GGDEF and EAL proteins play different roles in the control of Sinorhizobium meliloti growth, motility, exopolysaccharide production, and competitive nodulation on host alfalfa.

A new bacterial secondary messenger, bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), is usually synthesized or decomposed by proteins containing GGDEF or glutamate-alanine-leucine (EAL) domain. They often act as cyclase or phosphodiesterase of c-di-GMP and their genes are distributed among almost all bacteria according to known genomic DNA sequences. However, the systematic identification of GGDEF and EAL genes remains unclear in rhizobia, soil bacteria that interact with compatible legumes to form nitrogen-fixing nodules. In this study, 19 putative GGDEF and EAL genes were identified in a model rhizobium, Sinorhizobium meliloti, by bioinformatic analysis (encoding 5 GGDEF proteins, 4 EAL proteins, and 10 GGDEF and EAL double-domain proteins). Null mutants of 14 genes were constructed through systematic plasmid insertion. All 14 gene mutants showed deficient growth in minimal medium and defective motility, and 11 gene mutants produced a lot more exopolysaccharide and displayed less competitive nodulation on the host plant, alfalfa. Our results suggested that GGDEF and EAL proteins may play different roles in the control of S. meliloti physiology, although they contain conserved catalytic (GGDEF or EAL) domains. Our finding also implied that c-di-GMP may play an important role in the interactions between this rhizobium and its host plants to establish efficient symbiosis.

Characterization and expression analysis of Medicago truncatula ROP GTPase family during the early stage of symbiosis.

ROPs (Rho-related GTPases of plants) are small GTPases that are plant-specific signaling proteins. They act as molecular switches in a variety of developmental processes. In this study, seven cDNA clones coding for ROP GTPases have been isolated in Medicago truncatula, and conserved and divergent domains are identified in these predicted MtROP proteins. Phylogenetic analysis has indicated that MtROPs are distributed into groups II, III, IV but group I. MtROP genes are expressed in various tissues at different levels. A quantitative reverse transcription PCR analysis indicated that these MtROP genes have different expression profiles in the roots in response to infection with rhizobia. The expression of MtROP3, MtROP5 and MtROP6 are increased, as the expression of Nod factor or rhizobial-induced marker genes--NFP, Rip1 and Enod11; MtROP10 has showed enhanced expression at a certain post-inoculation time point. No significant changes in MtROP7 and MtROP9 expression have been detected and MtROP8 expression is dramatically decreased by about 80%-90%. Additionally, ROP promoter-GUS analysis has showed that MtROP3, MtROP5 and MtROP6 have elevated expression in transgenic root hairs after rhizobial inoculation. These results might suggest a role for some ROP GTPases in the regulation of early stages during rhizobial infection in symbiosis.

Maturation of the nodule-specific transcript MsHSF1c in Medicago sativa may involve interallelic trans-splicing.

In nonplant species, many heat-shock transcription factors (HSFs) undergo spatiotemporal-specific alternative splicing. However, little is known about the spatiotemporal-specific splicing of HSFs in plants. Previously, we reported that the alfalfa HSF gene MsHSF1 undergoes multiple alternative splicing events in various tissues. Here, we identified another spliced transcript isoform, MsHSF1c, containing a 177-base tandem repeat, and showed that the low-abundance MsHSF1c is a nodule-specific transcript of MsHSF1. We also found that MsHSF1 presents multiple alleles with single-base variations and the expression of MsHSF1 alleles has allele-specific differences in alfalfa nodules. Because single-base variations at position 1006 change the AT of MsHSF1b to GT in MsHSF1b-3, creating a pair of donor/acceptor sites with the AG of MsHSF1b/1b-1 at position 827-828 for pre-mRNA splicing, we suggest that MsHSF1c may be generated by trans-splicing between alleles MsHSF1b-3 and MsHSF1b or MsHSF1b-1. These results provide new insight into the role of tissue-specific contribution in the transcription of plant HSF genes.

Cloning and characterization of a heat shock protein 70 gene, MsHSP70-1, in Medicago sativa.

Some members of the heat shock protein 70 (HSP70) family have important functions in organism development. Here, we identified an alfalfa (Medicago sativa L. cv. Algonquin) HSP70 gene, MsHSP70-1, using cDNA array and reverse transcription-polymerase chain reaction. This gene contains a 1947 bp open reading frame encoding a protein of 649 amino acids. This protein contains all conserved domains, motifs, and characteristic sequences of plant HSP70s. The expression of MsHSP70-1 is enhanced in nodule compared with root, stem, leaf, and flower, and throughout the process of nodule development. Northern hybridization analysis indicated that the expression of MsHSP70-1 in nodule requires the active bacA gene of rhizobia. These results suggested that MsHSP70-1 might play an important role in alfalfa nodule development.

Characteristics of the LrhA subfamily of transcriptional regulators from Sinorhizobium meliloti.

In our previous work, we identified 94 putative genes encoding LysR-type transcriptional regulators from Sinorhizobium meliloti. All of these putative lysR genes were mutagenized using plasmid insertions to determine their phenotypes. Six LysR-type regulators, encoded by mutants SMa1979, SMb20715, SMc00820, SMc04163, SMc03975, and SMc04315, showed similar amino acid sequences (30%) and shared the conserved DNA-binding domain with LrhA, HexA, or DgdR. Phenotype analysis of these gene mutants indicated that the regulators control the swimming behaviors of the bacteria, production of quorum-sensing signals, and secretion of extracellular proteins. These characteristics are very similar to those of LrhA, HexA, and DgdR. Thus, we refer to this group as the LrhA subfamily. Sequence analysis showed that a great number of homologous genes of the LrhA subfamily were distributed in the alpha, beta, and gamma subdivisions of proteobacteria, and a few in actinobacteria. These findings could provide new clues to the roles of the LysR gene family.

Structure and alternative splicing of a heat shock transcription factor gene, MsHSF1, in Medicago sativa.

Plant heat shock transcription factors (HSF) are highly complex. In this study, we identified an alfalfa HSF gene MsHSF1 that is composed of four exons and three introns in the encoding region. The intron1-exon2-intron2-exon3-intron3 as an intervening sequence was inserted at the conserved position that separates the coding region for the DNA-binding domain by single intron in other known plant HSF genes. Alternative splicing of MsHSF1 has generated five transcript isoforms. Spliced transcript MsHSF1b consisted of exon1 and exon4, encodes a class A1 HSF protein that can specifically bind to the heat shock elements in vitro. Other four spliced transcripts (MsHSF1a-1 to 4) consist of exon1, part of the intervening sequence and exon4. These transcripts carry the premature termination codon and are low-abundant. Apparently these transcripts are the targets of nonsense-mediated mRNA decay (NMD). These results provide new insight into roles in the expression regulation of plant HSF genes.

[A LuxR family regulator, ExpR regulates the expression of motC operon from Sinorhizobium meliloti].

In Sinorhizobium meliloti strain Rm1021, ExpR+ mutation results in the overproduction of EPS II, which is required for efficient invasion of root nodules on the host plant alfalfa. When rhizobia were grown in LB/MC medium for 36-hour then placed at room temperature, most of ExpR+ mutant (Rm8530) cells aggregated at the bottom of the tubes, but ExpR+ sinR double mutant Rm11528 and wild type Rm1021 did not. The ExpR+ mutant was also found to swim slower than Rm1021 on swarming plates, but the ExpR+ sinR- mutant showed almost the same as wild type. The average diameter of swarming plaques for Rm8530, Rm11528 and Rm1021 was 13, 16 and 16 mm respectively, when bacteria were incubated at 28 degrees C for two days. After four days, the plaques enlarged to 17, 22 and 20 mm, respectively. These results indicate that ExpR+ mutation causes a serious defection of motility in this condition. By flagella staining with silver nitrate method, it was noted that all three strains had flagella. It suggests that the swimming behavior of Rm8530 may not result from the change in components and structures of flagella. Based on DNA microarray data, Hoang speculated that the Sin system seemed to regulate a multitude of genes in S. meliloti, including genes that participate in succinoglycan production, motility, and chemotaxis, as well as other cellular processes (Hoang et al. 2004). And most of the regulation by the Sin system was dependent on the presence of the ExpR regulator. Accordingly, the expression of the flaA, cheY1 and motC operon was determined using promoter: lacZ fusion in different genetic backgrounds. The results showed two fold lower of motC expression activity in ExpR+ mutant strain Rm8530 than in wild-type strain Rm1021 at early exponential phase and this decrease was hold back by further mutation of sinR. However, the beta-galactosidase activity of motC-lacZ fusion was almost the same in three strains at later exponential phase. These results suggest that ExpR may repress the expression of motC operon in a low cell density, but this repression can be deprived in a higher cell density. It may be a good explanation to the motility of Rm8530 on swarming plates, since it is lower cell density of bacteria on the swarming plates. Therefore, it may be concluded that ExpR both involved in the regulation of EPS II production and motility in S. meliloti.

Improvement of Bacterial Two Hybrid System.

Bacterial two-hybrid system is a newly developed method for studying protein-protein interactions, especially for effects of the environmental factors on the interaction. In our studies of the effect of NH(4)( ) or oxygen on the NifL-NifA interaction, it was found that E.coli strain DHP1 cya(-), when transformed by T18-NifL together with T25-NifA, exhibited high activity of beta-galactosidase in the presence of NH(4)( ), and appeared as red colonies when grown in MacConkey/maltose agar. This indicated the direct interaction of NifL and NifA. However,similar phenomena were also shown in the case of the DHP1 cya(-) strain harboring T18-NifL or T25-NifL alone, respectively, without its interacting counterpart T25-NifA or T18-NifA. In this paper, a brief method is presented to detect the protein-protein interaction using direct assay of the adenylate cyclase, thus minimizing the false positives produced by the beta-galactosidase activity assay or MacConkey/maltose agar plating.

Use of bacterial two-hybrid system to investigate the molecular interaction between the regulators NifA and NifL of Enterobacter cloacae.

Expression of the nitrogen fixation (nif) genes is tightly regulated by two proteins NifA and NifL in the gamma-subdivision of the proteobacteria. NifA is a transcriptional activator, which can be inactivated by NifL in the presence of oxygen or excess fixed nitrogen. A direct interaction between E. cloacae NifL and NifA was detected using the bacterial two-hybrid system. This interaction was accelerated in the presence of fixed nitrogen, while oxygen had no effect. NifL proteins, with their C-terminus being deleted, completely lost the ability to interact with NifA. The data suggest that the C-terminal domain of NifL acts as a sensor of the nitrogen status of the cell and mediates interaction with NifA.

Analysis of the downstream region of nodD3 P1 promoter by deletion and complementation tests in Sinorhizobium meliloti.

In Sinorhizobium meliloti, the nodD3 gene is transcriptionally controlled by two promoters, P1 and P2. Under P1, there is a 660 bp sequence including a small open reading frame, ORF2, followed by the nodD3 coding region. Genetic analysis using the different deletions on the 3' ends of P1 downstream sequence showed that the downstream sequence +1-+125nt is essential for P1 expression. Complementation, mutations and nodulation tests demonstrated that the ORF2 auto-represses P1 expression, while the P1 downstream sequence +1-+125nt counteracts it.

Identification of a TRAP transporter for malonate transport and its expression regulated by GtrA from Sinorhizobium meliloti.

Sinorhizobium meliloti can live as a saprophyte in soil or as a nitrogen-fixing symbiont inside the root nodule cells of alfalfa and related legumes by utilizing different organic compounds as its carbon source. Here we have identified the matPQMAB operon in S. meliloti 1021. Within this operon, matP, matQ and the M region of the fused gene matMA encode an extracytoplasmic solute receptor, a small transmembrane protein and a large transmembrane protein, consisting of three components of the tripartite ATP-independent periplasmic (TRAP) transporter for malonate transport. The A region of the fused gene matMA and matB encode malonate-metabolizing enzymes, malonyl-CoA decarboxylase and malonyl-CoA synthetase. The null mutant of each matPQMAB gene is unable to grow on M9 minimal medium containing malonate as the sole carbon source. However, these mutants can induce the formation of efficient nitrogen-fixing root nodules on alfalfa. The matPQMAB operon is expressed in free-living bacterial cells and symbiotic bacterial cells from infection threads and root nodules. The GntR family transcriptional regulator, GtrA, specifically binds the promoter of the matPQMAB operon, positively regulating its expression. Moreover, the matPQMAB can be transcriptionally induced by malonate. These results suggested that a C(3)-dicarboxylic acid TRAP transporter is responsible for malonate transport in S. meliloti.

Disruption of nifA gene influences multiple cellular processes in Sinorhizobium meliloti.

Sinorhizobium meliloti nifA is important in fixing nitrogen during symbiosis. A nifA null mutant induces small white invalid nodules in the roots of host plant. The additional phenotypic alterations associated with the disruption of the nifA gene are reported in this study. Under a free-living state, S. meliloti nifA mutant reduces its ability to swarm on a half-solid plate. Interestingly, the AHL (Acylhomoserine lactones) contents in the nifA mutant are lower than that of the wild type during the lag phase, whereas it is reversed in the logarithmic and stationary phases. Quantitative spectrophotometric assays reveal that the total amount of extracellular proteins of the nifA mutant are lower than that of the wild type. In addition, the mutant abolishes its nodulation competitive ability during symbiosis. These findings indicate that NifA plays a regulatory role in multiple cellular processes in S. meliloti.

nifH promoter activity is regulated by DNA supercoiling in Sinorhizobium meliloti.

In prokaryotes, DNA supercoiling regulates the expression of many genes; for example, the expression of Klebsiella pneumoniae nifLA operon depends on DNA negative supercoiling in anaerobically grown cells, which indicates that DNA supercoiling might play a role in gene regulation of the anaerobic response. Since the expression of the nifH promoter in Sinorhizobium meliloti is not repressed by oxygen, it is proposed that the status of DNA supercoiling may not affect the expression of the nifH promoter. We tested this hypothesis by analyzing nifH promoter activity in wild-type and gyr- Escherichia coli in the presence and absence of DNA gyrase inhibitors. Our results show that gene expression driven by the S. meliloti nifH promoter requires the presence of active DNA gyrase. Because DNA gyrase increases the number of negative superhelical turns in DNA in the presence of ATP, our data indicate that negative supercoiling is also important for nifH promoter activity. Our study also shows that the DNA supercoiling-dependent S. meliloti nifH promoter activity is related to the trans-acting factors NtrC and NifA that activate it. DNA supercoiling appeared to have a stronger effect on NtrC-activated nifH promoter activity than on NifA-activated promoter activity. Collectively, these results from the S. meliloti nifH promoter model system seem to indicate that, in addition to regulating gene expression during anaerobic signaling, DNA supercoiling may also provide a favorable topology for trans-acting factor binding and promoter activation regardless of oxygen status.

Two new Sinorhizobium meliloti LysR-type transcriptional regulators required for nodulation.

The establishment of an effective nitrogen-fixing symbiosis between Sinorhizobium meliloti and its legume host alfalfa (Medicago sativa) depends on the timely expression of nodulation genes that are controlled by LysR-type regulators. Ninety putative genes coding for LysR-type transcriptional regulators were identified in the recently sequenced S. meliloti genome. All 90 putative lysR genes were mutagenized using plasmid insertions as a first step toward determining their roles in symbiosis. Two new LysR-type symbiosis regulator genes, lsrA and lsrB, were identified in the screening. Both the lsrA and lsrB genes are expressed in free-living S. meliloti cells, but they are not required for cell growth. An lsrA1 mutant was defective in symbiosis and elicited only white nodules that exhibited no nitrogenase activity. Cells of the lsrA1 mutant were recovered from the white nodules, suggesting that the lsrA1 mutant was blocked early in nodulation. An lsrB1 mutant was deficient in symbiosis and elicited a mixture of pink and white nodules on alfalfa plants. These plants exhibited lower overall nitrogenase activity than plants inoculated with the wild-type strain, which is consistent with the fact that most of the alfalfa plants inoculated with the lsrB1 mutant were short and yellow. Cells of the lsrB1 mutant were recovered from both pink and white nodules, suggesting that lsrB1 mutants could be blocked at multiple points during nodulation. The identification of two new LysR-type symbiosis transcriptional regulators provides two new avenues for understanding the complex S. meliloti-alfalfa interactions which occur during symbiosis.

Role of oxyR from Sinorhizobium meliloti in regulating the expression of catalases.

The process of symbiotic nitrogen fixation results in the generation of reactive oxygen species such as the superoxide anion (O2-) and hydrogen peroxide (H2O2). The response of rhizobia to these toxic oxygen species is an important factor in nodulation and nitrogen fixation. In Sinorhizobium meliloti, one oxyR homologue and three catalase genes, katA, katB, and katC were detected by sequence analysis. This oxyR gene is located next to and divergently from katA on the chromosome. To investigate the possible roles of oxyR in regulating the expression of catalases at the transcriptional level in S. meliloti, an insertion mutant of this gene was constructed. The mutant was more sensitive and less adaptive to H2O2 than the wild type strain, and total catalase/peroxidase activity was reduced approximately fourfold with the OxyR mutation relative to controls. The activities of KatA and KatB and the expression of katA::lacZ and katB::lacZ promoter fusions were increased in the mutant strain compared with the parental strain grown in the absence of H2O2, indicating that katA and katB are repressed by OxyR. However, when exposed to H2O2, katA expression was also increased in both S. meliloti and Escherichia coli. When exposed to H2O2, OxyR is converted from a reduced to an oxidized form in E. coli. We concluded that the reduced form of OxyR functions as a repressor of katA and katB expression. Thus, in the presence of H2O2, reduced OxyR is converted to the oxidized form of OxyR that then results in increased katA expression. We further showed that oxyR expression is autoregulated via negative feedback.

Sinorhizobium meliloti ExoR and ExoS proteins regulate both succinoglycan and flagellum production.

The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa (Medicago sativa) by S. meliloti. Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction of succinoglycan and a reduction in symbiosis. Systematic analyses of the symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of root hair colonization. In addition, both the exoR95 and exoS96 mutations caused a marked reduction in the biosynthesis of flagella and consequent loss of ability of the cells to swarm and swim. Succinoglycan overproduction did not appear to be the cause of the suppression of flagellum biosynthesis. Further analysis indicated that both the exoR95 and exoS96 mutations affected the expression of the flagellum biosynthesis genes. These findings suggest that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis. These findings provide new avenues of understanding of the physiological changes S. meliloti cells go through during the early stages of symbiosis and of the signal transduction pathways that mediate such changes.