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We examined the bacterial endophyte-enriched root-associated microbiome within rice (Oryza sativa) 55 days after growth in soil with and without urea fertilizer and/or biofertilization with a growth-promotive bacterial strain (Rhizobium leguminosarum bv. trifolii E11). After treatment to deplete rhizosphere/rhizoplane communities, washed roots were macerated and their endophyte-enriched communities were analyzed by 16S ribosomal DNA 454 amplicon pyrosequencing. This analysis clustered 99,990 valid sequence reads into 1105 operational taxonomic units (OTUs) with 97% sequence identity, 133 of which represented a consolidated core assemblage representing 12.04% of the fully detected OTU richness. Taxonomic affiliations indicated Proteobacteria as the most abundant phylum (especially α- and γ-Proteobacteria classes), followed by Firmicutes, Bacteroidetes, Verrucomicrobia, Actinobacteria, and several other phyla. Dominant genera included Rheinheimera, unclassified Rhodospirillaceae, Pseudomonas, Asticcacaulis, Sphingomonas, and Rhizobium. Several OTUs had close taxonomic affiliation to genera of diazotrophic rhizobacteria, including Rhizobium, unclassified Rhizobiales, Azospirillum, Azoarcus, unclassified Rhizobiaceae, Bradyrhizobium, Azonexus, Mesorhizobium, Devosia, Azovibrio, Azospira, Azomonas, and Azotobacter. The endophyte-enriched microbiome was restructured within roots receiving growth-promoting treatments. Compared to the untreated control, endophyte-enriched communities receiving urea and/or biofertilizer treatments were significantly reduced in OTU richness and relative read abundances. Several unique OTUs were enriched in each of the treatment communities. These alterations in structure of root-associated communities suggest dynamic interactions in the host plant microbiome, some of which may influence the well-documented positive synergistic impact of rhizobial biofertilizer inoculation plus low doses of urea-N fertilizer on growth promotion of rice, considered as one of the world's most important food crops.

Key Message: The growth-promotion of rice seedling following inoculation with Sinorhizobium meliloti 1021 was a cumulative outcome of elevated expression of genes that function in accelerating cell division and enhancing cell expansion. Various endophytic rhizobacteria promote the growth of cereal crops. To achieve a better understanding of the cellular and molecular bases of beneficial cereal-rhizobia interactions, we performed computer-assisted microscopy and transcriptomic analyses of rice seedling shoots (Oryza sativa) during early stages of endophytic colonization by the plant growth-promoting Sinorhizobium meliloti 1021. Phenotypic analyses revealed that plants inoculated with live rhizobia had increased shoot height and dry weight compared to control plants inoculated with heat-killed cells of the same microbe. At 6 days after inoculation (DAI) with live cells, the fourth-leaf sheaths showed significant cytological differences including their enlargement of parenchyma cells and reduction in shape complexity. Transcriptomic analysis of shoots identified 2,414 differentially-expressed genes (DEGs) at 1, 2, 5 and 8 DAI: 195, 1390, 1025 and 533, respectively. Among these, 46 DEGs encoding cell-cycle functions were up-regulated at least 3 days before the rhizobia ascended from the roots to the shoots, suggesting that rhizobia are engaged in long-distance signaling events during early stages of this plant-microbe interaction. DEGs involved in phytohormone production, photosynthetic efficiency, carbohydrate metabolism, cell division and wall expansion were significantly elevated at 5 and 8 DAI, consistent with the observed phenotypic changes in rice cell morphology and shoot growth-promotion. Correlation analysis identified 104 height-related DEGs and 120 dry-weight-related DEGs that represent known quantitative-trait loci for seedling vigor and increased plant height. These findings provide multiple evidences of plant-microbe interplay that give insight into the growth-promotion processes associated with this rhizobia-rice beneficial association.

Image analysis of fractal geometry can be used to gain deeper insights into complex ecophysiological patterns and processes occurring within natural microbial biofilm landscapes, including the scale-dependent heterogeneities of their spatial architecture, biomass, and cell-cell interactions, all driven by the colonization behavior of optimal spatial positioning of organisms to maximize their efficiency in utilization of allocated nutrient resources. Here, we introduce CMEIAS JFrad, a new computing technology that analyzes the fractal geometry of complex biofilm architectures in digital landscape images. The software uniquely features a data-mining opportunity based on a comprehensive collection of 11 different mathematical methods to compute fractal dimension that are implemented into a wizard design to maximize ease-of-use for semi-automatic analysis of single images or fully automatic analysis of multiple images in a batch process. As examples of application, quantitative analyses of fractal dimension were used to optimize the important variable settings of brightness threshold and minimum object size in order to discriminate the complex architecture of freshwater microbial biofilms at multiple spatial scales, and also to differentiate the spatial patterns of individual bacterial cells that influence their cooperative interactions, resource use, and apportionment in situ. Version 1.0 of JFrad is implemented into a software package containing the program files, user manual, and tutorial images that will be freely available at http://cme.msu.edu/cmeias/. This improvement in computational image informatics will strengthen microscopy-based approaches to analyze the dynamic landscape ecology of microbial biofilm populations and communities in situ at spatial resolutions that range from single cells to microcolonies.

Colbricon Superiore and Inferiore are two small adjacent high-mountain lakes located in the Paneveggio Natural Park (Italy) that offer the rare opportunity to study two iso-ecologic water environments differing only by area and volume in a ratio of 2:1 and 3:1, respectively. We took advantage of this setting to investigate phytoplankton dynamics, compare variability and productivity differences between the two basins, and assess size-dependent issues. The phytoplankton group of the Dinophyceae was chosen as the indicator organisms of ecological perturbation owing to their high sensitivity to environmental variations, as well as their acknowledged nature of versatile proxy to report global climatic changes. The study was conducted for over 10 years with fortnightly samplings. Results indicated that (a) the Dinophyceae communities in the smaller lake were significantly more resistant to changes exerted by the fluctuation of lakewater transparency and pH; and (b) the smaller lake sustained a consistently higher production with an average Dinophyceae density 1.73 fold higher than that of the larger lake. The coefficients of variation show that the chemical parameters in the smaller lake display higher time-related fluctuation while being spatially homogeneous and that such conditions correlate with a higher stability of the Dinophyceae assemblage. The use of this setting is also proposed as a model to test relationships between ecosystem production and physical stability.

This paper describes how the quantitative analytical tools of CMEIAS image analysis software can be used to investigate in situ microbial interactions involving cell-to-cell communication within biofilms. Various spatial pattern analyses applied to the data extracted from the 2-dimensional coordinate positioning of individual bacterial cells at single-cell resolution indicate that microbial colonization within natural biofilms is not a spatially random process, but rather involves strong positive interactions between communicating cells that influence their neighbors' aggregated colonization behavior. Geostatistical analysis of the data provide statistically defendable estimates of the micrometer scale and interpolation maps of the spatial heterogeneity and local intensity at which these microbial interactions autocorrelate with their spatial patterns of distribution. Including in situ image analysis in cell communication studies fills an important gap in understanding the spatially dependent microbial ecophysiology that governs the intensity of biofilm colonization and its unique architecture.

Quantitative microscopy and digital image analysis are underutilized in microbial ecology largely because of the laborious task to segment foreground object pixels from background, especially in complex color micrographs of environmental samples. In this paper, we describe an improved computing technology developed to alleviate this limitation. The system's uniqueness is its ability to edit digital images accurately when presented with the difficult yet commonplace challenge of removing background pixels whose three-dimensional color space overlaps the range that defines foreground objects. Image segmentation is accomplished by utilizing algorithms that address color and spatial relationships of user-selected foreground object pixels. Performance of the color segmentation algorithm evaluated on 26 complex micrographs at single pixel resolution had an overall pixel classification accuracy of 99+%. Several applications illustrate how this improved computing technology can successfully resolve numerous challenges of complex color segmentation in order to produce images from which quantitative information can be accurately extracted, thereby gain new perspectives on the in situ ecology of microorganisms. Examples include improvements in the quantitative analysis of (1) microbial abundance and phylotype diversity of single cells classified by their discriminating color within heterogeneous communities, (2) cell viability, (3) spatial relationships and intensity of bacterial gene expression involved in cellular communication between individual cells within rhizoplane biofilms, and (4) biofilm ecophysiology based on ribotype-differentiated radioactive substrate utilization. The stand-alone executable file plus user manual and tutorial images for this color segmentation computing application are freely available at http://cme.msu.edu/cmeias/ . This improved computing technology opens new opportunities of imaging applications where discriminating colors really matter most, thereby strengthening quantitative microscopy-based approaches to advance microbial ecology in situ at individual single-cell resolution.

Rhizobia are well-known for their ability to infect and nodulate legume roots, forming a nitrogen-fixing symbiosis of agricultural importance. In addition, recent studies have shown that rhizobia can colonize roots and aerial plant tissues of rice as a model plant of the Graminaceae family. Here we show that rhizobia can invade tobacco, a model plant belonging to the Solanaceae family. Inoculation of seedling roots with five GFP-tagged rhizobial species followed by microscopy and viable plating analyses indicated their colonization of the surface and interior of the whole vegetative plant. Blockage of ascending epiphytic migration by coating the hypocotyls with Vaseline showed that the endophytic rhizobia can exit the leaf interior through stomata and colonize the external phyllosphere habitat. These studies indicate rhizobia can colonize both below and above-ground tissues of tobacco using a dynamic invasion process that involves both epiphytic and endophytic lifestyles.

A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is discussed.

We used computer-assisted microscopy at single cell resolution to quantify the in situ spatial scale of N-acylhomoserine lactone (AHL)-mediated cell-to-cell communication of Pseudomonas putida colonized on tomato and wheat root surfaces. The results of this in situ quantification study on close-to-natural surfaces challenge the conventional view of a quorum group requirement of high cell densities for this type of bacterial communication. In situ image analysis indicated that the effective 'calling distance' on root surfaces was most frequent at 4-5 microm, extended to 37 microm in the root tip/elongation zone and further out to 78 microm in the root hair zone. The spatial scale of these calling distances is very long-range in proportion to the size of individual bacteria. Geostatistical modeling analysis implicated the importance of AHL-gradients mediating effective communication between remote cells. We conclude that AHL-mediated cell-to-cell communication occurs not only within dense populations, but also in very small groups and over long ranges between individual bacteria, and therefore this cellular activity is more commonplace and effective than hitherto predicted. We propose that this cell-to-cell communication is governed more by the in situ spatial proximity of cells within AHL-gradients than the requirement for a quorum group of high population density.

Rhizobia, the root-nodule endosymbionts of leguminous plants, also form natural endophytic associations with roots of important cereal plants. Despite its widespread occurrence, much remains unknown about colonization of cereals by rhizobia. We examined the infection, dissemination, and colonization of healthy rice plant tissues by four species of gfp-tagged rhizobia and their influence on the growth physiology of rice. The results indicated a dynamic infection process beginning with surface colonization of the rhizoplane (especially at lateral root emergence), followed by endophytic colonization within roots, and then ascending endophytic migration into the stem base, leaf sheath, and leaves where they developed high populations. In situ CMEIAS image analysis indicated local endophytic population densities reaching as high as 9 x 10(10) rhizobia per cm3 of infected host tissues, whereas plating experiments indicated rapid, transient or persistent growth depending on the rhizobial strain and rice tissue examined. Rice plants inoculated with certain test strains of gfp-tagged rhizobia produced significantly higher root and shoot biomass; increased their photosynthetic rate, stomatal conductance, transpiration velocity, water utilization efficiency, and flag leaf area (considered to possess the highest photosynthetic activity); and accumulated higher levels of indoleacetic acid and gibberellin growth-regulating phytohormones. Considered collectively, the results indicate that this endophytic plant-bacterium association is far more inclusive, invasive, and dynamic than previously thought, including dissemination in both below-ground and above-ground tissues and enhancement of growth physiology by several rhizobial species, therefore heightening its interest and potential value as a biofertilizer strategy for sustainable agriculture to produce the world's most important cereal crops.

Neptunia natans is a unique aquatic legume indigenous to tropical and sub-tropical regions and is nodulated symbiotically by rhizobia using an unusual infection process unlike any previously described. Previously, isolates of neptunia-nodulating rhizobia from Senegal were characterized as Allorhizobium undicola. Here we report on a different group of neptunia-nodulating rhizobia isolated from India. Sequencing of the 16S rDNA gene from two of these Indian isolates (strains J1T and J2) show that they belong in the genus Devosia rather than Allorhizobium. Currently, the only described Devosia species is D. riboflavina (family Hyphomicrobiaceae, order Rhizobiales). The complete 16S rDNA sequences of strains J1T and J2 are 95.9% homologous to the type strain, D. riboflavina LMG 2277T, suggesting that these neptunia-nodulating strains from India belong to a new Devosia species. This hypothesis was confirmed by further studies of polyphasic taxonomy (DNA-DNA hybridisation, TP-RAPD patterns, SDS-PAGE of cellular proteins, 16S rDNA RFLP patterns, carbon source utilisation, cellular fatty acid analysis and other phenotypic characterisations), all of which support the proposal that these neptunia-nodulating strains constitute a new Devosia species, which we name Devosia neptuniae sp. nov. These gram negative, strictly aerobic short rods are motile by a subpolar flagellum, positive for catalase, oxidase, urease and beta-galactosidase, can utilise several carbohydrates (but not organic acids) as carbon sources and contain C18:0 3-OH, cis-7 C18:1 11-methyl and cis-7 C18:1 as their major cellular fatty acids. Unlike D. riboflavina, the longer-chain C24:1 3-OH and C26:1 3-OH hydroxy fatty acids are not detected. The type strain of D. neptuniae is LMG 21357T (CECT 5650T). Assignment of this new taxon represents the fourth example in the literature of a non-rhizobial genus of bacteria capable of forming a bonafide dinitrogen-fixing root-nodule symbiosis with legume plants.

Rhizobia are the common bacterial symbionts that form nitrogen-fixing root nodules in legumes. However, recently other bacteria have been shown to nodulate and fix nitrogen symbiotically with these plants. Neptunia natans is an aquatic legume indigenous to tropical and subtropical regions and in African soils is nodulated by Allorhizobium undicola. This legume develops an unusual root-nodule symbiosis on floating stems in aquatic environments through a unique infection process. Here, we analyzed the low-molecular-weight RNA and 16S ribosomal DNA (rDNA) sequence of the same fast-growing isolates from India that were previously used to define the developmental morphology of the unique infection process in this symbiosis with N. natans and found that they are phylogenetically located in the genus Devosia, not Allorhizobium or RHIZOBIUM: The 16S rDNA sequences of these two Neptunia-nodulating Devosia strains differ from the only species currently described in that genus, Devosia riboflavina. From the same isolated colonies, we also located their nodD and nifH genes involved in nodulation and nitrogen fixation on a plasmid of approximately 170 kb. Sequence analysis showed that their nodD and nifH genes are most closely related to nodD and nifH of Rhizobium tropici, suggesting that this newly described Neptunia-nodulating Devosia species may have acquired these symbiotic genes by horizontal transfer.

This work is the completion of a series of reports describing the nitrogen-fixing bacterial symbionts of sulla (Hedysarum coronarium L., Leguminosae) and providing the grounds for their proposal as a new taxon. The introduction summarizes a large amount of previous evidence gathered on the physiology, genetics and ecology of such organisms, which have in the past been referred to provisionally as 'Rhizobium hedysari'. Upon adding 16S RNA sequencing, amplified rDNA restriction analysis of the rrn operon, DNA-DNA hybridization homology and analysis of low-molecular-mass RNA species, it is concluded that the group of strains that specifically nodulate sulla consists of a coherent set of isolates that differ from previously described rhizobia to an extent that warrants the constitution of the species boundary. The name Rhizobium sullae sp. nov. is proposed, with isolate 1S123T (=USDA 4950T = DSM 14623T) as the type strain.

We compared carbon flow under constant low-substrate conditions (below 20 microM glucose in situ) in laboratory-scale glucose-fed methanogenic bioreactors containing two very different microbial communities that removed chemical oxygen demand at similar rates. One community contained approximately equal proportions of spiral and cocci morphologies, while the other community was dominated by cocci. In the former bioreactor, over 50% of the cloned SSU rRNA genes and the most common SSU rDNA terminal restriction fragment corresponded to Spirochaetaceae-related sequences, while in the latter bioreactor over 50% of the cloned SSU rRNA genes and the most common SSU rDNA terminal restriction fragment corresponded to Streptococcus-related sequences. Carbon flow was assessed by measuring 14C-labeled metabolites derived from a feeding of [U-14C]glucose that did not alter the concentration of glucose in the bioreactors. Acetate and ethanol were detected in the Spirochaetaceae-dominated reactor, whereas acetate and propionate were detected in the Streptococcus-dominated reactor. A spirochete isolated from a Spirochaetaceae-dominated reactor fermented glucose to acetate, ethanol, and small amounts of lactate. Maximum substrate utilization assays carried out on fluid from the same reactor indicated that acetate and ethanol were rapidly utilized by this community. These data indicate that an acetate- and ethanol-based food chain was present in the Spirochaetaceae-dominated bioreactor, while the typical acetate- and propionate-based food chain was prevalent in the Streptococcus-dominated bioreactor.

A central event of the infection process in the Rhizobium-legume symbiosis is the modification of the host cell wall barrier to form a portal of entry large enough for bacterial penetration. Transmission electron microscopy (TEM) indicates that rhizobia enter the legume root hair through a completely eroded hole that is slightly larger than the bacterial cell and is presumably created by localized enzymatic hydrolysis of the host cell wall. In this study, we have used microscopy and enzymology to further clarify how rhizobia modify root epidermal cell walls to shed new light on the mechanism of primary host infection in the Rhizobium-legume symbiosis. Quantitative scanning electron microscopy indicated that the incidence of highly localized, partially eroded pits on legume root epidermal walls that follow the contour of the rhizobial cell was higher in host than in nonhost legume combinations, was inhibited by high nitrate supply, and was not induced by immobilized wild-type chitolipooligosaccharide Nod factors reversibly adsorbed to latex beads. TEM examination of these partially eroded, epidermal pits indicated that the amorphous, noncrystalline portions of the wall were disrupted, whereas the crystalline portions remained ultrastructurally intact. Further studies using phase-contrast and polarized light microscopy indicated that (i) the structural integrity of clover root hair walls is dependent on wall polymers that are valid substrates for cell-bound polysaccharide-degrading enzymes from rhizobia, (ii) the major site where these rhizobial enzymes can completely erode the root hair wall is highly localized at the isotropic, noncrystalline apex of the root hair tip, and (iii) the degradability of clover root hair walls by rhizobial polysaccharide-degrading enzymes is enhanced by modifications induced during growth in the presence of chitolipooligosaccharide Nod factors from wild-type clover rhizobia. The results suggest a complementary role of rhizobial cell-bound glycanases and chitolipooligosaccharides in creating the localized portals of entry for successful primary host infection.

A major challenge in microbial ecology is to develop reliable and facile methods of computer-assisted microscopy that can analyze digital images of complex microbial communities at single cell resolution, and compute useful quantitative characteristics of their organization and structure without cultivation. Here we describe a computer-aided interactive system to analyze the high degree of morphological diversity in growing microbial communities revealed by phase-contrast microscopy. The system, called "CMEIAS" (Center for Microbial Ecology Image Analysis System) consists of several custom plug-ins for UTHSCSA ImageTool, a free downloadable image analysis program operating on a personal computer in a Windows NT environment. CMEIAS uses various measurement features and two object classifiers to extract size and shape measurements of segmented, digital images of microorganisms and classify them into their appropriate morphotype. The first object classifier uses a single measurement feature to analyze relatively simple communities containing only a few morphotypes (e.g., regular rods, cocci, filaments). A second new hierarchical tree classifier uses an optimized subset of multiple measurement features to analyze significantly more complex communities containing greater morphological diversity than ever before possible. This CMEIAS shape classifier automatically categorizes each cell into one of 11 predominant bacterial morphotypes, including cocci, spirals, curved rods, U-shaped rods, regular straight rods, unbranched filaments, ellipsoids, clubs, rods with extended prostheca, rudimentary branched rods, and branched filaments. The training and testing images for development and evaluation of the CMEIAS classifier were obtained from 1,937 phase-contrast grayscale digital images of various diverse communities. The CMEIAS shape classifier had an accuracy of 96.0% on a training set of 1,471 cells and 97.0% on a test set of 4,270 cells representing all 11 bacterial morphotype classes, indicating that accurate classification of rich morphological diversity in microbial communities is now possible. An interactive edit feature was added to address the main sources of error in automatic shape classification, enabling the operator to inspect the assigned morphotype of each bacterium based on visual recognition of its distinctive pseudocolor, reassign it to another morphotype class if necessary, and add up to five other morphotypes to the classification scheme. The shape classifier reports on the number and types of different morphotypes present and the abundance among each of them, thus providing the data needed to compute the morphological diversity within the microbial community. An example of how CMEIAS can augment the analysis of microbial community structure is illustrated by studies of morphological diversity as an indicator of dynamic ecological succession following a nutrient shift-up perturbation in two continuously fed, anaerobic bioreactors with morphologically distinct start communities. Various steps to minimize the limitations of computer-assisted microscopy to classify bacterial morphotypes using CMEIAS are described. In summary, CMEIAS is an accurate, robust, flexible semiautomatic computing tool that can significantly enhance the ability to quantitate bacterial morphotype diversity and should serve as a useful adjunct to the analysis of microbial community structure. This first version of CMEIAS will be released as free, downloadable plug-ins so it can provide wide application in studies of microbial ecology.

Methanogenic bioreactor communities were used as model ecosystems to evaluate the relationship between functional stability and community structure. Replicated methanogenic bioreactor communities with two different community structures were established. The effect of a substrate loading shock on population dynamics in each microbial community was examined by using morphological analysis, small-subunit (SSU) rRNA oligonucleotide probes, amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), and partial sequencing of SSU rDNA clones. One set of replicated communities, designated the high-spirochete (HS) set, was characterized by good replicability, a high proportion of spiral and short thin rod morphotypes, a dominance of spirochete-related SSU rDNA genes, and a high percentage of Methanosarcina-related SSU rRNA. The second set of communities, designated the low-spirochete (LS) set, was characterized by incomplete replicability, higher morphotype diversity dominated by cocci, a predominance of Streptococcus-related and deeply branching Spirochaetales-related SSU rDNA genes, and a high percentage of Methanosaeta-related SSU rRNA. In the HS communities, glucose perturbation caused a dramatic shift in the relative abundance of fermentative bacteria, with temporary displacement of spirochete-related ribotypes by Eubacterium-related ribotypes, followed by a return to the preperturbation community structure. The LS communities were less perturbed, with Streptococcus-related organisms remaining prevalent after the glucose shock, although changes in the relative abundance of minor members were detected by morphotype analysis. A companion paper demonstrates that the more stable LS communities were less functionally stable than the HS communities (S. A. Hashsham, A. S. Fernandez, S. L. Dollhopf, F. B. Dazzo, R. F. Hickey, J. M. Tiedje, and C. S. Criddle, Appl. Environ. Microbiol. 66:4050-4057, 2000).

Parallel processing is more stable than serial processing in many areas that employ interconnected activities. This hypothesis was tested for microbial community function using two quadruplicate sets of methanogenic communities, each set having substantially different populations. The two communities were maintained at a mean cell residence time of 16 days and a mean glucose loading rate of 0.34 g/liter-day in variable-volume reactors. To test stability to perturbation, they were subjected to an instantaneous glucose pulse that resulted in a 6.8-g/liter reactor concentration. The pattern of accumulated products in response to the perturbation was analyzed for various measures of functional stability, including resistance, resilience, and reactivity for each product. A new stability parameter, "moment of amplification envelope," was used to compare the soluble compound stability. These parameters indicated that the communities with predominantly parallel substrate processing were functionally more stable in response to the perturbation than the communities with predominantly serial substrate processing. The data also indicated that there was good replication of function under perturbed conditions; the degrees of replication were 0.79 and 0.83 for the two test communities.

Legume plants carefully control the extent of nodulation in response to rhizobial infection. To examine the mechanism underlying this process we conducted a detailed analysis of the Lotus japonicus hypernodulating mutants, har1-1, 2 and 3 that define a new locus, HYPERNODULATION ABERRANT ROOT FORMATION (Har1), involved in root and symbiotic development. Mutations in the Har1 locus alter root architecture by inhibiting root elongation, diminishing root diameter and stimulating lateral root initiation. At the cellular level these developmental alterations are associated with changes in the position and duration of root cell growth and result in a premature differentiation of har1-1 mutant root. No significant differences between har1-1 mutant and wild-type plants were detected with respect to root growth responses to 1-aminocyclopropane1-carboxylic acid, the immediate precursor of ethylene, and auxin; however, cytokinin in the presence of AVG (aminoetoxyvinylglycine) was found to stimulate root elongation of the har1-1 mutant but not the wild-type. After inoculation with Mesorhizobium loti, the har1 mutant lines display an unusual hypernodulation (HNR) response, characterized by unrestricted nodulation (hypernodulation), and a concomitant drastic inhibition of root and shoot growth. These observations implicate a role for the Har1 locus in both symbiotic and non-symbiotic development of L. japonicus, and suggest that regulatory processes controlling nodule organogenesis and nodule number are integrated in an overall mechanism governing root growth and development.

Rhizobium chitolipooligosaccharides (CLOSs) are heterogeneous fatty acylated N-acetyl glucosamine oligomers with variations in both the polar (hydrophilic) oligosaccharide head group and the non-polar (hydrophobic) fatty acyl chain. They trigger root hair deformation and cortical cell divisions in legume roots during development of the nitrogen-fixing root-nodule symbiosis. It has been proposed that only certain unique molecular species of CLOSs made by a particular rhizobia can elicit these responses on the corresponding legume host, suggesting that receptor-mediated perception of CLOSs serves as a basis of symbiotic specificity. We evaluated the relative symbiotic importance of the hydrophilic and hydrophobic structural domains of CLOSs by comparing the biological activities of CLOSs from wild type R. leguminosarum bv. trifolii ANU843 with that of various synthetic analogs. These tests were performed in axenic bioassays on the compatible symbiotic host, white clover (Trifolium repens) and the incompatible non-host legume, alfalfa (Medicago sativa). Fluorochrome-tagged derivatives of the native CLOSs and the analogs were also prepared in order to evaluate the uptake and localization patterns of these molecules within host root cells. The results indicate a direct link between uptake and biological activities of Rhizobium CLOSs on legume roots. The smallest CLOS analog taken up and biologically active on white clover and alfalfa was a N-fatty acylglucosamine, without an essential requirement of oligomerization, fatty N-acyl unsaturation, or acetate/sulfate functionalization. This suggests that N-fattyacylglucosamine is the common minimum structure required and sufficient for uptake and biological activity of CLOS glycolipids in these legumes, and that the various specific modifications of its polar head group and hydrophobic tail modulate its inherent ability to further express these activities, thus influencing which legumes are capable of responding to CLOSs rather than dictating their biological activities per se.

The Rhizobium leguminosarum bv trifolii exoB gene has been isolated by heterologous complementation of an exoB mutant of R. meliloti. We have cloned a chromosomal DNA fragment from the R. leguminosarum bv trifolii genome that contains an open reading frame of 981 bp showing 80% identity at the amino acid level to the UDP-glucose 4-epimerase of R. meliloti. This enzyme produces UDP-galactose, the donor of galactosyl residues for the lipid-linked oligosaccharide repeat units of various heteropolysaccharides of rhizobia. An R. leguminosarum bv trifolii exoB disruption mutant differed from the wild type in the structure of both the acidic exopolysaccharide and the lipopolysaccharide. The acidic exopolysaccharide made by our wild-type strain is similar to the Type 2 exopolysaccharide made by other R. leguminosarum bv trifolii wild types. The exopolysaccharide made by the exoB mutant lacked the galactose residue and the substitutions attached to it. The exoB mutant induced the development of abnormal root nodules and was almost completely unable to invade plant cells. Our results stress the importance of exoB in the Rhizobium-plant interaction.

We used bright-field, time-lapse video, cross-polarized, phase-contrast, and fluorescence microscopies to examine the influence of isolated chitolipooligosaccharides (CLOSs) from wild-type Rhizobium leguminosarum bv. trifolii on development of white clover root hairs, and the role of these bioactive glycolipids in primary host infection. CLOS action caused a threefold increase in the differentiation of root epidermal cells into root hairs. At maturity, root hairs were significantly longer because of an extended period of active elongation without a change in the elongation rate itself. Time-series image analysis showed that the morphological basis of CLOS-induced root hair deformation is a redirection of tip growth displaced from the medial axis as previously predicted. Further studies showed several newly described infection-related root hair responses to CLOSs, including the localized disruption of the normal crystallinity in cell wall architecture and the induction of new infection sites. The application of CLOS also enabled a NodC- mutant of R. leguminosarum bv. trifolii to progress further in the infection process by inducing bright refractile spot modifications of the deformed root hair walls. However, CLOSs did not rescue the ability of the NodC- mutant to induce marked curlings or infection threads within root hairs. These results indicate that CLOS Nod factors elicit several host responses that modulate the growth dynamics and symbiont infectibility of white clover root hairs but that CLOSs alone are not sufficient to permit successful entry of the bacteria into root hairs during primary host infection in the Rhizobium-clover symbiosis.

The bacterial gene nodE is the key determinant of host specificity in the Rhizobium leguminosarum-legume symbiosis and has been proposed to determined unique polyunsaturated fatty acyl moieties in chitolipooligosaccharides (CLOS) made by the bacterial symbiont. We evaluated nodE function by examining CLOS structures made by wild-type R. leguminosarum bv. trifolii ANU843, an isogenic nodE::Tn5 mutant, and a recombinant strain containing multiple copies of the pSym nod region of ANU843. 1H-NMR, electrospray ionization mass spectrometry, fast atom bombardment mass spectrometry, flame ionization detection-gas chromatography, gas chromatography/mass spectrometry, and high performance liquid chromatography/UV photodiode array analyses revealed that these bacterial strains made the same spectrum of CLOS species. We also found that ions in the mass spectra which were originally assigned to nodE-dependent CLOS species containing unique polyunsaturated fatty acids (Spaink, H. P., Bloemberg, G. V., van Brussel, A. A. N., Lugtenberg, B. J. J., van der Drift, K. M. G. M., Haverkamp, J., and Thomas-Oates, J. E. (1995) Mol. Plant-Microbe Interact. 8, 155-164) were actually due to sodium adducts of the major nodE-independent CLOS species. No evidence for nodE-dependent CLOSs was found for these strains. These results indicate a need to revise the current model to explain how nodE determines host range in the R. leguminosarum-legume symbiosis.

Several wild-type strains of Rhizobium meliloti isolated from alfalfa nodules exhibited different plasmid profiles, yet did not differ in growth rate in yeast-mannitol medium, utilization of 43 different carbon sources, intrinsic resistance to 14 antibiotics, or detection of 16 enzyme activities. In contrast, three measures of effectiveness in symbiotic nitrogen fixation with alfalfa (shoot length, dry weight, and nitrogen content) indicated that R. meliloti SAF22, whose plasmid profile differs from those of the other strains tested, is significantly less effective than other wild-type strains in symbiotic nitrogen fixation. Light microscopy of nodules infected with strain SAF22 showed an abnormal center of nitrogen fixation zone III, with bacteria occupying a smaller portion of the infected host cells and vacuoles occupying a significantly larger portion of adjacent uninfected host cells. In contrast, the effective nodules infected with other wild types or plasmid pRmSAF22c-cured segregants of SAF22 did not display this cytological abnormality.

The general view on Rhizobium chitolipooligosaccharides (CLOS) is that they are made in very low levels as diffusible molecules and are primarily secreted by the bacteria into the extracellular milieu where they interact with the host. However, the structural and predicted physicochemical properties of these amphiphilic molecules led us to postulate that they should normally be targeted to bacterial membranes after synthesis. Thus, we analyzed membrane lipid extracts of Rhizobium leguminosarum bv. trifolii wild-type strain ANU843 cells and the corresponding culture supernatants for CLOS-type glycolipids. As predicted, fractionation of the membrane extracts from pelleted cells led to the isolation of a diverse family of CLOS in high yield (> or = 15 mg/L of culture), whereas all attempts to isolate CLOS from the corresponding culture supernatant failed. Structural analyses reveal that the membrane CLOS of ANU843 consist of a complex mixture of O-acetylated or non-O-acetylated chito- tri-, -tetra-, and pentasaccharides bearing an N-acyl moiety at the nonreducing glucosamine residue. cis-Vaccenic acid was the predominant acyl substituent (> 70%), but several other saturated, unsaturated, and 3-hydroxy fatty acids were found in the CLOS glycolipids. Membrane accumulation of CLOS in ANU843 is promoted by the presence of 4',7-dihydroxyflavone and pSym nod genes. Potential host-selective biological activity host-selective biological activity of the purified membrane CLOS fraction from ANU843 was indicated by its ability to elicit meristems resembling rudimentary nodule primordia in the root cortex of axenic seedlings of the host legume, white clover, but not of the nonhost legumes hairy vetch or alfalfa.(ABSTRACT TRUNCATED AT 250 WORDS)

Two transposon Tn5-induced mutants of wild-type broad-host-range Rhizobium sp. GRH2 were isolated and found to harbour different alterations in surface polysaccharides. These mutants, designated GRH2-14 and GRH2-50, induced a few, empty nodules on Acacia and lost the ability to nodulate most host herbaceous legumes. Whereas mutant GRH2-14 produces an acidic exopolysaccharide (EPS) similar to the wild-type, the acidic EPS of mutant GRH2-50 lacks galactose and the pyruvyl and 3-hydroxybutyryl substituents attached to this sugar moiety. In addition, both mutants GRH2-50 and GRH2-14 were altered in smooth lipopolysaccharides (LPS). DNA sequence analyses of the corresponding Tn5 insertions revealed that strain GRH2-50 was mutated in a DNA locus homologous to galE, and in vitro enzyme assays indicated that the UDPglucose 4-epimerase (GalE) activity was missing in this mutant strain. DNA hybridization studies showed that the GRH2-50 mutant DNA has homologous sequences within the different biovars of Rhizobium leguminosarum. However, no DNA homology to GRH2-14 altered DNA was found in those rhizobial strains, indicating that it represents a new chromosomal lps locus in Rhizobium sp. (Acacia) involved in symbiotic development.

Rhizobium leguminosarum bv. trifolii is the bacterial symbiont which induces nitrogen-fixing root nodules on the leguminous host, white clover (Trifolium repens L.). In this plant-microbe interaction, the host plant excretes a flavone, 4',7-dihydroxyflavone (DHF), which activates expression of modulation genes, enabling the bacterial symbiont to elicit various symbiosis-related morphological changes in its roots. We have investigated the accumulation of a diglycosyl diacylglycerol (BF-7) in wild-type R. leguminosarum bv. trifolii ANU843 when grown with DHF and the biological activities of this glycolipid bacterial factor on host and nonhost legumes. In vivo labeling studies indicated that wild-type ANU843 cells accumulate BF-7 in response to DHF, and this flavone-enhanced alteration in membrane glycolipid composition was suppressed in isogenic nodA::Tn5 and nodD::Tn5 mutant derivatives. Seedling bioassays performed under microbiologically controlled conditions indicated that subnanomolar concentrations of purified BF-7 elicit various symbiosis-related morphological responses on white clover roots, including thick short roots, root hair deformation, and foci of cortical cell divisions. Roots of the nonhost legumes alfalfa and vetch were much less responsive to BF-7 at these low concentrations. A structurally distinct diglycosyl diacylglycerol did not induce these responses on white clover, indicating structural constraints in the biological activity of BF-7 on this legume host. In bioassays using aminoethoxyvinylglycine to suppress plant production of ethylene, BF-7 elicited a meristematic rather than collaroid type of mitogenic response in the root cortex of white clover. These results indicate an involvement of flavone-activated nod expression in membrane accumulation of BF-7 and a potent ability of this diglycosyl diacylglycerol glycolipid to perform as a bacterial factor enabling R. leguminosarum bv. trifolii to activate segments of its host's symbiotic program during early development of the root nodule symbiosis.

The phospholipid and associated fatty acid compositions of the bacterial symbiont of clover, Rhizobium leguminosarum biovar trifolii wild-type ANU843, was analyzed by two-dimensional silica thin-layer chromatography, fast atom bombardment-mass spectrometry, flame-ionization detection gas-liquid chromatography and combined gas-liquid chromatography/mass spectrometry. The phospholipid composition included phosphatidylethanolamine (15%), N-methylphosphatidylethanolamine (47%), N,N-dimethylphosphatidylethanolamine (9%), phosphatidylglycerol (19%), cardiolipin (5%) and phosphatidylcholine (2%). Fatty acid composition included predominantly cis-11-octadecenoic acid, lower levels of cis-9-hexadecenoic acid, hexadecanoic acid, 11-methyl-11-octadecenoic acid, octadecanoic acid, 11,12-methyleneoctadecanoic acid, eicosanoic acid and traces of branched, and di- and triunsaturated fatty acids. The influence of expression of the "nodulation" genes encoding symbiotic functions on the composition of these membrane lipids was examined in wild-type cells grown with or without the flavone inducer, 4',7-dihydroxyflavone and in mutated cells lacking the entire symbiotic plasmid where these genes reside, or containing single transposon insertions in selected nodulation genes. No significant changes in phospholipid or associated fatty acid compositions were detected by the above methods of analysis.

We obtained from a phospholipid extract of wild-type Rhizobium leguminosarum bv. trifolii ANU843 methoxylated fatty acids that had been previously reported as constitutive unusual Rhizobium fatty acids. The use of deuterated reagents and subsequent gas-liquid chromatography-mass spectrometry analyses showed that these methoxylated fatty acid derivatives are the products of chemical alterations of common cyclopropane-containing and unsaturated fatty acids occurring during various acid-catalyzed transesterification treatments aimed at producing the methyl ester derivatives. Similar results were obtained from a phospholipid extract of Escherichia coli K-12. In contrast, these chemical alterations were not induced by an alkaline methanolysis method of transesterification. If an acidic treatment is needed to release the fatty acids from the source molecule, the finding of unusual methoxylated fatty acids should be carefully confirmed with deuterated reagents.

Rhizobium sp. wild-type strain GRH2 was originally isolated from root nodules of the leguminous tree Acacia cyanophylla and has a broad host range which includes herbaceous legumes, e.g., Trifolium spp. We examined the extracellular exopolysaccharides (EPSs) produced by strain GRH2 and found three independent glycosidic structures: a high-molecular-weight acidic heteropolysaccharide which is very similar to the acidic EPS produced by Rhizobium leguminosarum biovar trifolii ANU843, a low-molecular-weight native heterooligosaccharide resembling a dimer of the repeat unit of the high-molecular-weight EPS, and low-molecular-weight neutral beta (1,2)-glucans. A Tn5 insertion mutant derivative of GRH2 (exo-57) that fails to form acidic heteropolysaccharides was obtained. This Exo- mutant formed nitrogen-fixing nodules on Acacia plants but infected a smaller proportion of cells in the central zone of the nodules than did wild-type GRH2. In addition, the exo-57 mutant failed to nodulate several herbaceous legume hosts that are nodulated by wild-type strain GRH2.

The activities of salt-elutable peroxidases from roots of white clover and pea were examined during the early interaction of these legume hosts with strains of Rhizobium leguminosarum in homologous and heterologous combination. Peroxidase-specific activity from clover root hairs began to increase 6 hr after inoculation with R. l. bv. viciae RL300 and was localized over the entire area of their deformations. In contrast, the onset of elicitation of peroxidase activity from root hairs was delayed after inoculation with R. l. bv. trifolii ANU843 and was localized only at the site of infection thread initiation. Three wild-type strains (R. l. bv. trifolii ANU843, R. l. bv. viciae RL300 and 1003) and one hybrid transconjugant strain of R. leguminosarum containing pSym from R. l. bv. viciae 248 (RBL5715) elicited increased specific activity of peroxidases eluted from pea and clover roots in heterologous combination. A comparison of peroxidase activity eluted from pea roots inoculated with ANU843 or its pSym-cured derivative indicated that pSym is required for elicitation of peroxidase on this heterologous host. The level of peroxidase activity elicited by nodE mutants (which have extended host range) is decreased on their new host. An extracellular fraction of RL300 contained flavonoid-dependent, heat-stable, and ethanol-soluble elicitor(s) of peroxidase activity. Treatment of clover seedlings with this cell-free fraction decreased the number of root hairs infected by ANU843. We propose that elicitation of root hair peroxidase may contribute to the infection process in this Rhizobium-legume symbiosis by altering root hair wall structure at sites of incipient penetration.

A novel glycolipid was isolated by chloroform-methanol extraction of Rhizobium trifolii ANU843 cells. Compositional analysis, methylation studies, 1H NMR and spectroscopies led to the identification of a diglycosyl diacylglyceride: 1,2-di-O-acyl-3-O-[alpha-D-glucopyranosyl-(1----3)-O-alpha-D- mannopyranosyl]glycerol. Iso-hexadecanoic and anteiso-heptadecanoic acids were the predominant fatty acids esterifying the glyceryl moiety, but a microheterogeneity in fatty acid composition was found, resulting in at least five distinct molecular species of the glycolipid. Although widespread in plants, animals and Gram-positive bacteria, glycosyl glycerides have been seldom reported in Gram-negative bacteria and this work is the first evidence of their occurrence in the bacterial family Rhizobiaceae.

The involvement of Rhizobium enzymes that degrade plant cell wall polymers has long been an unresolved question about the infection process in root nodule symbiosis. Here we report the production of enzymes from Rhizobium leguminosarum bv. trifolii that degrade carboxymethyl cellulose and polypectate model substrates with sensitive methods that reliably detect the enzyme activities: a double-layer plate assay, quantitation of reducing sugars with a bicinchoninate reagent, and activity gel electrophoresis-isoelectric focusing. Both enzyme activities were (i) produced commonly by diverse wild-type strains, (ii) cell bound with at least some of the activity associated with the cell envelope, and (iii) not changed appreciably by growth in the presence of the model substrates or a flavone that activates expression of nodulation (nod) genes on the resident symbiotic plasmid (pSym). Equivalent levels of carboxymethyl cellulase activity were found in wild-type strain ANU843 and its pSym-cured derivative, ANU845, consistent with previous results of Morales et al. (V. Morales, E. Martínez-Molina, and D. Hubbell, Plant Soil 80:407-415, 1984). However, polygalacturonase activity was lower in ANU845 and was not restored to wild-type levels in the recombinant derivative of pSym- ANU845 containing the common and host-specific nod genes within a 14-kb HindIII DNA fragment of pSym from ANU843 cloned on plasmid pRt032. Activity gel electrophoresis resolved three carboxymethyl cellulase isozymes of approximately 102, 56, and 33 kDa in cell extracts from ANU843. Isoelectric focusing activity gels revealed one ANU843 polygalacturonase isozyme with a pI of approximately 7.2. These studies show that R. leguminosarum bv. trifolii produces multiple enzymes that cleave glycosidic bonds in plant cell walls and that are cell bound.

An extracellular metabolite purified from Rhizobium trifolii ANU843 was established as N-acetylglutamic acid (GluNAc) by 1H NMR and Fourier transform IR spectroscopy, gas chromatography/mass spectrometry of its methylated product, and organic synthesis. TLC analyses indicated that extracellular accumulation of GluNAc by R. trifolii ANU843 grown in defined BIII culture medium was dependent on induction of its bacterial nodulation (nod) genes and the positive regulatory gene nodD on its symbiotic plasmid. 1H NMR analyses showed less GluNAc in fractionated culture supernatants of nodL and nodM mutant derivatives of R. trifolii ANU843. GluNAc induced three morphological responses on axenic roots of white clover seedlings: (i) root hair branching; (ii) tip swelling followed by resumed elongation of root hairs; and (iii) a slight increase in foci of cortical cell divisions, which developed into nodule-like primordia. These biological activities of extracellular GluNAc from R. trifolii ANU843 were confirmed with authentic standards of GluNAc. These results indicate that extracellular accumulation of N-acetylglutamic acid is linked to flavone-dependent metabolism involving nodD, nodL, and nodM in R. trifolii ANU843. This constitutes the first report on the structure of a nod-dependent extracellular signal from R. trifolii that can affect root hair and nodule development in white clover and whose biological activity on this host has been confirmed with authentic standards.

The interaction between Rhizobium lipopolysaccharide (LPS) and white clover roots was examined. The Limulus lysate assay indicated that Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) released LPS into the external root environment of slide cultures. Immunofluorescence and immunoelectron microscopy showed that purified LPS from R. trifolii 0403 bound rapidly to root hair tips and infiltrated across the root hair wall. Infection thread formation in root hairs was promoted by preinoculation treatment of roots with R. trifolii LPS at a low dose (up to 5 micrograms per plant) but inhibited at a higher dose. This biological activity of LPS was restricted to the region of the root present at the time of exposure to LPS, higher with LPS from cells in the early stationary phase than in the mid-exponential phase, incubation time dependent, incapable of reversing inhibition of infection by NO3- or NH4+, and conserved among serologically distinct LPSs from several wild-type R. trifolii strains (0403, 2S-2, and ANU843). In contrast, infections were not increased by preinoculation treatment of roots with LPSs from R. leguminosarum bv. viciae strain 300, R. meliloti 102F28, or members of the family Enterobacteriaceae. Most infection threads developed successfully in root hairs pretreated with R. trifolii LPS, whereas many infections aborted near their origins and accumulated brown deposits if pretreated with LPS from R. meliloti 102F28. LPS from R. leguminosarum 300 also caused most infection threads to abort. Other specific responses of root hairs to infection-stimulating LPS from R. trifolii included acceleration of cytoplasmic streaming and production of novel proteins. Combined gas chromatography-mass spectroscopy and proton nuclear magnetic resonance analyses indicated that biologically active LPS from R. trifolii 0403 in the early stationary phase had less fucose but more 2-O-methylfucose, quinovosamine, 3,6-dideoxy-3-(methylamino)galactose, and noncarbohydrate substituents (O-methyl, N-methyl, and acetyl groups) on glycosyl components than did inactive LPS in the mid-exponential phase. We conclude that LPS-root hair interactions trigger metabolic events that have a significant impact on successful development of infection threads in this Rhizobium-legume symbiosis.

A polysaccharide depolymerase isolated from the phage lysate of Rhizobium trifolii 4S was used to fragment capsular polysaccharides (CPS) and extracellular polysaccharides (EPS) of R. trifolii 0403 into oligosaccharides. These products were analyzed for clover lectin (trifoliin A)-binding ability, effect on infection of white clover root hairs, and changes in glycosyl and noncarbohydrate composition with culture age. The oligosaccharides from CPS of cultures grown on agar plates for 3, 5, and 7 days exhibited lectin-binding ability at levels similar to those of the corresponding intact CPS. The intact EPS did not bind to clover lectin, although the oligosaccharide fragments from EPS did. In contrast, oligosaccharides from deacetylated CPS had less than half the lectin-binding ability of the native polysaccharide substrate. The CPS from 5-day-old cultures, its corresponding oligosaccharide fragments, and the oligosaccharide fragments of EPS from 5-day-old cultures, all at a concentration of 2.5 micrograms per seedling, stimulated infection thread formation in root hairs of clover seedlings inoculated with R. trifolii 0403. Thus, this bacteriophage-induced polysaccharide depolymerase converted the acidic CPS and EPS of R. trifolii 0403 into biologically active oligosaccharides capable of binding trifoliin A and stimulating root hair infection. The amount of the noncarbohydrate substitutions (pyruvate, acetate, and ether-linked 3-hydroxybutyrate) in the CPS oligosaccharides changed with culture age as shown by 1H-nuclear magnetic resonance spectroscopy. The binding of trifoliin A, therefore, appears to be sensitive to changes in the degree of substitution of noncarbohydrate substitutions in the CPS of R. trifolii 0403.

In-vivo synthesis of the white-clover lectin, trifoliin A, was examined by the incorporation of labeled amino acids into protein during heterotrophic growth of intact Trifolium repens L. seedlings. Lectin synthesis was quantified by measuring the level of labeled protein immunoprecipitated from root exudate, from the hapten (2-deoxyglucose) eluate of the roots, and from root and shoot homogenates. The presence of labeled trifoliin A was confirmed by non-denaturing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by fluorography and comparison with trifoliin A standards. In-vivo-labeled trifoliin A was detected in seedling root homogenate 2 h after the addition of labeled amino acids and on the root surface by 8 h. Incorporation of labeled amino acids into protein and trifoliin A was greatest with 2-d-old seedlings and was greater when the plants were grown continuously in the dark than when they were exposed to 14 h light daily. Significantly more labeled lectin accumulated on the root surface of seedlings grown with 1.5 mM KNO3 than of seedlings grown either without N or with 15.0 mM KNO3. The labeled lectin from the root surface in all nitrate treatments and from the rootexudate samples of seedlings grown N-free and with 1.5 mM KNO3 was fully able to bind to Rhizobium trifolii. In contrast, only 2% of the immunoprecipitable protein found in the root exudate of seedlings grown with 15.0 mM KNO3 was able to bind to the bacteria. Thus, excess nitrate does not repress the synthesis of trifoliin A in the root, but does affect the distribution and activity of this newly synthesized lectin in a way which reduces its ability to interact with R. trifolii. By using Western blot analysis, much more total trifoliin A is detected in the homogenates of shoots than roots. However, greater than 80% of the total labeled protein and 85-90% of the total labeled lectin were found in the root homogenates of 2-d-old dark-grown seedlings incubated for 5 h with labeled amino acids. In addition, Western blot analysis indicated that the shoot homogenate contained smaller-molecular-weight peptides which reacted with the specific anti-trifoliin A antibody. These studies indicate that stored trifoliin A in the seed is degraded in the shoots during seedling development, while newly synthesized trifoliin A in the roots is excreted to the root surface and external environment.

The complete structure of the acidic, extracellular, capsular polysaccharide of Rhizobium trifolii 843 has been elucidated by a combination of chemical, enzymic, and spectroscopic methods, confirming an earlier proposed sugar sequence and assigning the locations of the acyl substituents. The polysaccharide was depolymerized by a lyase into octasaccharide units which were uniform in carbohydrate composition and linkage. These units also contained a uniform distribution of acetyl and pyruvic acetal [O-(1-carboxyethylidene)] groups, and half of them were further acylated with D-3-hydroxybutanoyl groups. A much smaller proportion (less than 5%) of the oligomers was further acylated by a second D-3-hydroxybutanoyl group. The locations of the subtituents were determined chemically and by J-correlated, 1H-n.m.r. spectroscopy, proton nuclear Overhauser effect (n.O.e.) measurements, double-rd structure of the carbohydrate chain were determined by methylation analysis using g.l.c.-m.s. fast-atom-bombardment mass spectrometry, and n.m.r. studies on the reduced, deacylated oligomer. Structural studies were supplemented by n.m.r. analyses on the original polymer. The oligosaccharides were found to be branched octasaccharides with four sugar residues in each branch, and the carbohydrate sequence agreed well with that expected from earlier work. In the abbreviated sequence and structure (1a), the sugar residues are labelled "a" through "h". The main chain (a-d) is composed of a 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid group (a) that is linked to O-4 of a 3-O-acetyl-D-glucosyluronic acid residue (b) which is beta-linked to O-4 of a D-glucosyl residue (c). Residue c is beta-linked to O-4 of the branching D-glucose residue (d). The side chain consists of a substituted D-galactosyl group (h) which is beta-linked to O-3 of residue 9 of a beta-(1----4)-linked D-glucose trisaccharide (fragment e-f-g). The reducing end of the resulting tetrasaccharide (e-f-g-h) is beta-linked to O-6 of the branching D-glucose residue (d). In the native polymer, this branching residue is alpha-linked to O-4 of the modified D-glucuronic acid residue (a) which is the unsaturated sugar in the oligomer. A small proportion of the O-2 atoms of the acetylated D-glucosyluronic acid residues is acetylated because of ester migration. The two terminal sugars (g and h) of the branch chain bear 4,6-O-(1-carboxyethylidene) groups. The D-galactosyl groups of half of the oligomers are acylated by D-3-hydroxybutanoyl groups at O-3.(ABSTRACT TRUNCATED AT 400 WORDS)

Trifoliin A, a Rhizobium-binding glycoprotein from white clover, was detected in sterile clover root exudate by a sensitive immunofluorescence assay employing encapsulated cells of Rhizobium trifolii 0403 heat-fixed to microscope slides. Its presence in root exudate was further examined by immunoaffinity chromatography. The binding of trifoliin A to cells was specifically inhibited by the hapten, 2-deoxyglucose. Significantly higher quantities of trifoliin A were detected in root exudate of seedlings grown hydroponically in nitrogen-free medium than in rooting medium containing 15 mM NO-3, a concentration which completely suppressed root hair infection by the nitrogen-fixing symbiont. The presence of trifoliin A in root exudate may make it possible for recognition processes to occur before the microsymbiont attaches to its plant host.

Morphological changes which accompany nutrient enrichment of Rhizobium trifolii 0403 were studied. Assays of cell number and size coupled with scanning electron microscopy and immunofluorescence microscopy showed that succinate induces cells to stop dividing in vitro and to swell either in the cell center or at one cell pole. The extent and frequency of in vitro cell swelling were in direct relation to the concentration of succinate added to the enrichment medium. The in vitro swelling of cells in 16.6 mM succinate plus Casamino Acids, glucose, and yeast extract closely resembled that of bacteroids of R. trifolii 0403 in nitrogen-fixing nodules of white clover. We hypothesize that succinate may be involved in the transformation of vegetative bacteria into the bacteroid morphology found in nitrogen-fixing nodules.

Bacterial attachment is viewed as a cellular recognition event during the infection of legumes by the nitrogen-fixing symbiont, Rhizobium. Studies on the biochemical basis of selective attachment are reviewed, and suggest that this recognition process is accomplished by specific glycoprotein lectin-polysaccharide interactions on the surfaces of the symbionts. An understanding of host specificity may lead to ways to broaden the host range of nitrogen-fixing symbioses.

Genes that seem to be involved in the initial steps of infection of a legume by Rhizobium have been transferred, by transformation, to mutant strains of Azotobacter vinelandii that are unable to fix nitrogen. These genes code for a surface antigen that binds specifically to a protein from the host plant.

The validity of obtaining soil water for fecal coliform analyses by porcelain cup soil water samplers was examined. Numbers from samples of manure slurry drawn through porcelain cups were reduced 100- to 10,000,000-fold compared to numbers obtained from the external manure slurry, and 65% of the cups yielded coliform-free samples. Fecal coliforms adsorbed to cups apparently were released, thus influencing the counts of subsequent samples. Fecal coliforms persisted in soil water samplers buried in soil and thus could significantly influence the coliform counts of water samples obtained a month later. These studies indicate that porcelain cup soil water samplers do not yield valid water samples for fecal coliform analyses.

The activity of capsular polysaccharide pyruvyltransferase catalyzing the pyruvylation of acidic heteropolysaccharide was measured in Rhizobium trifolii 843 and 0403 rif. This enzyme activity was determined with EDTA-treated cells, uridine diphosphate-sugar precursors, and phosphoenol [1-14C]pyruvate. Activity was measured by the incorporation of radioactivity into organic solvent-soluble glycoconjugates. Enzymatic pyruvylation of capsular polysaccharide occurred from phosphoenolpyruvate at the lipid-bound saccharide stage.

Transmission electron microscopy was used to study the cellular morphologies of a wild-type Rhizobium meliloti strain (L5-30), a nitrogen fixation-ineffective (Fix-) succinate dehydrogenase mutant (Sdh-) strain, and a Fix+ Sdh+ revertant strain within alfalfa nodules and after free-living growth in a minimal medium containing 27 mM mannitol plus 20 mM succinate. The results showed a requirement of succinate dehydrogenase activity for symbiotic differentiation and maintenance of R. meliloti bacteroids within alfalfa nodules and for succinate-induced cellular pleomorphism in free-living cultures. Also, the Sdh- strain had a 3.5-fold lower rate of oxygen consumption in the defined medium than did the wild type.