurred at frequencies of approximately 10(-3) in plate matings of strains bearing as many as five dissimilar genetic markers. In matings with R1822 on membrane filters, recombinants were formed at incidences as high as 4%.     

Fingerprinting bacterial chromosomal DNA with restriction endonuclease EcoRI: comparison of Rhizobium spp. and identification of mutants.

Total cellular DNA from Rhizobium trifolii, R. melitoti, and R. japonicum strains 110 and 117 were prepared. DNA fragments generated with restriction endonuclease EcoRI from these DNA samples were compared in agarose gels after electrophoresis. DNA cleavage patterns generated from R. japonicum strain 110, R. trifolii, and R. meliloti were clearly distinguishable from each other. Restriction endonuclease cleavage patterns of DNA from R. japonicum strain 110 and presumptive R. trifolii mutant strains that nodulate soybean were found to be similar. Rhizobium trifolii mutant strains were also lysed by a phage specific for R. japonicum strain 110. These results show that "R. trifolii mutant strains" are indeed derivatives of R. japonicum strain 110 and not R. trifolii.     

Role of lectins in plant-microorganism interactions: I. Binding of soybean lectin to rhizobia

Highly purified soybean lectin (SBL) was labeled with fluorescein isothiocyanate (FITC-SBL) or tritium ((3)H-SBL) and repurified by affinity chromatography. FITC-SBL was found to bind to living cells of 15 of the 22 Rhizobium japonicum strains tested. The lectin did not bind to cells of the other seven R. japonicum strains, or to cells of any of the nine Rhizobium strains tested which do not nodulate soybean. The binding of the lectin to the SBL-positive strains of R. japonicum was shown to be specific and reversible by hapten inhibition with d-galactose or N-acetyl-d-galactosamine.The lectin-binding properties of the SBL-positive R. japonicum strains were found to change substantially with culture age. The percentage of cells in a population exhibiting fluorescence after exposure to FITC-SBL varied between 0 and 70%. The average number of SBL molecules bound per cell varied between 0 and 2 x 10(6). While most strains had their highest percentage of SBL-positive cells and maximum number of SBL-binding sites per cell in the early and midlog phases of growth, one strain had a distinctly different pattern. The SBL-negative strains did not bind lectin at any stage of growth.Quantitative binding studies with (3)H-SBL indicated that the affinity constant for binding of SBL to its receptor sites on R. japonicum is approximately 4 x 10(7)m(-1). Many of the binding curves were biphasic. An inhibitor of SBL binding was found to be present in R. japonicum culture filtrates.     

Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.     

Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum.

Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.     

Flow-microfluorometric analysis of Escherichia coli, Rhizobium meliloti, and Rhizobium japonicum at different stages of the growth cycle.

The applicability of flow-microfluorometry (FMF) to the study of bacterial samples was investigated on cultures of Rhizobium meliloti, Rhizobium japonicum, and Escherichia coli using fluorescent and light-scattering signals. This technique which analyzes individual bacterial cells in a population was used to monitor the relative change in nucleic acid content and cell size during the growth cycle of the three microorganisms which were known to have different growth rates. Early log-phase E. coli cells contained at least eightfold more nucleic acid and were significantly larger than the stationary-phase cells. Cultures of early log-phase R. meliloti cells contained three to four-fold more nucleic acid and were slightly larger than cells in the stationary phase. Rhizobium japonicum had very little change in either parameter. In general, the amount of change in both cell size and nucleic acid content upon initiation of log-phase growth was related to the overall growt rate of the organisms, with E. coli experiencing the greatest change and R. japonicum the least. Results obtained by FMF analysis, therefore, were consistent with observations reported by earlier workers. Cultures of R. meliloti also were used to demonstrate that the intensity of the fluorescent signals was sensitive to digestion by DNase and RNase and to prolonged storage and fixation. The potential use of FMF in the study of microorganisms is discussed.     

Reiteration of genes involved in symbiotic nitrogen fixation by fast-growing Rhizobium japonicum.

By using cloned Rhizobium meliloti nodulation (nod) genes and nitrogen fixation (nif) genes, we found that the genes for both nodulation and nitrogen fixation were on a plasmid present in fast-growing Rhizobium japonicum strains. Two EcoRI restriction fragments from a plasmid of fast-growing R. japonicum hybridized with nif structural genes of R. meliloti, and three EcoRI restriction fragments hybridized with the nod clone of R. meliloti. Cross-hybridization between the hybridizing fragments revealed a reiteration of nod and nif DNA sequences in fast-growing R. japonicum. Both nif structural genes D and H were present on 4.2- and 4.9-kilobase EcoRI fragments, whereas nifK was present only on the 4.2-kilobase EcoR2 fragment. These results suggest that the nif gene organizations in fast-growing and in slow-growing R. japonicum strains are different.     

Nitrogen fixation (nif) genes and large plasmids of Rhizobium japonicum.

The location of structural nitrogen-fixation genes was determined for the slow- and fast-growing types of Rhizobium japonicum. Slow-growing R. japonicum strains do not harbor structural nif genes, homologous to nifD and nifH, on large plasmids (100 to 200 megadaltons). In contrast, all fast-growing R. japonicum strains, except PRC194, contain structural nif genes on large plasmids.     

Glutamate as a differential nitrogen source for the characterization of acetylene-reducing Rhizobium strains

A majority (36 of 44) of Rhizobium japonicum strains tested reduced acetylene asymbiotically when grown on an agar medium containing 0.1% (w/v) L-glutamate as a sole nitrogen (N) source. Glutamate as N source led to pinpoint colonies and uniform glutamine synthetase activity of three selected, slow-growing acetylene-reducing strains ( R. japonicum L-259 and 110 and a cowpea-type Rhizobium 32H1). The three test strains were characterized further by antibiotic resistance, colony type, cellular morphology, and differential growth on different N sources. The evidence suggests that, in an agar medium, glutamate creates a growth condition leading to acetylene reduction activity, pinpoint colonies and pleomorphism.     

Immunofluorescent polar tips of Rhizobium japonicum: possible site of attachment or lectin binding.

Rhizobium japonicum USDA 31 demonstrated marked polarity by binding homologous fluorescent antibody (FA) heavily on one end of the cell. FA prepared against R. japonicum strains 110 and 138, and against R. trifolii TA1 cross-reacted with strain 31 only in the polar tip region. No polar immunofluorescing tips could be seen with FA against two other strains of R. japonicum or with those against several unrelated microorganisms. Common antigens localized only in a polar region were seen in many rhizobia stained with R. japonicum 31 FA: 22 of 23 strains of R. japonicum, 10 of 17 strains of R. trifolii, 3 of 7 strains of R. melitolii, 3 of 6 strains of R. phaseoli, and 3 of 9 strains of R. leguminosarum had some cells with detectable polar tips. The proportion of R. japonicum 31 cells with polar tips was high throughout the growth cycle. Polar tip staining was not affected by drastic cell treatments. A function was proposed for the polar tip region as a site for attachment. R. japonicum 31 cells attached to each other in a tip-to-tip fashion and endwise to fungal hyphae with the polar tip in contact with the hyphal wall. Binding of fluorescein isothiocyanate-labeled soybean lectin to certain strains of R. japonicum gave additional evidence of polarity. Polar binding of both antibody and lectin may provide insights into relationships between rhizobia and roots of host legumes.     

Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp

Some properties of glutamine synthetase I (GSI) and GSII are described for a fast-growing Rhizobium sp. (Rhizobium trifolii T1), a slow-growing Rhizobium sp. (Rhizobium japonicum USDA 83), and Agrobacterium tumefaciens C58. GSII of the fast-growing Rhizobium sp. and GSII of the Agrobacterium sp. were considerably more heat labile than GSII of the slow-growing Rhizobium sp. As previously shown in R. japonicum 61A76, GSI became adenylylated rapidly in all species tested in response to ammonium. GSII activity disappeared within one generation of growth in two of the strains, but the disappearance of GSII activity required two generations in another. Isoactivity points for transferase assay, which were derived from the pH curves of adenylylated GSI and deadenylylated GSI, were approximately pH 7.8 for both R. trifolii and A. tumefaciens. No isoactivity point was found for R. japonicum under the standard assay conditions used. When the feedback inhibitor glycine was used to inhibit differentially the adenylylated GSI and deadenylylated GSI of R. japonicum, an isoactivity point was observed at pH 7.3. Thus, the transferase activity of GSI could be determined independent of the state of adenylation. A survey of 23 strains of bacteria representing 11 genera indicated that only Rhizobium spp. and Agrobacterium spp. contained GSII. Thus, this enzyme appears to be unique for the Rhizobiaceae.     

The Rhizobial hemA Gene Is Required for Symbiosis in Species with Deficient [delta]-Aminolevulinic Acid Uptake Activity

Most rhizobial hemA mutants induce root nodules on their respective legume hosts that lack nitrogen fixation activity and leghemoglobin expression. However, a Bradyrhizobium japonicum hemA mutant elicits effective nodules on soybean, and we proposed previously that synthesis and uptake of the heme precursor [delta]-aminolevulinic acid (ALA) by the plant and bacterial symbiont, respectively, allow mutant rescue (I. Sangwan, M.R. O'Brian [1991] Science 251: 1220-1222). In the present work, the B. japonicum hemA mutant MLG1 elicited normal nodules on three hosts, including cowpea, a plant that is not effectively nodulated by a hemA mutant of Rhizobium sp. These data indicate that B. japonicum rather than soybean possesses the unique trait that allows normal nodule development by a hemA mutant. Cowpea expressed glutamate-dependent ALA formation activity in nodules induced by B. japonicum strains I110 or MLG1 and by Rhizobium sp. ANU240. Exogenous ALA was taken up by B. japonicum bacteroids isolated from soybean or cowpea nodules, and the kinetics of uptake were biphasic. By comparison, Rhizobium sp. ANU240 had very low ALA uptake activity. In addition, ALA uptake was observed in cultured cells of B. japonicum but not in cultured cells of three other rhizobial species tested. We suggest that the differential success of legume-rhizobial hemA symbioses is due to an ALA uptake activity in B. japonicum that is deficient in other rhizobia, thereby further validating the ALA rescue hypothesis.     

Cell adsorption and selective desorption for separation of microbial cells by using chitosan-immobilized silica

Cell adsorption and selective desorption for separation of microbial cells were conducted by using chitosan-immobilized silica (CIS). When chitosan was immobilized onto silica surfaces with glutaraldehyde, bacterial cells adsorbed well and retained viability. Testing of the adsorption and desorption ability of CIS using various microbes such as Escherichia coli, Aeromonas hydrophila, Pseudomonas aeruginosa, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillus casei, Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Saccharomyces cerevisiae, Saccharomyces ludwigii, and Schizosaccharomyces pombe revealed that most microbes could be adsorbed and selectively desorbed under different conditions. In particular, recovery was improved when L-cysteine was added. A mixture of two bacterial strains adsorbed onto CIS could also be successfully separated by use of specific solutions for each strain. Most of the desorbed cells were alive. Thus, quantitative and selective fractionation of cells is readily achievable by employing chitosan, a known antibacterial material.     

Conditional change of DNA replication control in an RNA polymerase mutant of Escherichia coli

The infectivity of the soybean symbiont Rhizobium japonicum changed two- to fivefold with culture age for strains 110 ARS, 138 Str Spc, and 123 Spc, whereas culture age had relatively little effect on the infectivity of strains 83 Str and 61A76 Str. Infectivity was measured by determining the number of nodules which developed on soybean primary roots in the zone which contained developing and preemergent root hairs at the time of inoculation. Root cells in this region of the host root are susceptible to Rhizobium infection, but this susceptibility is lost during acropetal development and maturation of the root cells within a period of 4 to 6 h (T. V. Bhuvaneswari, B. G. Turgeon, and W. D. Bauer, Plant Physiol. 66:1027-1031, 1980). Profiles of nodulation frequency at different locations on the root were not affected by the age of the R. japonicum cultures, indicating that culture age affected the efficiency of Rhizobium infection rather than how soon infections were initiated after inoculation. Inoculum dose-response experiments also indicated that culture age affected the efficiency of infection. Two strains, 61A76 Str and 83 Str, were relatively inefficient at all culture ages, particularly at low inoculum doses. Changes in infectivity with culture age were reasonably well correlated with changes in the proportion of cells in a culture capable of binding soybean lectin. Suspensions of R. japonicum in water were found to retain their viability and infectivity.     

Nitrate reductase activities of rhizobia and the correlation between nitrate reduction and nitrogen fixation.

All species of Rhizobium except R. lupini had nitrate reductase activity. Only R. lupini was incapable of growth with nitrate as the sole source of nitrogen. However, the conditions necessary for the induction of nitrate reductase varied among species of Rhizobium. Rhizobium japonicum and some Rhizobium species of the cowpea strains expressed nitrate reductase activities both in the root nodules of appropriate leguminous hosts and when grown in the presence of nitrate. Rhizobium trifolii, R. phaseoli, and R. leguminosarum did not express nitrate reductase activities in the root nodules, but they did express them when grown in the presence of nitrate. In bacteroids of R. japonicum and some strains of cowpea Rhizobium, high N2 fixation activities were accompanied by high nitrate reductase activities. In bacteroids of R. trifolii, R. leguminosarum, and R. phaseoli, high N2 fixation activities were not accompanied by high nitrate reductase activities.