Macromolecular crowding regulates matrix composition and gene expression in human gingival fibroblast cultures.

Standard cell cultures are performed in aqueous media with a low macromolecule concentration compared to tissue microenvironment. In macromolecular crowding (MMC) experiments, synthetic polymeric crowders are added into cell culture media to better mimic macromolecule concentrations found in vivo. However, their effect on cultured cells is incompletely understood and appears context-dependent. Here we show using human gingival fibroblasts, a cell type associated with fast and scarless wound healing, that MMC (standard medium supplemented with Ficoll 70/400) potently modulates fibroblast phenotype and extracellular matrix (ECM) composition compared to standard culture media (nMMC) over time. MMC significantly reduced cell numbers, but increased accumulation of collagen I, cellular fibronectin, and tenascin C, while suppressing level of SPARC (Secreted Protein Acidic and Cysteine Rich). Out of the 75 wound healing and ECM related genes studied, MMC significantly modulated expression of 25 genes compared to nMMC condition. MMC also suppressed myofibroblast markers and promoted deposition of basement membrane molecules collagen IV, laminin 1, and expression of LAMB3 (Laminin Subunit Beta 3) gene. In cell-derived matrices produced by a novel decellularization protocol, the altered molecular composition of MMC matrices was replicated. Thus, MMC may improve cell culture models for research and provide novel approaches for regenerative therapy.

Leucocyte- and platelet-rich fibrin regulates expression of genes related to early wound healing in human gingival fibroblasts.

BACKGROUND: Leucocyte- and platelet-rich fibrin (L-PRF) is a blood-derived biomaterial rich in leucocytes and platelets embedded in a high-density fibrin network that can be compressed into a membrane and used in surgical applications to stimulate tissue regeneration and wound healing, especially in oral cavity. This study aimed to determine the combined effects of the growth factors and cells present in L-PRF on fibroblasts that directly face the L-PRF membranes placed during surgical procedures. METHODS: The effect of L-PRF from six donors on the expression of 84 key wound healing genes in normal human gingival fibroblasts was tested by RT-qPCR. RESULTS: L-PRF significantly regulated the expression of 33 fibroblast genes (39%), including interleukins, myofibroblast-, extracellular matrix- and angiogenesis-associated genes, and matrix metalloproteinase-1 and -3. L-PRF regulated fibroblast gene expression both time- and dose-dependently, and the effects were mediated by mitogen-activated protein kinases ERK1/2, JNK and p38. L-PRF also stimulated fibroblast wound closure and promoted the ability of fibroblasts to induce endothelial tube formation. L-PRF-induced gene expression changes in fibroblast were similar to those observed in early human and pig wounds. CONCLUSIONS: This study provides new insights into the biological mechanism by which L-PRF regulates key gingival fibroblast functions important in wound healing.

Epidermal growth factor receptor signaling suppresses αvß6 integrin and promotes periodontal inflammation and bone loss.

In periodontal disease (PD), bacterial biofilms cause gingival inflammation, leading to bone loss. In healthy individuals, αvß6 integrin in junctional epithelium maintains anti-inflammatory transforming growth factor-ß1 (TGF-ß1) signaling, whereas its expression is lost in individuals with PD. Bacterial biofilms suppress ß6 integrin expression in cultured gingival epithelial cells (GECs) by attenuating TGF-ß1 signaling, leading to an enhanced pro-inflammatory response. In the present study, we show that GEC exposure to biofilms induced activation of mitogen-activated protein kinases and epidermal growth factor receptor (EGFR). Inhibition of EGFR and ERK stunted both the biofilm-induced ITGB6 suppression and IL1B stimulation. Furthermore, biofilm induced the expression of endogenous EGFR ligands that suppressed ITGB6 and stimulated IL1B expression, indicating that the effects of the biofilm were mediated by autocrine EGFR signaling. Biofilm and EGFR ligands induced inhibitory phosphorylation of the TGF-ß1 signaling mediator Smad3 at S208. Overexpression of a phosphorylation-defective mutant of Smad3 (S208A) reduced the ß6 integrin suppression. Furthermore, inhibition of EGFR signaling significantly reduced bone loss and inflammation in an experimental PD model. Thus, EGFR inhibition may provide a target for clinical therapies to prevent inflammation and bone loss in PD.

Connexin 43 regulates the expression of wound healing-related genes in human gingival and skin fibroblasts.

Fibroblasts are the most abundant connective tissue cells and play an important role in wound healing. It is possible that faster and scarless wound healing in oral mucosal gingiva relative to skin may relate to the distinct phenotype of the fibroblasts residing in these tissues. Connexin 43 (Cx43) is the most ubiquitous Cx in skin (SFBLs) and gingival fibroblasts (GFBLs), and assembles into hemichannels (HCs) and gap junctions (GJs) on the cell membrane. We hypothesized that SFBLs and GFBLs display distinct expression or function of Cx43, and that this may partly underlie the different wound healing outcomes in skin and gingiva. Here we show that Cx43 distinctly formed Cx43 GJs and HCs in human skin and gingiva in vivo. However, in SFBLs, in contrast to GFBLs, only a small proportion of total Cx43 assembled into HC plaques. Using an in vivo-like 3D culture model, we further show that the GJ, HC, and channel-independent functions of Cx43 distinctly regulated wound healing-related gene expression in GFBLs and SFBLs. Therefore, the distinct wound healing outcomes in skin and gingiva may partly relate to the inherently different assembly and function of Cx43 in the resident fibroblasts.

Connexin 43 Hemichannels Regulate the Expression of Wound Healing-Associated Genes in Human Gingival Fibroblasts.

Connexin 43 (Cx43) is the most ubiquitous connexin in various cells, and presents as hemichannels (HCs) and gap junctions (GJs) on the cell membrane. We have recently shown that Cx43 abundance was strongly reduced in fibroblasts of human gingival wounds, and blocking Cx43 function in cultured human gingival fibroblasts (GFBLs) strongly regulated the expression of wound healing-related genes. However, it is not known whether these responses involved Cx43 HCs or GJs. Here we show that Cx43 assembled into distinct GJ and HC plaques in GFBLs both in vivo and in vitro. Specific blockage of Cx43 HC function by TAT-Gap19, a Cx43 mimetic peptide, significantly upregulated the expression of several MMPs, TGF-ß signaling molecules, Tenascin-C, and VEGF-A, while pro-fibrotic molecules, including several extracellular matrix proteins and myofibroblast and cell contractility-related molecules, were significantly downregulated. These changes were linked with TAT-Gap19-induced suppression of ATP signaling and activation of the ERK1/2 signaling pathway. Collectively, our data suggest that reduced Cx43 HC function could promote fast and scarless gingival wound healing. Thus, selective suppression of Cx43 HCs may provide a novel target to modulate wound healing.

Suppression of αvß6 Integrin Expression by Polymicrobial Oral Biofilms in Gingival Epithelial Cells.

Periodontal diseases manifest by the formation of deep pockets between the gingiva and teeth where multispecies bacterial biofilms flourish, causing inflammation and bone loss. Epithelial cell receptor αvß6 integrin that regulates inflammation by activating the anti-inflammatory cytokine transforming growth factor-ß1, is highly expressed in healthy junctional epithelium that connects the gingiva to the tooth enamel. However, its expression is attenuated in human periodontal disease. Moreover, Itgb6 -/- mice display increased periodontal inflammation compared to wild-type mice. We hypothesized that bacterial biofilms present in the periodontal pockets suppress αvß6 integrin levels in periodontal disease and that this change aggravates inflammation. To this end, we generated three-week-old multi-species oral biofilms in vitro and treated cultured gingival epithelial cells (GECs) with their extracts. The biofilm extracts caused suppression of ß6 integrin expression and upregulation of pro-inflammatory cytokines, including interleukin-1ß and -6. Furthermore, GECs with ß6 integrin siRNA knockdown showed increased interleukin-1ß expression, indicating that αvß6 integrin-deficiency is associated with pro-inflammatory cytokine responsiveness. FSL-1, a synthetic bacterial lipopeptide, also suppressed ß6 integrin expression in GECs. Therefore, biofilm components, including lipopeptides, may downregulate αvß6 integrin expression in the pocket epithelium and thus promote epithelial cell-driven pro-inflammatory response in periodontal disease.

Elevated CD26 Expression by Skin Fibroblasts Distinguishes a Profibrotic Phenotype Involved in Scar Formation Compared to Gingival Fibroblasts.

Compared to skin, wound healing in oral mucosa is faster and produces less scarring, but the mechanisms involved are incompletely understood. Studies in mice have linked high expression of CD26 to a profibrotic fibroblast phenotype, but this has not been tested in models more relevant for humans. We hypothesized that CD26 is highly expressed by human skin fibroblasts (SFBLs), and this associates with a profibrotic phenotype distinct from gingival fibroblasts (GFBLs). We compared CD26 expression in human gingiva and skin and in gingival and hypertrophic-like scar-forming skin wound healing in a pig model, and used three-dimensional cultures of human GFBLs and SFBLs. In both humans and pigs, nonwounded skin contained abundantly CD26-positive fibroblasts, whereas in gingiva they were rare. During skin wound healing, CD26-positive cells accumulated over time and persisted in forming hypertrophic-like scars, whereas few CD26-positive cells were present in the regenerated gingival wounds. Cultured human SFBLs displayed significantly higher levels of CD26 than GFBLs. This was associated with an increased expression of profibrotic genes and transforming growth factor-ß signaling in SFBLs. The profibrotic phenotype of SFBLs partially depended on expression of CD26, but was independent of its catalytic activity. Thus, a CD26-positive fibroblast population that is abundant in human skin but not in gingiva may drive the profibrotic response leading to excessive scarring.

Expression and function of connexin 43 in human gingival wound healing and fibroblasts.

Connexins (C×s) are a family of transmembrane proteins that form hemichannels and gap junctions (GJs) on the cell membranes, and transfer small signaling molecules between the cytoplasm and extracellular space and between connecting cells, respectively. Among C×s, suppressing C×43 expression or function promotes skin wound closure and granulation tissue formation, and may alleviate scarring, but the mechanisms are not well understood. Oral mucosal gingiva is characterized by faster wound closure and scarless wound healing outcome as compared to skin wounds. Therefore, we hypothesized that C×43 function is down regulated during human gingival wound healing, which in fibroblasts promotes expression of genes conducive for fast and scarless wound healing. Cultured gingival fibroblasts expressed C×43 as their major connexin. Immunostaining of unwounded human gingiva showed that C×43 was abundantly present in the epithelium, and in connective tissue formed large C×43 plaques in fibroblasts. At the early stages of wound healing, C×43 was strongly down regulated in wound epithelial cells and fibroblasts, returning to the level of normal tissue by day 60 post-wounding. Blocking of C×43 function by C×43 mimetic peptide Gap27 suppressed GJ-mediated dye transfer, promoted migration, and caused significant changes in the expression of wound healing-associated genes in gingival fibroblasts. In particular, out of 54 genes analyzed, several MMPs and TGF-ß1, involved in regulation of inflammation and extracellular matrix (ECM) turnover, and VEGF-A, involved in angiogenesis, were significantly upregulated while pro-fibrotic ECM molecules, including Collagen type I, and cell contractility-related molecules were significantly down regulated. These responses involved MAPK, GSK3α/ß and TGF-ß signaling pathways, and AP1 and SP1 transcription factors. Thus, suppressed function of C×43 in fibroblasts promotes their migration, and regulates expression of wound healing-associated genes via AP1, SP1, MAPK, GSK3α/ß and TGF-ß signaling pathways, and may promote fast and scarless wound healing in human gingiva.

Human gingival fibroblasts display a non-fibrotic phenotype distinct from skin fibroblasts in three-dimensional cultures.

Scar formation following skin injury can be a major psychosocial and physiological problem. However, the mechanisms of scar formation are still not completely understood. Previous studies have shown that wound healing in oral mucosa is faster, associates with a reduced inflammatory response and results to significantly reduced scar formation compared with skin wounds. In the present study, we hypothesized that oral mucosal fibroblasts from human gingiva are inherently distinct from fibroblasts from breast and abdominal skin, two areas prone to excessive scar formation, which may contribute to the preferential wound healing outcome in gingiva. To this end, we compared the phenotype of human gingival and skin fibroblasts cultured in in vivo-like three-dimensional (3D) cultures that mimic the cells' natural extracellular matrix (ECM) niche. To establish 3D cultures, five parallel fibroblast lines from human gingiva (GFBLs) and breast skin (SFBLs) were seeded in high density, and cultured for up to 21 days in serum and ascorbic acid containing medium to induce expression of wound-healing transcriptome and ECM deposition. Cell proliferation, morphology, phenotype and expression of wound healing and scar related genes were analyzed by real-time RT-PCR, Western blotting and immunocytochemical methods. The expression of a set of genes was also studied in three parallel lines of human abdominal SFBLs. Findings showed that GFBLs displayed morphologically distinct organization of the 3D cultures and proliferated faster than SFBLs. GFBLs expressed elevated levels of molecules involved in regulation of inflammation and ECM remodeling (MMPs) while SFBLs showed significantly higher expression of TGF-ß signaling, ECM and myofibroblast and cell contractility-related genes. Thus, GFBLs display an inherent phenotype conducive for fast resolution of inflammation and ECM remodeling, characteristic for scar-free wound healing, while SFBLs have a profibrotic, scar-prone phenotype.

Critical role for αvß6 integrin in enamel biomineralization.

Tooth enamel has the highest degree of biomineralization of all vertebrate hard tissues. During the secretory stage of enamel formation, ameloblasts deposit an extracellular matrix that is in direct contact with the ameloblast plasma membrane. Although it is known that integrins mediate cell-matrix adhesion and regulate cell signaling in most cell types, the receptors that regulate ameloblast adhesion and matrix production are not well characterized. We hypothesized that αvß6 integrin is expressed in ameloblasts where it regulates biomineralization of enamel. Human and mouse ameloblasts were found to express both ß6 integrin mRNA and protein. The maxillary incisors of Itgb6(-/-) mice lacked yellow pigment and their mandibular incisors appeared chalky and rounded. Molars of Itgb6(-/-) mice showed signs of reduced mineralization and severe attrition. The mineral-to-protein ratio in the incisors was significantly reduced in Itgb6(-/-) enamel, mimicking hypomineralized amelogenesis imperfecta. Interestingly, amelogenin-rich extracellular matrix abnormally accumulated between the ameloblast layer of Itgb6(-/-) mouse incisors and the forming enamel surface, and also between ameloblasts. This accumulation was related to increased synthesis of amelogenin, rather than to reduced removal of the matrix proteins. This was confirmed in cultured ameloblast-like cells, in which αvß6 integrin was not an endocytosis receptor for amelogenins, although it participated in cell adhesion on this matrix indirectly via endogenously produced matrix proteins. In summary, integrin αvß6 is expressed by ameloblasts and it plays a crucial role in regulating amelogenin deposition and/or turnover and subsequent enamel biomineralization.

Localization and potential function of kindlin-1 in periodontal tissues.

Kindlin-1 is an intracellular focal adhesion protein that regulates the actin cytoskeleton. Patients suffering from Kindler syndrome have a homologous mutation of the kindlin-1 gene and develop skin blisters, periodontal disease, and intestinal complications because of deficient adhesion of the basal epithelial cells. We investigated kindlin-1 localization in periodontal tissue and its functions in cultured keratinocytes and showed that kindlin-1 co-localizes with migfilin and paxillin in the basal epithelial cells of oral mucosa and in cultured keratinocytes. The kindlin-1-deficient oral mucosal tissue from a patient with Kindler syndrome showed a complete lack of paxillin and reduced migfilin immunostaining in the basal keratinocytes. Co-immunoprecipitation showed that migfilin directly interacted with kindlin-1. RNA interference-induced kindlin-1 deficiency in keratinocytes led to an altered distribution of migfilin-containing focal adhesions, reduced cell spreading, decreased cell proliferation, and decelerated cell migration. Disruption of microtubules in the kindlin-1-deficient cells further reduced cell spreading, suggesting that microtubules can partially compensate for kindlin-1 deficiency. Kindlin-1 supported mature cell-extracellular matrix adhesions of keratinocytes, as downregulation of kindlin-1 expression significantly reduced the cell-adhesion strength. In summary, kindlin-1 interacts with migfilin and plays a crucial role in actin-dependent keratinocyte cell adhesion essential for epidermal and periodontal health.

Kindler syndrome and periodontal disease: review of the literature and a 12-year follow-up case.

BACKGROUND: The association of aggressive periodontitis with Kindler syndrome was based on a single case in 1996 and later confirmed with a larger population. Since then, significant research has greatly increased our understanding of the molecular pathology of this disorder. We review recent advances in the molecular mechanisms of the syndrome and present a maintenance case report of a patient who has been followed in our clinic. METHODS: A female patient who was diagnosed with Kindler syndrome and aggressive periodontitis at the age of 16 years has been followed and treated in our clinic for 12 years. Her main treatment has been maintenance therapy following her initial treatment and restorative work previously documented. Gingival biopsies obtained during the recent extraction of hopeless maxillary molars were used for histologic assessment of gingival tissue attachment apparatus and to isolate gingival fibroblasts. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using these cells to confirm the lack of expression of kindlin-1. RESULTS: RT-PCR showed the total loss of kindlin-1 mRNA in cultured gingival fibroblasts, supporting the clinical diagnosis of Kindler syndrome. Tissue biopsies revealed atypical pocket epithelium. Maintenance therapy has been moderately successful. Teeth that were recently lost had a poor prognosis at the initial assessment. The patient's gingiva and oral mucosa continue to be fragile with episodes of sloughing and inflammation. CONCLUSIONS: Periodontitis in Kindler syndrome responds to maintenance therapy, but the gingiva and oral mucosa continue to display an abnormal appearance with white patches. Histologic findings suggest that the junctional epithelium in Kindler syndrome may be abnormal and could explain why these patients have periodontal disease. Attachment loss progressed around teeth with an initial guarded or poor prognosis. Teeth that started with a good or fair prognosis continue to have a fair prognosis. Limited dental implant treatment is being considered.

Absence of alphavbeta6 integrin is linked to initiation and progression of periodontal disease.

Integrin alphavbeta6 is generally not expressed in adult epithelia but is induced in wound healing, cancer, and certain fibrotic disorders. Despite this generalized absence, we observed that alphavbeta6 integrin is constitutively expressed in the healthy junctional epithelium linking the gingiva to tooth enamel. Moreover, expression of alphavbeta6 integrin was down-regulated in human periodontal disease, a common medical condition causing tooth loss and also contributing to the development of cardiovascular diseases by increasing the total systemic inflammatory burden. Remarkably, integrin beta6 knockout mice developed classic signs of spontaneous, chronic periodontal disease with characteristic inflammation, epithelial down-growth, pocket formation, and bone loss around the teeth. Integrin alphavbeta6 acts as a major activator of transforming growth factor-beta1 (TGF-beta1), a key anti-inflammatory regulator in the immune system. Co-expression of TGF-beta1 and alphavbeta6 integrin was observed in the healthy junctional epithelium. Moreover, an antibody that blocks alphavbeta6 integrin-mediated activation of TGF-beta1 initiated inflammatory periodontal disease in a rat model of gingival inflammation. Thus, alphavbeta6 integrin is constitutively expressed in the epithelium sealing the gingiva to the tooth and plays a central role in protection against inflammatory periodontal disease through activation of TGF-beta1.

Mating factor linkage and genome evolution in basidiomycetous pathogens of cereals.

Sex in basidiomycete fungi is controlled by tetrapolar mating systems in which two unlinked gene complexes determine up to thousands of mating specificities, or by bipolar systems in which a single locus (MAT) specifies different sexes. The genus Ustilago contains bipolar (Ustilago hordei) and tetrapolar (Ustilago maydis) species and sexual development is associated with infection of cereal hosts. The U. hordei MAT-1 locus is unusually large (approximately 500 kb) and recombination is suppressed in this region. We mapped the genome of U. hordei and sequenced the MAT-1 region to allow a comparison with mating-type regions in U. maydis. Additionally the rDNA cluster in the U. hordei genome was identified and characterized. At MAT-1, we found 47 genes along with a striking accumulation of retrotransposons and repetitive DNA; the latter features were notably absent from the corresponding U. maydis regions. The tetrapolar mating system may be ancestral and differences in pathogenic life style and potential for inbreeding may have contributed to genome evolution.

HaCaT keratinocyte migration is dependent on epidermal growth factor receptor signaling and glycogen synthase kinase-3alpha.

After epithelial disruption by tissue injury, keratinocytes migrate from the wound edge into a provisional matrix. This process is stimulated by growth factors that signal through epidermal growth factor (EGF) receptor, including EGF, heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor-alpha (TGF-alpha), and by for example keratinocyte growth factor (KGF) and TGF-beta1 that function through different receptors. We have previously shown that keratinocyte migration induced by EGF or staurosporine is dependent on the activity of glycogen synthase kinase-3 (GSK-3). In the present study, we show that keratinocyte migration induced by TGF-beta1, KGF, EGF, TGF-alpha and staurosporine depends on EGFR signaling, involves autocrine HB-EGF expression and is potently blocked by GSK-3 inhibitors SB-415286 and LiCl. Inhibition of GSK-3 also retards wound reepithelialization in vivo in mice. Moreover, inhibition of GSK-3 activity prevented cell rounding that is an early event in EGFR-mediated keratinocyte migration. Isoform-specific GSK-3alpha and GSK-3beta knockdown and overexpression experiments with siRNAs and adenoviral constructs, respectively, revealed that GSK-3alpha is required for keratinocyte migration, whereas excessive activity of GSK-3beta is inhibitory. Thus, induction of keratinocyte migration is conveyed through EGFR, promoted by endogenous HB-EGF and requires GSK-3alpha activity.

Extracellular proteins involved in soybean cultivar-specific nodulation are associated with pilus-like surface appendages and exported by a type III protein secretion system in Sinorhizobium fredii USDA257.

Several gram-negative plant and animal pathogenic bacteria have evolved a type III secretion system (TTSS) to deliver effector proteins directly into the host cell cytosol. Sinorhizobium fredii USDA257, a symbiont of soybean and many other legumes, secretes proteins called Nops (nodulation outer proteins) into the extracellular environment upon flavonoid induction. Mutation analysis and the nucleotide sequence of a 31.2-kb symbiosis (sym) plasmid DNA region of USDA257 revealed the existence of a TTSS locus in this symbiotic bacterium. This locus includes rhc (rhizobia conserved) genes that encode components of a TTSS and proteins that are secreted into the environment (Nops). The genomic organization of the TTSS locus of USDA257 is remarkably similar to that of another broad-host range symbiont, Rhizobium sp. strain NGR234. Flavonoids that activate the transcription of the nod genes of USDA257 also stimulate the production of novel filamentous appendages known as pili. Electron microscope examination of isolated pili reveals needle-like filaments of 6 to 8 nm in diameter. The production of the pili is dependent on a functional nodD1 and the presence of a nod gene-inducing compound. Mutations in several of the TTSS genes negate the ability of USDA257 to elaborate pili. Western blot analysis using antibodies raised against purified NopX, Nop38, and Nop7 reveals that these proteins were associated with the pili. Mutations in rhcN, rhcJ, rhcC, and ttsI alter the ability of USDA257 to form nodules on Glycine max and Macroptilium atropurpureum.

Citrate synthase mutants of Sinorhizobium fredii USDA257 form ineffective nodules with aberrant ultrastructure.

The tricarboxylic acid (TCA) cycle plays an important role in generating the energy required by bacteroids to fix atmospheric nitrogen. Citrate synthase is the first enzyme that controls the entry of carbon into the TCA cycle. We cloned and determined the nucleotide sequence of the gltA gene that encodes citrate synthase in Sinorhizobium fredii USDA257, a symbiont of soybeans (Glycine max [L.] Merr.) and several other legumes. The deduced citrate synthase protein has a molecular weight of 48,198 and exhibits sequence similarity to citrate synthases from several bacterial species, including Sinorhizobium meliloti and Rhizobium tropici. Southern blot analysis revealed that the fast-growing S. fredii strains and Rhizobium sp. strain NGR234 contained a single copy of the gene located in the bacterial chromosome. S. fredii USDA257 gltA mutant HBK-CS1, which had no detectable citrate synthase activity, had diminished nodulation capacity and produced ineffective nodules on soybean. Light and electron microscopy observations revealed that the nodules initiated by HBK-CS1 contained very few bacteroids. The infected cells contained large vacuoles and prominent starch grains. Within the vacuoles, membrane structures that appeared to be reminiscent of disintegrating bacteroids were detected. The citrate synthase mutant had altered cell surface characteristics and produced three times more exopolysaccarides than the wild type produced. A plasmid carrying the USDA257 gltA gene, when introduced into HBK-CS1, was able to restore all of the defects mentioned above. Our results demonstrate that a functional citrate synthase gene of S. fredii USDA257 is essential for efficient soybean nodulation and nitrogen fixation.