Characterization of the Sinorhizobium meliloti sinR/sinI locus and the production of novel N-acyl homoserine lactones

Sinorhizobium meliloti is a soil bacterium which can establish a nitrogen-fixing symbiosis with the legume Medicago sativa. Recent work has identified a pair of genes, sinR and sinI, which represent a potential quorum-sensing system and are responsible for the production of N-acyl homoserine lactones (AHLs) in two S. meliloti strains, Rm1021 and Rm41. In this work, we characterize the sinRI locus and show that these genes are responsible for the synthesis of several long-chain AHLs ranging from 12 to 18 carbons in length. Four of these, 3-oxotetradecanoyl HL, 3-oxohexadecenoyl HL, hexadecenoyl HL, and octadecanoyl HL, have novel structures. This is the first report of AHLs having acyl chains longer than 14 carbons. We show that a disruption in sinI eliminates these AHLs and that a sinR disruption results in only basal levels of the AHLs. Moreover, the same sinI and sinR mutations also lead to a decrease in the number of pink nodules during nodulation assays, as well as a slight delay in the appearance of pink nodules, indicating a role for quorum sensing in symbiosis. We also show that sinI and sinR mutants are still capable of producing several short-chain AHLs, one of which was identified as octanoyl HL. We believe that these short-chain AHLs are evidence of a second quorum-sensing system in Rm1021, which we refer to here as the mel system, for "S. meliloti."     

Chemical identification of<Emphasis Type="Italic"> N</Emphasis>-acyl homoserine lactone quorum-sensing signals produced by<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis> strains in defined medium

The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and electrospray ionization tandem mass spectrometry (ESI MS/MS). Both strains synthesized several long-chain AHLs. Defined medium cultures of strain AK631 synthesized a complex mixture of AHLs with short acyl side chains. Strain 1021 produced no short-chain AHLs when grown on defined medium and made a somewhat different set of long-chain AHLs than previously reported for cultures in rich medium. While the two strains produced several AHLs in common, the differences in AHLs produced suggest that there may be significant differences in their patterns of quorum-sensing regulation.     

Suppression of the Fix- phenotype of Rhizobium meliloti exoB mutants by lpsZ is correlated to a modified expression of the K polysaccharide.

The rhizobial production of extracellular polysaccharide (EPS) is generally required for the symbiotic infection of host plants that form nodules with an apical meristem (indeterminate nodules). One exception is Rhizobium meliloti AK631, an exoB mutant of Rm41, which is deficient in EPS production yet infects and fixes nitrogen (i.e., is Fix+) on alfalfa, an indeterminate nodule-forming plant. A mutation of lpsZ in AK631 results in a Fix- strain with altered phage sensitivity, suggesting that a cell surface factor may substitute for EPS in the alfalfa-AK631 symbiosis. Biochemical analyses of the cell-associated polysaccharides of AK631 and Rm5830 (AK631 lpsZ) demonstrated that the lpsZ mutation affected the expression of a surface polysaccharide that is analogous to the group II K polysaccharides of Escherichia coli; the polysaccharide contains 3-deoxy-D-manno-2-octulosonic acid or a derivative thereof in each repeating unit. Rm5830 produced a polysaccharide with altered chromatographic and electrophoretic properties, indicating a difference in the molecular weight range. Similar results were obtained in a study of Rm1021, a wild-type isolate that lacks the lpsZ gene: the introduction of lpsZ into Rm1021 exoB (Rm6903) both suppresses the Fix- phenotype and results in a modified expression of the K polysaccharide. Chromatography and electrophoresis analysis showed that the polysaccharide extracted from Rm6903 lpsZ+ differed from that of Rm6903 in molecular weight range. Importantly, the effect of LpsZ is not structurally specific, as the introduction lpsZ+ into Rhizobium fredii USDA257 also resulted in a molecular weight range change in the structurally distinct K polysaccharide produced by that strain. This evidence suggests that LpsZ has a general effect on the size-specific expression of rhizobial K polysaccharides.     

The rkp-3 gene region of Sinorhizobium meliloti Rm41 contains strain-specific genes that determine K antigen structure.

The rkp-3 region is indispensable for capsular polysaccharide (K antigen) synthesis in Sinorhizobium meliloti Rm41. Strain Rm41 produces a K antigen of strain-specific structure, designated as the KR5 antigen. The data in this report show that the rkp-3 gene region comprises 10 open reading frames involved in bacterial polysaccharide synthesis and export. The predicted amino acid sequences for the rkpL-Q gene products are homologous to enzymes involved in the production of specific sugar moieties, while the putative products of the rkpRST genes show a high degree of similarity to proteins required for transporting polysaccharides to the cell surface. Southern analysis experiments using gene-specific probes suggest that genes involved in the synthesis of the precursor sugars are unique in strain Rm41, whereas sequences coding for export proteins are widely distributed among Sinorhizobium species. Mutations in the rkpL-Q genes result in a modified K antigen pattern and impaired symbiotic capabilities. On this basis, we suggest that these genes are required for the production of the KR5 antigen that is necessary for S. meliloti Rm41 exoB (AK631)-alfalfa (Medicago sativa) symbiosis.     

Quorum sensing controls exopolysaccharide production in Sinorhizobium meliloti

Sinorhizobium meliloti is a soil bacterium capable of invading and establishing a symbiotic relationship with alfalfa plants. This invasion process requires the synthesis, by S. meliloti, of at least one of the two symbiotically important exopolysaccharides, succinoglycan and EPS II. We have previously shown that the sinRI locus of S. meliloti encodes a quorum-sensing system that plays a role in the symbiotic process. Here we show that the sinRI locus exerts one level of control through regulation of EPS II synthesis. Disruption of the autoinducer synthase gene, sinI, abolished EPS II production as well as the expression of several genes in the exp operon that are responsible for EPS II synthesis. This phenotype was complemented by the addition of acyl homoserine lactone (AHL) extracts from the wild-type strain but not from a sinI mutant, indicating that the sinRI-specified AHLs are required for exp gene expression. This was further confirmed by the observation that synthetic palmitoleyl homoserine lactone (C(16:1)-HL), one of the previously identified sinRI-specified AHLs, specifically restored exp gene expression. Most importantly, the absence of symbiotically active EPS II in a sinI mutant was confirmed in plant nodulation assays, emphasizing the role of quorum sensing in symbiosis.     

Effector-stimulated single molecule protein-DNA interactions of a quorum-sensing system in Sinorhizobium meliloti

Intercellular communication by means of small signal molecules coordinates gene expression among bacteria. This population density-dependent regulation is known as quorum sensing. The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti Rm1021 possesses the Sin quorum sensing system based on N-acyl homoserine lactones (AHL) as signal molecules. Here, we demonstrate that the LuxR-type regulator ExpR binds specifically to a target sequence in the sinRI locus in the presence of different AHLs with acyl side chains from 8 to 20 carbons. Dynamic force spectroscopy based on the atomic force microscope provided detailed information about the molecular mechanism of binding upon activation by six different AHLs. These single molecule experiments revealed that the mean lifetime of the bound protein-DNA complex varies depending on the specific effector molecule. The small differences between individual AHLs also had a pronounced influence on the structure of protein-DNA interaction: The reaction length of dissociation varied from 2.6 to 5.8 A. In addition, dynamic force spectroscopy experiments indicate that N-heptanoyl-DL-homoserine lactone binds to ExpR but is not able to stimulate protein-DNA interaction.     

The acetyl substituent of succinoglycan is not necessary for alfalfa nodule invasion by Rhizobium meliloti Rm1021.

Rhizobium meliloti Rm1021 requires a Calcofluor-binding exopolysaccharide, termed succinoglycan or EPS I, to invade alfalfa nodules. We have determined that a strain carrying a mutation in the exoZ locus produces succinoglycan that lacks the acetyl substituent. The exoZ mutant nodules alfalfa normally.     

Recombinant Rhizobium meliloti strains with extra biotin synthesis capability.

The growth of Rhizobium meliloti 1021 in an experimental alfalfa (Medicago sativa L.) rhizosphere was stimulated by adding nanomolar amounts of biotin. To overcome this biotin limitation, R. meliloti strains were constructed by conjugating the Escherichia coli biotin synthesis operon into biotin auxotroph R. meliloti 1021-B3. Transconjugant strains Rm1021-WS10 and Rm1021-WS11 grew faster in vitro and achieved a higher cell density than did R. meliloti 1021 and overproduced biotin on a defined medium. The increase in cell yield was associated with as much as a 99% loss in viability for Rm1021-WS11, but data suggested that a separate stabilizing factor in the E. coli DNA reduced cell death in Rm1021-WS10. In rhizosphere tests, the recombinant strains showed delayed growth and competed poorly against Rm1021.     

Proteomic analysis of wild-type Sinorhizobium meliloti responses to N-acyl homoserine lactone quorum-sensing signals and the transition to stationary phase

Proteome analysis revealed that two long-chain N-acyl homoserine lactones (AHLs) produced by Sinorhizobium meliloti 1021 induced significant differences in the accumulation of more than 100 polypeptides in early-log-phase cultures of the wild type. Fifty-six of the corresponding proteins have been identified by peptide mass fingerprinting. The proteins affected by addition of these two AHLs had diverse functions in carbon and nitrogen metabolism, energy cycles, metabolite transport, DNA synthesis, and protein turnover. Two hours of exposure to 3-oxo-C(16:1)-homoserine lactone (3-oxo-C(16:1)-HL) affected the accumulation of 40 of the 56 identified proteins, whereas comparable exposure to C(14)-HL affected 13 of the 56 proteins. Levels of four proteins were affected by both AHLs. Exposure to 3-oxo-C(16:1)-HL for 8 h affected the accumulation of 17 proteins, 12 of which had reduced accumulation. Of the 80 proteins identified as differing in accumulation between early-log- and early-stationary-phase cultures, only 13 were affected by exposure to 3-oxo-C(16:1)-HL or C(14)-HL. These results provide a foundation for future studies of the functions regulated by AHL quorum sensing in S. meliloti and help to establish proteomic analysis as a powerful global approach to the identification of quorum-sensing regulatory patterns in wild-type bacteria.     

Overproduction and increased molecular weight account for the symbiotic activity of the rkpZ-modified K polysaccharide from Sinorhizobium meliloti Rm1021

K polysaccharides (KPSs) of Sinorhizobium meliloti strains are strain-specific surface polysaccharides analogous to the group II K antigens of Escherichia coli. The K(R)5 antigen of strain AK631 is a highly polymerized disaccharide of pseudaminic and glucuronic acids. During invasion of host plants, this K antigen is able to replace the structurally different exopolysaccharide succinoglycan (EPS I) and promotes the formation of a nitrogen-fixing (Fix(+)) symbiosis. The KPS of strain Rm1021 is a homopolymer of 3-deoxy-D-manno-2 octulosonic acid (Kdo). The Kdo polysaccharide is covalently linked to the lipid anchor, has a low molecular weight (LMW), and is symbiotically inactive. On introduction of the Rm41-specific rkpZ gene into strain Rm1021, a modified KPS is expressed that is able to substitute EPS I during symbiosis with the host plant. To better understand the nature of modification conferred by rkpZ, we performed a structural analysis of the KPS using nuclear magnetic resonance (NMR), electrospray ionization-mass spectrometry (ESI-MS), and gas chromatography (GC-MS). The modified KPS retained primary polyKdo structure, but its degree of polymerization (DP) and level of production were increased significantly. In contrast to the wild-type polyKdo, only a part of polyKdo was lipidated. Shorter polysaccharide chains were lipid-free, whereas longer polysaccharide chains were lipidated. Sinorhizobium meliloti Rm1021 was found to carry two paralogs of rkpZ. Both genes are involved in polyKdo production, but they only show partial functional activity as compared with the rkpZ of Rm41.     

Recipient-induced transfer of the symbiotic plasmid pRL1JI in Rhizobium leguminosarum bv. viciae is regulated by a quorum-sensing relay

Analysis of the regulation of plasmid transfer genes on the symbiotic plasmid pRL1JI in Rhizobium leguminosarum bv. viciae has revealed a novel regulatory relay that is specifically poised to detect an N-acyl-homoserine lactone (AHL) made by different cells (potential recipients of pRL1JI). Adjacent to the traI-trbBCDEJKLFGHI plasmid transfer operon on pRL1JI are two regulatory genes, bisR and traR, which encode LuxR-type quorum-sensing regulators required for conjugation. Potential recipients of pRL1JI induce the traI-trb operon and plasmid transfer via a quorum-sensing relay involving BisR, TraR and the traI-trb operon in donor cells. BisR induces expression of traR in response to N-(3-hydroxy-7-cis-tetradecenoyl)-l-homoserine lactone (3-OH-C14:1-HSL), which is produced by CinI in potential recipient strains. In donor strains (carrying pRL1JI), BisR represses the expression of the chromosomal gene cinI; this repression results in a very low level of formation of 3-OH-C14:1-HSL and hence relatively low levels of expression of traR and the traI-trb operon in strains carrying pRL1JI. However, if 3-OH-C14:1-HSL from potential recipients is present, then traR and plasmid transfer are induced. The induction of traR occurs at very low concentrations of 3-OH-C14:1-HSL (around 1 nm). TraR then induces the traI-trb operon in a quorum-sensing dependent manner in re-sponse to the TraI-made AHLs, N-(3-oxo-octanoyl)-l-homoserine lactone and N-(octanoyl)-l-homoserine lactone. The resulting autoinduction results in high levels of expression of the traI-trb operon. Premature expression of the traI-trb operon is reduced by TraM, which probably titres out TraR preventing expression of traI when there are low levels of traR expression. Expression of traR in stationary phase cells is limited by feedback inhibition mediated by TraI-made AHLs.     

ExpR is not required for swarming but promotes sliding in Sinorhizobium meliloti

Swarming is a mode of translocation dependent on flagellar activity that allows bacteria to move rapidly across surfaces. In several bacteria, swarming is a phenotype regulated by quorum sensing. It has been reported that the swarming ability of the soil bacterium Sinorhizobium meliloti Rm2011 requires a functional ExpR/Sin quorum-sensing system. However, our previous published results demonstrate that strains Rm1021 and Rm2011, both known to have a disrupted copy of expR, are able to swarm on semisolid minimal medium. In order to clarify these contradictory results, the role played by the LuxR-type regulator ExpR has been reexamined. Results obtained in this work revealed that S. meliloti can move over semisolid surfaces using at least two different types of motility. One type is flagellum-independent surface spreading or sliding, which is positively influenced by a functional expR gene mainly through the production of exopolysaccharide II (EPS II). To a lesser extent, EPS II-deficient strains can also slide on surfaces by a mechanism that is at least dependent on the siderophore rhizobactin 1021. The second type of surface translocation shown by S. meliloti is swarming, which is greatly dependent on flagella and rhizobactin 1021 but does not require ExpR. We have extended our study to demonstrate that the production of normal amounts of succinoglycan (EPS I) does not play a relevant role in surface translocation but that its overproduction facilitates both swarming and sliding motilities.     

Plasmid-associated genes in the model micro-symbiont Sinorhizobium meliloti 1021 affect the growth and development of young rice seedlings

Sinorhizobium meliloti strain 1021 and its closely related strain Rm2011 inhibit rice seedling (Oryza sativa L. cv. Pelde) growth and development under certain rice-growing conditions. Experiments showed that inoculation of seedlings with approximately less than 10 cells of 1021 was sufficient to cause this inhibition. By using a series of plasmid-cured and plasmid-deleted derivatives of Rm2011, it was found that interactions between genes encoded on pSymA, and possibly pSymB, of Rm2011, affected rice growth and development by affecting both/either the plant and/or the bacteria. Further studies found that genes potentially related to indole-3-acetic acid (IAA) synthesis and nitrate metabolism, encoded on pSymA, were involved in rice growth inhibition in Sm1021- and Sm2011-treated rice seedlings. We conclude that the rice growth inhibition by S. meliloti Sm1021 is pSymA-associated and is induced by environmental nitrate.     

Novel DNA sequences from natural strains of the nitrogen-fixing symbiotic bacterium Sinorhizobium meliloti

Variation in genome size and content is common among bacterial strains. Identifying these naturally occurring differences can accelerate our understanding of bacterial attributes, such as ecological specialization and genome evolution. In this study, we used representational difference analysis to identify potentially novel sequences not present in the sequenced laboratory strain Rm1021 of the nitrogen-fixing bacterium Sinorhizobium meliloti. Using strain Rm1021 as the driver and the type strain of S. meliloti ATCC 9930, which has a genome size approximately 370 kilobases bigger than that of strain Rm1021, as the tester, we identified several groups of sequences in the ATCC 9930 genome not present in strain Rm1021. Among the 85 novel DNA fragments examined, 55 showed no obvious homologs anywhere in the public databases. Of the remaining 30 sequences, 24 contained homologs to the Rm1021 genome as well as unique segments not found in Rm1021, 3 contained sequences homologous to those published for another S. meliloti strain but absent in Rm1021, 2 contained sequences homologous to other symbiotic nitrogen-fixing bacteria (Rhizobium etli and Bradyrhizobium japonicum), and 1 contained a sequence homologous to a gene in a non-nitrogen-fixing species, Pseudomonas sp. NK87. Using PCR, we assayed the distribution of 12 of the above 85 novel sequences in a collection of 59 natural S. meliloti strains. The distribution varied widely among the 12 novel DNA fragments, from 1.7% to 72.9%. No apparent correlation was found between the distribution of these novel DNA sequences and their genotypes obtained using multilocus enzyme electrophoresis. Our results suggest potentially high rates of gene gain and loss in S. meliloti genomes.     

Utilization of acyl-homoserine lactone quorum signals for growth by a soil pseudomonad and Pseudomonas aeruginosa PAO1

Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42 degrees C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other gamma-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.