A rhizobial homolog of IHF stimulates transcription of dctA in Rhizobium leguminosarum but not in Sinorhizobium meliloti

ABSTRACT LysR-type transcriptional regulators represent one of the largest groups of prokaryotic regulators described to date. In the gram-negative legume endosymbiont Sinorhizobium meliloti, enzymes involved in the protocatechuate branch of the β-ketoadipate pathway are encoded within the pcaDCHGB operon, which is subject to regulation by the LysR-type protein PcaQ. In this work, purified PcaQ was shown to bind strongly (equilibrium dissociation constant, 0.54 nM) to a region at positions −78 to −45 upstream of the pcaD transcriptional start site. Within this region, we defined a PcaQ binding site with dyad symmetry that is required for regulation of pcaD expression in vivo and for binding of PcaQ in vitro. We also demonstrated that PcaQ participates in negative autoregulation by monitoring expression of pcaQ via a transcriptional fusion to lacZ. Although pcaQ homologues are present in many α-proteobacteria, this work describes the first reported purification of this regulator, as well as characterization of its binding site, which is conserved in Agrobacterium tumefaciens, Rhizobium leguminosarum, Rhizobium etli, and Mesorhizobium loti.

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Succinate Transport Is Not Essential for Symbiotic Nitrogen Fixation by Sinorhizobium meliloti or Rhizobium leguminosarum

Applied and Environmental Microbiology ◽

10.1128/aem.01561-17 ◽

2017 ◽

Vol 84 (1) ◽

Cited By ~ 10

Author(s):

Michael J. Mitsch ◽

George C. diCenzo ◽

Alison Cowie ◽

Turlough M. Finan

Keyword(s):

Nitrogen Fixation ◽

Carbon Source ◽

Sinorhizobium Meliloti ◽

Rhizobium Leguminosarum ◽

Symbiotic Nitrogen Fixation ◽

Sequencing Analysis ◽

Wild Type ◽

Content Type ◽

Transcriptional Changes ◽

Symbiotic Properties

ABSTRACTSymbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria during endosymbiotic relationships with plants. The bacteria require the plant to provide a carbon source for the generation of reductant to power SNF. While C4-dicarboxylates (succinate, fumarate, and malate) appear to be the primary, if not sole, carbon source provided to the bacteria, the contribution of each C4-dicarboxylate is not known. We address this issue using genetic and systems-level analyses. Expression of a malate-specific transporter (MaeP) inSinorhizobium melilotiRm1021dctmutants unable to transport C4-dicarboxylates resulted in malate import rates of up to 30% that of the wild type. This was sufficient to support SNF withMedicago sativa, with acetylene reduction rates of up to 50% those of plants inoculated with wild-typeS. meliloti.Rhizobium leguminosarumbv. viciae 3841dctmutants unable to transport C4-dicarboxylates but expressing themaePtransporter had strong symbiotic properties, withPisum sativumplants inoculated with these strains appearing similar to plants inoculated with wild-typeR. leguminosarum. This was despite malate transport rates by the mutant bacteroids being 10% those of the wild type. An RNA-sequencing analysis of the combinedP. sativum-R. leguminosarumnodule transcriptome was performed to identify systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were detected. Overall, these data illustrated that succinate and fumarate are not essential for SNF and that, at least in specific symbioses,l-malate is likely the primary C4-dicarboxylate provided to the bacterium.IMPORTANCESymbiotic nitrogen fixation (SNF) is an economically and ecologically important biological process that allows plants to grow in nitrogen-poor soils without the need to apply nitrogen-based fertilizers. Much research has been dedicated to this topic to understand this process and to eventually manipulate it for agricultural gains. The work presented in this article provides new insights into the metabolic integration of the plant and bacterial partners. It is shown that malate is the only carbon source that needs to be available to the bacterium to support SNF and that, at least in some symbioses, malate, and not other C4-dicarboxylates, is likely the primary carbon provided to the bacterium. This work extends our knowledge of the minimal metabolic capabilities the bacterium requires to successfully perform SNF and may be useful in further studies aiming to optimize this process through synthetic biology approaches. The work describes an engineering approach to investigate a metabolic process that occurs between a eukaryotic host and its prokaryotic endosymbiont.

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Heterologous Complementation Reveals a Specialized Activity for BacA in the Medicago–Sinorhizobium meliloti Symbiosis

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-02-17-0030-r ◽

2017 ◽

Vol 30 (4) ◽

pp. 312-324 ◽

Cited By ~ 13

Author(s):

George C. diCenzo ◽

Maryam Zamani ◽

Hannah N. Ludwig ◽

Turlough M. Finan

Keyword(s):

Pisum Sativum ◽

Sinorhizobium Meliloti ◽

Rhizobium Leguminosarum ◽

Root Nodules ◽

Specific Interaction ◽

Model System ◽

Single Model ◽

Leguminous Plants ◽

Melilotus Alba ◽

Developmental Progression

The bacterium Sinorhizobium meliloti Rm2011 forms N2-fixing root nodules on alfalfa and other leguminous plants. The pSymB chromid contains a 110-kb region (the ETR region) showing high synteny to a chromosomally located region in Sinorhizobium fredii NGR234 and related rhizobia. We recently introduced the ETR region from S. fredii NGR234 into the S. meliloti chromosome. Here, we report that, unexpectedly, the S. fredii NGR234 ETR region did not complement deletion of the S. meliloti ETR region in symbiosis with Medicago sativa. This phenotype was due to the bacA gene of NGR234 not being functionally interchangeable with the S. meliloti bacA gene during M. sativa symbiosis. Further analysis revealed that, whereas bacA genes from S. fredii or Rhizobium leguminosarum bv. viciae 3841 failed to complement the Fix− phenotype of a S. meliloti bacA mutant with M. sativa, they allowed for further developmental progression prior to a loss of viability. In contrast, with Melilotus alba, bacA from S. fredii and R. leguminosarum supported N2 fixation by a S. meliloti bacA mutant. Additionally, the S. meliloti bacA gene can support N2 fixation of a R. leguminosarum bacA mutant during symbiosis with Pisum sativum. A phylogeny of BacA proteins illustrated that S. meliloti BacA has rapidly diverged from most rhizobia and has converged toward the sequence of pathogenic genera Brucella and Escherichia. These data suggest that the S. meliloti BacA has evolved toward a specific interaction with Medicago and highlights the limitations of using a single model system for the study of complex biological topics.

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Pathways for phosphatidylcholine biosynthesis in bacteria

Microbiology ◽

10.1099/mic.0.26522-0 ◽

2003 ◽

Vol 149 (12) ◽

pp. 3461-3471 ◽

Cited By ~ 65

Author(s):

Fernando Martínez-Morales ◽

Max Schobert ◽

Isabel M. López-Lara ◽

Otto Geiger

Keyword(s):

Borrelia Burgdorferi ◽

Legionella Pneumophila ◽

Bradyrhizobium Japonicum ◽

Sinorhizobium Meliloti ◽

Rhizobium Leguminosarum ◽

Brucella Melitensis ◽

Membrane Lipid ◽

Mesorhizobium Loti ◽

Methylation Pathway ◽

Phosphatidylcholine Biosynthesis

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes with important structural and signalling functions. Although many prokaryotes lack PC, it can be found in significant amounts in membranes of rather diverse bacteria. Two pathways for PC biosynthesis are known in bacteria, the methylation pathway and the phosphatidylcholine synthase (PCS) pathway. In the methylation pathway, phosphatidylethanolamine is methylated three times to yield PC, in reactions catalysed by one or several phospholipid N-methyltransferases (PMTs). In the PCS pathway, choline is condensed directly with CDP-diacylglyceride to form PC in a reaction catalysed by PCS. Using cell-free extracts, it was demonstrated that Sinorhizobium meliloti, Agrobacterium tumefaciens, Rhizobium leguminosarum, Bradyrhizobium japonicum, Mesorhizobium loti and Legionella pneumophila have both PMT and PCS activities. In addition, Rhodobacter sphaeroides has PMT activity and Brucella melitensis, Pseudomonas aeruginosa and Borrelia burgdorferi have PCS activities. Genes from M. loti and L. pneumophila encoding a Pmt or a Pcs activity and the genes from P. aeruginosa and Borrelia burgdorferi responsible for Pcs activity have been identified. Based on these functional assignments and on genomic data, one might predict that if bacteria contain PC as a membrane lipid, they usually possess both bacterial pathways for PC biosynthesis. However, important pathogens such as Brucella melitensis, P. aeruginosa and Borrelia burgdorferi seem to be exceptional as they possess only the PCS pathway for PC formation.

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Conservation of noIR in the Sinorhizobium and Rhizobium Genera of the Rhizobiaceae Family

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.12.1186 ◽

1998 ◽

Vol 11 (12) ◽

pp. 1186-1195 ◽

Cited By ~ 28

Author(s):

Ernö Kiss ◽

Peter Mergaert ◽

Boglàrka Olàh ◽

Attila Kereszt ◽

Christian Staehelin ◽

...

Keyword(s):

Dna Binding ◽

Primary Structure ◽

Dna Hybridization ◽

Sinorhizobium Meliloti ◽

Rhizobium Leguminosarum ◽

Negative Regulation ◽

Regulatory Proteins ◽

Dna Binding Domain ◽

Nod Factor ◽

Homologous Sequences

In Sinorhizobium meliloti the NolR repressor displays differential negative regulation of nodulation genes and is required for optimal nodulation. Here, we demonstrate that the NolR function is not unique to S. meliloti but is also present in other species of the Rhizobiaceae family. DNA hybridization indicates the presence of nolR homologous sequences in species belonging to the Rhizobium and Sinorhizobium genera while no hybridization signal was detected in species from the Mesorhizobium, Bradyrhizo-bium, Azorhizobium, and Agrobacterium genera. We isolated the nolR gene from the Rhizobium leguminosarum bv. viciae strain TOM and showed that the TOM nolR gene acts similarly to S. meliloti nolR by repressing the expression of both the nodABCIJ and the nodD genes, resulting in decreased Nod factor production. The presence of a functional nolR gene in R. leguminosarum is correlated with an increased rate and extent of nodulation of pea. The conserved primary structure, the location of the DNA-binding domain, and the similar size of NolR proteins, compared with a family of small bacterial regulatory proteins including HlyU, SmtB, and the ArsR-type regulators, revealed that NolR belongs to this family.

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Identification of soil bacteria expressing a symbiotic plasmid from Rhizobium leguminosarum bv. trofolii

Canadian Journal of Microbiology ◽

10.1139/m97-022 ◽

1997 ◽

Vol 43 (2) ◽

pp. 164-177 ◽

Cited By ~ 7

Author(s):

S. Sivakumaran ◽

B. D. W. Jarvis ◽

P. J. Lockhart

Keyword(s):

Dna Hybridization ◽

Sinorhizobium Meliloti ◽

Rhizobium Leguminosarum ◽

Root Nodule ◽

Sandy Loam ◽

Rhizobium Tropici ◽

Rhizobium Loti ◽

Rrna Sequencing ◽

Reference Strains ◽

Good Agreement

A hundred strains of non-nodulating, Gram-negative, rod-shaped bacteria were isolated from clover–ryegrass pastures on three different soil types and from a sandy loam under lupins. When crossed with Escherichia coli PN200 containing the cointegrate plasmid pPN1, 11 transconjugants gained the ability to form nodules on the roots of white clover (Trifolium repens cv. Grasslands Huia). A nodA probe indicated that they had gained nodulation genes. The identities of these 11 strains and 4 others derived from earlier work on non-nodulating root nodule bacteria, were determined by ribotyping, DNA – DNA hybridization, and partial 16S rRNA sequencing. Good agreement was obtained between the three methods, and 11 of the strains were identified as Rhizobium leguminosarum (6), Rhizobium loti (2), Rhizobium etli (1), Rhizobium tropici (1), and Sinorhizobium meliloti (1). DNA –DNA hybridization indicated that the remaining four strains were related to the Rhizobium leguminosarum reference strains. The existence of several species of non-nodulating rhizobia in pasture soil, including species for which the normal host plant was absent, is discussed in relation to the fate of symbiotic plasmids from Rhizobium seed inoculants. It is also suggested that new species should be named for the geographical region from which they are first isolated rather than the host plant.Key words: Rhizobium, non-nodulating, nonsymbiotic, isolation, identification.

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Cloning and Characterization of a Rhizobium leguminosarum Gene Encoding a Bacteriocin with Similarities to RTX Toxins

Applied and Environmental Microbiology ◽

10.1128/aem.65.7.2833-2840.1999 ◽

1999 ◽

Vol 65 (7) ◽

pp. 2833-2840 ◽

Cited By ~ 51

Author(s):

Ivan J. Oresnik ◽

Sunny Twelker ◽

Michael F. Hynes

Keyword(s):

Amino Acid ◽

Sinorhizobium Meliloti ◽

Insertional Mutagenesis ◽

Rhizobium Leguminosarum ◽

Nodule Occupancy ◽

Bacteriocin Activity ◽

Gene Encoding ◽

Reading Frame ◽

Rtx Toxins ◽

Culture Supernatants

ABSTRACT A 3-kb region containing the determinant for bacteriocin activity from Rhizobium leguminosarum 248 was isolated and characterized by Tn5 insertional mutagenesis and DNA sequencing. Southern hybridizations showed that this bacteriocin was encoded on the plasmid pRL1JI and that homologous loci were not found in other unrelated R. leguminosarum strains. Tn5 insertional mutagenesis showed that mutations in the C-terminal half of the bacteriocin open reading frame apparently did not abolish bacteriocin activity. Analysis of the deduced amino acid sequence revealed that, similarly to RTX proteins (such as hemolysin and leukotoxin), this protein contains a characteristic nonapeptide repeated up to 18 times within the protein. In addition, a novel 19- to 25-amino-acid motif that occurred every 130 amino acids was detected. Bacteriocin bioactivity was correlated with the presence of a protein of approximately 100 kDa in the culture supernatants, and the bacteriocin bioactivity demonstrated a calcium dependence in bothR. leguminosarum and Sinorhizobium meliloti. A mutant of strain 248 unable to produce this bacteriocin was found to have a statistically significant reduction in competitiveness for nodule occupancy compared to two test strains in coinoculation assays. However, this strain was unable to compete any more successfully with a third test strain, 3841, than was wild-type 248.

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Diversity and numbers of root-nodule bacteria (rhizobia) in Polish soils

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.2005.012 ◽

2011 ◽

Vol 74 (1) ◽

pp. 83-86 ◽

Cited By ~ 8

Author(s):

Stefan Martyniuk ◽

Jadwiga Oroń ◽

Maria Martyniuk

Keyword(s):

Sinorhizobium Meliloti ◽

Rhizobium Leguminosarum ◽

Root Nodule ◽

Chemical Properties ◽

Physical And Chemical Properties ◽

Nodule Bacteria ◽

Root Nodule Bacteria ◽

Plant Infection ◽

Physical And Chemical ◽

And Chemical Properties

Using a sand pouch-plant infection method, populations of several species of root-nodule bacteria (rhizobia) were enumerated in eighty soils collected throughout Poland. Rhizobium leguminosarum bv. viciae (symbionts of pea, faba bean, vetch) and R. leguminosarum bv. trifolii (symbionts of clover) were detected in 77 and 76 soils, respectively. Most of these soils contained moderate and high numbers of these species of the rhizobia. Symbionts of beans, R. leguminosarum bv. phaseoli, were assessed in 76 soils; of this number 15 soils had no detectable populations of bean rhizobia and in 40 soils high or moderate numbers of these bacteria were found. Bradyrhizobium sp. (Lupinus), root-nodule bacteria of lupine and serradella, were absent in 19 soils, out of 80 tested, and 34 soils were colonised by high or moderate populations of bradyrhizobia. Sinorhizobium meliloti, rhizobia nodulating alfalfa, were sparse in the examined soils; with 56 soil containing no detectable numbers of S. meliloti and only 6 soils harbouring high or moderate populations of this species. The estimated numbers of the rhizobia in the studied soils were also related to some physical and chemical properties of these soils.

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Cloning, Sequencing, and Characterization of thecgmB Gene of Sinorhizobium meliloti Involved in Cyclic β-Glucan Biosynthesis

Journal of Bacteriology ◽

10.1128/jb.181.15.4576-4583.1999 ◽

1999 ◽

Vol 181 (15) ◽

pp. 4576-4583 ◽

Cited By ~ 20

Author(s):

Ping Wang ◽

Cheryl Ingram-Smith ◽

Jill A. Hadley ◽

Karen J. Miller

Keyword(s):

Sinorhizobium Meliloti ◽

Rhizobium Leguminosarum ◽

Genomic Library ◽

Rhizobium Meliloti ◽

Functional Expression ◽

Open Reading Frames ◽

Inverse Pcr ◽

Insertion Mutant ◽

Overlapping Open Reading Frames ◽

Reading Frames

ABSTRACT Periplasmic cyclic β-glucans of Rhizobium species provide important functions during plant infection and hypo-osmotic adaptation. In Sinorhizobium meliloti (also known asRhizobium meliloti), these molecules are highly modified with phosphoglycerol and succinyl substituents. We have previously identified an S. meliloti Tn5 insertion mutant, S9, which is specifically impaired in its ability to transfer phosphoglycerol substituents to the cyclic β-glucan backbone (M. W. Breedveld, J. A. Hadley, and K. J. Miller, J. Bacteriol. 177:6346–6351, 1995). In the present study, we have cloned, sequenced, and characterized this mutation at the molecular level. By using the Tn5 flanking sequences (amplified by inverse PCR) as a probe, an S. meliloti genomic library was screened, and two overlapping cosmid clones which functionally complement S9 were isolated. A 3.1-kb HindIII-EcoRI fragment found in both cosmids was shown to fully complement mutant S9. Furthermore, when a plasmid containing this 3.1-kb fragment was used to transformRhizobium leguminosarum bv. trifolii TA-1JH, a strain which normally synthesizes only neutral cyclic β-glucans, anionic glucans containing phosphoglycerol substituents were produced, consistent with the functional expression of an S. meliloti phosphoglycerol transferase gene. Sequence analysis revealed the presence of two major, overlapping open reading frames within the 3.1-kb fragment. Primer extension analysis revealed that one of these open reading frames, ORF1, was transcribed and its transcription was osmotically regulated. This novel locus of S. meliloti is designated thecgm (cyclic glucan modification) locus, and the product encoded by ORF1 is referred to as CgmB.

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Nickel Availability and hupSL Activation by Heterologous Regulators Limit Symbiotic Expression of theRhizobium leguminosarum bv. Viciae Hydrogenase System in Hup− Rhizobia

Applied and Environmental Microbiology ◽

10.1128/aem.66.3.937-942.2000 ◽

2000 ◽

Vol 66 (3) ◽

pp. 937-942 ◽

Cited By ~ 17

Author(s):

Belén Brito ◽

Jorge Monza ◽

Juan Imperial ◽

Tomás Ruiz-Argüeso ◽

Jose Manuel Palacios

Keyword(s):

Transcriptional Activation ◽

Sinorhizobium Meliloti ◽

Rhizobium Leguminosarum ◽

Transcriptional Regulator ◽

Hydrogen Uptake ◽

Hydrogenase Activity ◽

Structural Genes ◽

Mesorhizobium Loti ◽

Genes Expression ◽

Relevant Factors

ABSTRACT A limited number of Rhizobium andBradyrhizobium strains possess a hydrogen uptake (Hup) system that recycles the hydrogen released from the nitrogen fixation process in legume nodules. To extend this ability to rhizobia that nodulate agronomically important crops, we investigated factors that affect the expression of a cosmid-borne Hup system from Rhizobium leguminosarum bv. viciae UPM791 in R. leguminosarumbv. viciae, Rhizobium etli, Mesorhizobium loti, and Sinorhizobium meliloti Hup− strains. After cosmid pAL618 carrying the entire hup system of strain UPM791 was introduced, all recipient strains acquired the ability to oxidize H2 in symbioses with their hosts, although the levels of hydrogenase activity were found to be strain and species dependent. The levels of hydrogenase activity were correlated with the levels of nickel-dependent processing of the hydrogenase structural polypeptides and with transcription of structural genes. Expression of the NifA-dependent hupSL promoter varied depending on the genetic background, while the hyp operon, which is controlled by the FnrN transcriptional regulator, was expressed at similar levels in all recipient strains. With the exception of theR. etli-bean symbiosis, the availability of nickel to bacteroids strongly affected hydrogenase processing and activity in the systems tested. Our results indicate that efficient transcriptional activation by heterologous regulators and processing of the hydrogenase as a function of the availability of nickel to the bacteroid are relevant factors that affect hydrogenase expression in heterologous rhizobia.

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