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At least 19 recordsLinked to original sources

PubMed1997

The Sesbania Root Symbionts Sinorhizobium saheli and S. teranga bv. sesbaniae Can Form Stem Nodules on Sesbania rostrata, although They Are Less Adapted to Stem Nodulation than Azorhizobium caulinodans.

Sesbania species can establish symbiotic interactions with rhizobia from two taxonomically distant genera, including the Sesbania rostrata stem-nodulating Azorhizobium sp. and Azorhizobium caulinodans and the newly described Sinorhizobium saheli and Sinorhizobium teranga bv. sesbaniae, isolated from the roots of various Sesbania species. A collection of strains from both groups were analyzed for their symbiotic properties with different Sesbania species. S. saheli and S. teranga bv. sesbaniae strains were found to effectively stem nodulate Sesbania rostrata, showing that stem nodulation is not restricted to Azorhizobium. Sinorhizobia and azorhizobia, however, exhibited clear differences in other aspects of symbiosis. Unlike Azorhizobium, S. teranga bv. sesbaniae and S. saheli did not induce effective stem nodules on plants previously inoculated on the roots, although stem nodulation was arrested at different stages. For Sesbania rostrata root nodulation, Sinorhizobium appeared more sensitive than Azorhizobium to the presence of combined nitrogen. S. saheli and S. teranga bv. sesbaniae were effective symbionts with all Sesbania species tested, while Azorhizobium strains fixed nitrogen only in symbiosis with Sesbania rostrata. In a simple screening test, S. saheli and S. teranga bv. sesbaniae were incapable of asymbiotic nitrogenase activity. Thus, Azorhizobium can easily be distinguished from Sinorhizobium among Sesbania symbionts on the basis of symbiotic and free-living nitrogen fixation. The ability of Azorhizobium to overcome the systemic plant control appears to be a stem adaptation function. This last property, together with its host-specific symbiotic nitrogen fixation, makes Azorhizobium highly specialized for stem nodulation of the aquatic legume Sesbania rostrata.

Journal Article↗

PubMed1996

Azorhizobium caulinodans uses both cytochrome bd (quinol) and cytochrome cbb3 (cytochrome c) terminal oxidases for symbiotic N2 fixation.

Azorhizobium caulinodans employs both cytochrome bd (cytbd; quinol oxidase) and cytcbb3 (cytc oxidase) as terminal oxidases in environments with very low O2 concentrations. To investigate physiological roles of these two terminal oxidases both in microaerobic culture and in symbiosis, knockout mutants were constructed. As evidenced by visible absorbance spectra taken from mutant bacteria carrying perfect gene replacements, both the cytbd- and cytcbb3- mutations were null alleles. In aerobic culture under 2% O2 atmosphere, Azorhizobium cytbd- and cytcbb3- single mutants both fixed N2 at 70 to 90% of wild-type rates; in root nodule symbiosis, both single mutants fixed N2 at 50% of wild-type rates. In contrast, Azorhizobium cytbd- cytcbb3-double mutants, which carry both null alleles, completely lacked symbiotic N2 fixation activity. Therefore, both Azorhizobium cytbd and cytcbb3 oxidases drive respiration in environments with nanomolar O2 concentrations during symbiotic N2 fixation. In culture under a 2% O2 atmosphere, Azorhizobium cytbd- cytcbb3- double mutants fixed N2 at 70% of wild-type rates, presumably reflecting cytaa3 and cytbo (and other) terminal oxidase activities. In microaerobic continuous cultures in rich medium, Azorhizobium cytbd- and cytcbb3- single mutants were compared for their ability to deplete a limiting-O2 sparge; cytbd oxidase activity maintained dissolved O2 at 3.6 microM steady state, whereas cytcbb3 oxidase activity depleted O2 to submicromolar levels. Growth rates reflected this difference; cytcbb3 oxidase activity disproportionately supported microaerobic growth. Paradoxically, in O2 limited continuous culture, Azorhizobium cytbd oxidase is inactive below 3.6 microM dissolved O2 whereas in Sesbania rostrata symbiotic nodules, in which physiological, dissolved O2 is maintained at 10 to 20 nM, both Azorhizobium cytbd and cytcbb3 seem to contribute equally as respiratory terminal oxidases.

Aerobiosis↗

PubMed1994

Azorhizobium caulinodans respires with at least four terminal oxidases.

In culture, Azorhizobium caulinodans used at least four terminal oxidases, cytochrome aa3 (cytaa3), cytd, cyto, and a second a-type cytochrome, which together mediated general, respiratory electron (e-) transport to O2. To genetically dissect physiological roles for these various terminal oxidases, corresponding Azorhizobium apocytochrome genes were cloned, and three cytaa3 mutants, a cytd mutant, and a cytaa3, cytd double mutant were constructed by reverse genetics. These cytochrome oxidase mutants were tested for growth, oxidase activities, and N2 fixation properties both in culture and in symbiosis with the host plant Sesbania rostrata. The cytaa3 mutants grew normally, fixed N2 normally, and remained fully able to oxidize general respiratory e- donors (NADH, succinate) which utilize a cytc-dependent oxidase. By difference spectroscopy, a second, a-type cytochrome was detected in the cytaa3 mutants. This alternative a-type cytochrome (Amax = 610 nm) was also present in the wild type but was masked by bona fide cytaa3 (Amax = 605 nm). In late exponential-phase cultures, the cytaa3 mutants induced a new, membrane-bound, CO-binding cytc550, which also might serve as a cytc oxidase (a fifth terminal oxidase). The cloned Azorhizobium cytaa3 genes were strongly expressed during exponential growth but were deactivated prior to onset of stationary phase. Azorhizobium cytd mutants showed 40% lower N2 fixation rates in culture and in planta, but aerobic growth rates were wild type. The cytaa3, cytd double mutant showed 70% lower N2 fixation rates in planta. Pleiotropic cytc mutants were isolated by screening for strains unable to use N,N,N',N'-tetramethyl-p-phenylenediamine as a respiratory e- donor. These mutants synthesized no detectable cytc, excreted coproporphyrin, grew normally in aerobic minimal medium, grew poorly in rich medium, and fixed N2 poorly both in culture and in planta. Therefore, while aerobic growth was sustained by quinol oxidases alone, N2 fixation required cytc oxidase activities. Assuming that the terminal oxidases function as do their homologs in other bacteria, Azorhizobium respiration simultaneously employs both quinol and cytc oxidases. Because Azorhizobium terminal oxidase mutants were able to reformulate their terminal oxidase mix and grow more or less normally in aerobic culture, these terminal oxidases are somewhat degenerate. Its extensive terminal oxidase repertoire might allow Azorhizobium spp. to flourish in wide-ranging O2 environments.

Aerobiosis↗

PubMed1992

Conserved motifs in a divergent nod box of Azorhizobium caulinodans ORS571 reveal a common structure in promoters regulated by LysR-type proteins.

Nodulation of leguminous plants by Rhizobium, Bradyrhizobium, and Azorhizobium spp. is dependent on the induction by the plant host of different bacterial nodulation (nod) loci. The transcription of these nod loci is activated in the presence of plant-produced flavonoids upon binding of the NodD protein--a LysR-type activator--to specific sequences present in the nod promoters. Originally, a 47-base-pair (bp) region called the nod box was shown to be the target sequence for binding of NodD. From the comparison of the nod box sequences of (brady)rhizobia with a more divergent nod box from Azorhizobium caulinodans, we now propose a modular build-up of the nod box with the sequence A-T-C-N9-G-A-T as the binding target of the NodD protein (the NodD box). More generally, we show that LysR-type-regulated promoters contain the characteristic sequence T-N11-A as the core of an inverted repeat and propose this to be the "LysR motif" involved in specific binding to LysR-type proteins. Data obtained upon site-specific mutagenesis of this motif in the NodD box sustains this proposal. Further, we provide strong arguments that the inducer flavonoid, involved in transcriptional activation of Azorhizobium nod genes, interacts directly with the NodD protein, thereby increasing its binding affinities for the NodD box.

Base Sequence↗

PubMed1995

Interactive regulation of Azorhizobium nifA transcription via overlapping promoters.

The Azorhizobium nifA promoter (PnifA) is positively regulated by two physiological signal transduction pathways, NtrBC, which signals anabolic N status, and FixLJK, which signals prevailing O2 status. Yet, PnifA response (gene product per unit time) to these two activating signals together is more than twice that of the summed, individual signals. In the absence of NIFA, a negative PnifA autoregulator, the fully induced PnifA response is more than 10-fold greater than that of summed, individual signals. Given this synergism, these two signal transduction pathways must interactively regulate PnifA activity. PnifA carries three cis-acting elements, an anaerobox, which presumably binds FIXK, a NIFAbox, which presumably binds NIFA itself, and a sigma 54 box, which presumably binds sigma 54 initiator, a subunit of RNA polymerase. For combinatorial analysis, single, double, and triple promoter mutations were constructed in these cis-acting elements, and PnifA activities were measured in six different trans-acting background, i.e., fixK, fixJ, nifA, ntrC, rpoF, and wild type. Under all physiological conditions studied, high-level PnifA activity required both FIXK in trans and the anaerobox element in cis. Surprisingly, because PnifA was hyperactive with a mutated sigma 54box, this cis-acting element mediates both negative and positive control. Because PnifA hyperactivity also required a wild-type upstream NIFAbox element, even in the absence of NIFA, a second upstream nifA transcription start superimposed on the NIFAbox element was hypothesized. When nifA mRNA 5' start points were mapped by primer extension, both a minor upstream transcript(s) starting 45 bp distal to the anaerobox and a major downstream transcript starting 10 bp distal to the sigma 54 box were observed. In Azorhizobium, RNA polymerase sigma 54 initiator subunits are encoded by a multigene family, which includes rpoF and rpoN genes. Because rpoF mutants show an Ntr+ phenotype, whereas rpoN mutants are Ntr-, multiple sigma 54 initiators are functionally distinct. Two independent rpoF mutants both show a tight Nif- phenotype. Moreover, rpoF product sigma 54F is absolutely required for high-level PnifA activity. In summary, the Azorhizobium nifA gene carries overlapping housekeeping-type and sigma 54-type promoters which interactively respond to different signals. Effectively, the upstream, housekeeping-type promoter responds to FIXK and positively regulates the downstream, sigma 54-type promoter. The downstream, sigma 54-type promoter responds to NTRC and negatively regulates the upstream, housekeeping-type promoter. In terms of transcript yield, the upstream, housekeeping-type promoter is therefore weak, and the downstream, sigma 54-type promoter is strong.

Bacterial Proteins↗

PubMed2005

Azorhizobium doebereinerae sp. Nov. Microsymbiont of Sesbania virgata (Caz.) Pers.

Thirty-four rhizobium strains were isolated from root nodules of the fast-growing woody native species Sesbania virgata in different regions of southeast Brazil (Minas Gerais and Rio de Janeiro States). These isolates had cultural characteristics on YMA quite similar to Azorhizobium caulinodans (alkalinization, scant extracellular polysaccharide production, fast or intermediate growth rate). They exhibited a high similarity of phenotypic and genotypic characteristics among themselves and to a lesser extent with A. caulinodans. DNA:DNA hybridization and 16SrRNA sequences support their inclusion in the genus Azorhizobium, but not in the species A. caulinodans. The name A. doebereinerae is proposed, with isolate UFLA1-100 (=BR5401, =LMG9993=SEMIA 6401) as the type strain.

Azorhizobium↗

PubMed2003

Isolation and characterization of periplasmic cyclic beta-glucans of Azorhizobium caulinodans.

Oligoglucose molecules isolated from Azorhizobium caulinodans were characterized by compositional analysis, Smith degradation, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and (1)H and (13)C nuclear magnetic resonance analysis. A. caulinodans produced nonbranched and unsubstituted cyclic glucans composed solely of glucose, with the degree of polymerization ranging from 10 to 13. A major fraction of the periplasmic glucans contains 11 glucose residues within rings. The glucose residues are linked by beta-(1,3) and beta-(1,6) glycosidic bonds. These molecules seem to be quite similar to the periplasmic beta-(1,3);(1,6)-glucans synthesized by the Bradyrhizobium strain and are substantially different from the cyclic beta-(1,2)-glucans produced by Agrobacterium and Sinorhizobium species. Azorhizobial cyclic glucan synthesis is not osmoregulated. The response to the osmotic stress in Azorhizobium can be regulated similarly to Brucella spp. It is probable that the biosynthesis of beta-glucans is subject to the feedback control mechanism.

Azorhizobium caulinodans↗

PubMed2000

The xylem of rice (Oryza sativa) is colonized by Azorhizobium caulinodans.

Following inoculation with Azorhizobium caulinodans ORS571 (pXLGD4), lateral root development of rice and colonization of lateral root cracks by bacteria were shown to be stimulated by the flavonoid naringenin. Rice seedlings growing aseptically in the presence of naringenin were inoculated with ORS571 (pXLGD4), carrying the lacZ reporter gene. By microscopic analysis of sections of inoculated rice roots, it has been demonstrated that the xylem of rice roots can be colonized by Azorhizobium caulinodans. We discuss whether this colonization of the xylem of rice roots by azorhizobia could provide a suitable niche for endophytic nitrogen fixation.

Azorhizobium caulinodans↗

PubMed2001

Xylem colonization of tomato by Azorhizobium caulinodans ORS571.

Tomato seedlings growing aseptically in Murashige and Skoog Medium were inoculated with Azorhizobium caulinodans ORS571 (pXLGD4), carrying the lacZ reporter gene. By microscopic analyses of inoculated tomato roots, it has been demonstrated that the xylem of tomato roots can be colonized by Azorhizobium. We discuss whether this colonization of the xylem of tomato roots by diazotrophic azorhizobia might provide a suitable niche for endophytic nitrogen fixation.

Azorhizobium caulinodans↗

PubMed1993

Enzyme polymorphism of Azorhizobium strains and other stem- and root-nodulating bacteria isolated from Sesbania rostrata.

Relationships between bacterial groups nodulating Sesbania rostrata were evaluated through examination of electrophoretic polymorphism of esterases and metabolic enzymes. The following conclusions were drawn: (i) the differentiation of two genomic species within Azorhizobium strains and a group of non-identified strains (probably Rhizobium) was strongly supported by enzyme electrophoresis; (ii) esterases were more electrophoretically polymorphic than metabolic enzymes, since 35 and 11 electrophoretic types, respectively, were detected within the 57 strains studied; (iii) strains isolated from stem or root nodules were genetically very similar and could not be differentiated; (iv) six Azorhizobium strains isolated from plants growing in saline soils could not be grouped separately from the other strains, which might be attributed to the adaptation of azorhizobia to epiphytic conditions; and (v) a comparative study of esterase patterns of azorhizobia showed that strains isolated in the Philippines probably originated in northern Senegal, but did not reveal a clear separation between strains originating from northern and central Senegal.

Electrophoresis, Agar Gel↗

PubMed1994

The NodC protein of Azorhizobium caulinodans is an N-acetylglucosaminyltransferase.

Nod factors are signal molecules produced by Azorhizobium, Bradyrhizobium, and Rhizobium species that trigger nodule formation in leguminous host plants. The backbone of Nod factors consists of a beta-1,4-N-acetylglucosamine oligosaccharide from which the N-acetyl group at the nonreducing end is replaced by a fatty acid. The nodABC gene products are necessary for backbone biosynthesis. By incubation of cell extracts from Azorhizobium caulinodans with radioactive uridine diphosphate-N-acetylglucosamine, Nod factor precursors were identified and characterized as beta-1,4-N-acetylglucosamine oligosaccharides. By analysis of different nod gene mutants and by expression of nodC in Escherichia coli, the N-acetylglucosaminyltransferase activity was ascribed to the NodC protein. The results suggest that the first step in biosynthesis of Nod factors is the assembly of the oligosaccharide chain.

Bacteria↗

PubMed1996

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 DH5 alpha. The strain DH5 alpha (pUCNABCSU) synthesized LCOs, but they were only secreted when a plasmid containing both nodl and nodJ was supplied in trans. nodl or nodJ alone was not sufficient. In E. coli as well as in Azorhizobium, the nodlJ-encoded transporter showed a specificity for more hydrophilic LCOs.

ATP-Binding Cassette Transporters↗

PubMed1992

Elucidation of the complete Azorhizobium nicotinate catabolism pathway.

A complete pathway for Azorhizobium caulinodans nicotinate catabolism has been determined from mutant phenotype analyses, isolation of metabolic intermediates, and structural studies. Nicotinate serves as a respiratory electron donor to O2 via a membrane-bound hydroxylase and a specific c-type cytochrome oxidase. The resulting oxidized product, 6-hydroxynicotinate, is next reduced to 1,4,5,6-tetrahydro-6-oxonicotinate. Hydrolytic ring breakage follows, with release of pyridine N as ammonium. Decarboxylation then releases the nicotinate C-7 carboxyl group as CO2, and the remaining C skeleton is then oxidized to yield glutarate. Transthioesterification with succinyl coenzyme A (succinyl-CoA) yields glutaryl-CoA, which is then oxidatively decarboxylated to yield crotonyl-CoA. As with general acyl beta oxidation, L-beta-hydroxybutyryl-CoA, acetoacetyl-CoA, and finally two molecules of acetyl-CoA are produced. In sum, nicotinate is catabolized to yield two CO2 molecules, two acetyl-CoA molecules, and ammonium. Nicotinate catabolism stimulates Azorhizobium N2 fixation rates in culture. Nicotinate catabolism mutants still able to liberate pyridine N as ammonium retain this capability, whereas mutants so blocked do not. From, mutant analyses and additional physiological tests, N2 fixation stimulation is indirect. In N-limited culture, nicotinate catabolism augments anabolic N pools and, as a consequence, yields N2-fixing cells with higher dinitrogenase content.

Acyl Coenzyme A↗

PubMed2002

pMH11, A tool for gene disruption and expression analysis in Azorhizobium caulinodans.

Tools for mutagenesis and expression analyses are needed to study the role of bacterial genes. Here, we report the construction of pMH11, a small, mobilizable plasmid that replicates in Escherichia coli, but not in Azorhizobium caulinodans, a nodulating microsymbiont of Sesbania rostrata, and that contains a unique BamHI restriction site upstream of a promoterless lacZ gene. pMH11 and two derivatives with the multiple cloning site of pBluescript (KS(II)) are useful for mutagenesis by gene disruption and for expression analyses after selection for cointegration by kanamycin resistance. Weakly constitutive promoter activity from the vector allowed transcription of genes downstream of the integration site, so that no polar effects were caused by gene disruption.

Azorhizobium caulinodans↗

PubMed2001

Azide-resistant mutants of Azorhizobium caulinodans with enhanced symbiotic effectiveness.

Azide-resistant mutants of Azorhizobium caulinodans strains Sb3, S78, SrR13 and SrS8 were isolated and screened for nitrate reductase activity. Selected nitrate reductase negative mutants were inoculated on Sesbania bispinosa and S. rostrata under sterile conditions in chillum jars to study their symbiotic behavior. Azide-resistant mutants exhibited either similar or higher symbiotic effectiveness than the parent strain after 30 d of plant growth. Nodule mass, nitrogenase activity and uptake hydrogenase activity of the mutants varied depending on the host as well as on the plant growth stage. In comparison to wild-type parent strains, four azide-resistant mutants, Sb3Az18, S78Az21, SrR13Az17 and SrS8Az6 showed significant increase in nodulation and nitrogen fixation as well as shoot dry mass of the inoculated plants.

Azorhizobium caulinodans↗

PubMed2005

Responses of Azorhizobium caulinodans to cadmium stress.

The symbiosis of Azorhizobium caulinodans and an annul legume Sesbania rostrata was recently found to be tolerant to cadmium pollution by an unknown mechanism. In this study, A. caulinodans ORS571 and ZY-20 showed much stronger tolerance to cadmium than a mutant ORS571-X15 and a common Rhizobium sp., with minimum inhibitory concentration values as high as 4 and 5 mM (versus 1 and 0.1 mM) on yeast extract mannitol agar medium, respectively. Although Cd uptake by all three strains of A. caulinodans were mostly from absorption rather than binding (both loosely or tightly) on cell surface, in resistant strains a higher portion of extractable Cd was bound on the cell surface vs. absorbed (about 1:2.5 ratio) compared to the sensitive mutant (about 1:35.1 ratio). These results suggest that certain level of metal exclusion by a permeability barrier was involved in the mechanism of resistance to Cd by A. caulinodans ORS571 and ZY-20. Over the 12-h period of cultivation in yeast extract mannitol agar medium with Cd addition, the Cd concentrations in the outer membrane and periplasm and spheroplast were the highest at the first 3 h, and declined steadily over time. The fact that Cd concentrations in spheroplast of all three strains were many folds higher than those in outer membrane and periplasm, suggests that extracellular sequestration was not the only mechanism of Cd tolerance in A. caulinodans. The decline of Cd concentrations was significantly faster and started earlier in strains ORS571 and ZY-20 than in ORS571-X15. This suggests a second, probably more substantial, mechanism involves active transport of the metal from the cell, e.g., some efflux system for maintaining homeostasis under cadmium stress.

Analysis of Variance↗

PubMed2004

Molecular and functional characterization of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster.

In this work, we report the cloning and sequencing of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster. Sequence analysis revealed the presence of 20 open reading frames hupTUVhypFhupSLCDFGHJK hypABhupRhypCDEhupE. The physical and genetic organization of A. caulinodans ORS571 hydrogenase system suggests a close relatedness to that of Rhodobacter capsulatus. In contrast to the latter species, a gene homologous to Rhizobium leguminosarum hupE was identified downstream of the hyp operon. A hupSL mutation drastically reduced the high levels of hydrogenase activity induced by the A. caulinodans ORS571 wild-type strain in symbiosis with Sesbania rostrata plants. However, no significant effects on dry weight and nitrogen content of S. rostrata plants inoculated with the hupSL mutant were observed in plant growth experiments.

Amino Acid Sequence↗

PubMed2004

A gfp reporter plasmid to visualize Azorhizobium caulinodans during nodulation of Sesbania rostrata.

Compared with other labeling techniques, the use of the green fluorescent protein (GFP) is advantageous to visualize bacteria because observations can be performed in real time. This feature is particularly interesting to study invasion events of rhizobia during nodule development on their legume host plant. To investigate the symbiotic interaction between Azorhizobium caulinodans ORS571 and Sesbania rostrata, we constructed two plasmids, pMP220-hem-gfp5 and pBBR5-hem-gfp5-S65T, that carry a modified gfp gene, the expression of which is controlled by the constitutive hem promoter. Introduction of either of these plasmids into A. caulinodans allowed the visualization of single bacteria. Determination of the plasmid stability in cultured bacteria and in nodules demonstrated that pBBR5-hem-gfp5-S65T is more stable than pMP220-hem-gfp5. The plasmid pBBR5-hem-gfp5-S65T can be used to study early invasion events during nodule development on hydroponic roots of S. rostrata.

Azorhizobium caulinodans↗