The ++Sinorhizobium meliloti lon protease is involved in regulating exopolysaccharide synthesis and is required for nodulation of alfalfa

While screening for Sinorhizobium meliloti Pho regulatory mutants, a transposon mutant was isolated that constitutively expressed higher levels of acid and alkaline phosphatase enzymes. This mutant was also found to form pseudonodules on alfalfa that were delayed in appearance relative to those formed by the wild-type strain, it contained few bacteroids, and it did not fix nitrogen. Sequence analysis of the transposon insertion site revealed the affected gene to have high homology to Lon proteases from a number of organisms. In minimal succinate medium, the mutant strain was found to grow more slowly, reach lower maximal optical density, and produce more extracellular polysaccharide (EPS) than the wild-type strain. The mutant fluoresced brightly on minimal succinate agar containing calcofluor (which binds to EPSI, a constitutively expressed succinoglycan), and gas chromotographic analysis of purified total EPS showed that the glucose-to-galactose ratio in the lon mutant total EPS was 5.0 +/- 0.2 (mean +/- standard error), whereas the glucose-to-galactose ratio in the wild-type strain was 7.1 +/- 0.5. These data suggested that in addition to EPSI, the lon mutant also constitutively synthesized EPSII, a galactoglucan which is the second major EPS known to be produced by S. meliloti, but typically is expressed only under conditions of phosphate limitation. (13)C nuclear magnetic resonance analysis showed no major differences between EPS purified from the mutant and wild-type strains. Normal growth, EPS production, and the symbiotic phenotype were restored in the mutant strain when the wild-type lon gene was present in trans. The results of this study suggest that the S. meliloti Lon protease is important for controlling turnover of a constitutively expressed protein(s) that, when unregulated, disrupts normal nodule formation and normal growth.     

Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti

Flavonoids play critical roles in legume–rhizobium symbiosis. However, the role of individual flavonoid compounds in this process has not yet been clearly established. We silenced different flavonoid-biosynthesis enzymes to generate transgenic Medicago truncatula roots with different flavonoid profiles. Silencing of chalcone synthase, the key entry-point enzyme for flavonoid biosynthesis led to flavonoid-deficient roots. Silencing of isoflavone synthase and flavone synthase led to roots deficient for a subset of flavonoids, isoflavonoids (formononetin and biochanin A) and flavones (7,4'-dihydroxyflavone), respectively. When tested for nodulation by Sinorhizobium meliloti , flavonoid-deficient roots had a near complete loss of nodulation, whereas flavone-deficient roots had reduced nodulation. Isoflavone-deficient roots nodulated normally, suggesting that isoflavones might not play a critical role in M. truncatula nodulation, even though they are the most abundant root flavonoids. Supplementation of flavone-deficient roots with 7, 4'-dihydroxyflavone, a major inducer of S. meliloti nod genes, completely restored nodulation. However, the same treatment did not restore nodulation in flavonoid-deficient roots, suggesting that other non- nod gene-inducing flavonoid compounds are also critical to nodulation. Supplementation of roots with the flavonol kaempferol (an inhibitor of auxin transport), in combination with the use of flavone pre-treated S. meliloti cells, completely restored nodulation in flavonoid-deficient roots. In addition, S. meliloti cells constitutively producing Nod factors were able to nodulate flavone-deficient roots, but not flavonoid-deficient roots. These observations indicated that flavones might act as internal inducers of rhizobial nod genes, and that flavonols might act as auxin transport regulators during nodulation. Both these roles of flavonoids appear critical for symbiosis in M. truncatula .     

Increase in alfalfa nodulation, nitrogen fixation, and plant growth by specific DNA amplification in Sinorhizobium meliloti.

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.     

Construction and environmental release of a Sinorhizobium meliloti strain genetically modified to be more competitive for alfalfa nodulation

Highly efficient nitrogen-fixing strains selected in the laboratory often fail to increase legume production in agricultural soils containing indigenous rhizobial populations because they cannot compete against these populations for nodule formation. We have previously demonstrated, with a Sinorhizobium meliloti PutA- mutant strain, that proline dehydrogenase activity is required for colonization and therefore for the nodulation efficiency and competitiveness of S. meliloti on alfalfa roots (J. I. Jiménez-Zurdo, P. van Dillewijn, M. J. Soto, M. R. de Felipe, J. Olivares, and N. Toro, Mol. Plant-Microbe Interact. 8:492-498, 1995). In this work, we investigated whether the putA gene could be used as a means of increasing the competitiveness of S. meliloti strains. We produced a construct in which a constitutive promoter was placed 190 nucleotides upstream from the start codon of the putA gene. This resulted in an increase in the basal expression of this gene, with this increase being even greater in the presence of the substrate proline. We found that the presence of multicopy plasmids containing this putA gene construct increased the competitiveness of S. meliloti in microcosm experiments in nonsterile soil planted with alfalfa plants subjected to drought stress only during the first month. We investigated whether this construct also increased the competitiveness of S. meliloti strains under agricultural conditions by using it as the inoculum in a contained field experiment at León, Spain. We found that the frequency of nodule occupancy was higher with inoculum containing the modified putA gene for samples that were analyzed after 34 days but not for samples that were analyzed later.     

Increased production of the exopolysaccharide succinoglycan enhances Sinorhizobium meliloti 1021 symbiosis with the host plant Medicago truncatula

The nitrogen-fixing rhizobial symbiont Sinorhizobium meliloti 1021 produces acidic symbiotic exopolysaccharides that enable it to initiate and maintain infection thread formation on host legume plants. The exopolysaccharide that is most efficient in mediating this process is succinoglycan (exopolysaccharide I [EPSI]), a polysaccharide composed of octasaccharide repeating units of 1 galactose and 7 glucose residues, modified with succinyl, acetyl, and pyruvyl substituents. Previous studies had shown that S. meliloti 1021 mutants that produce increased levels of succinoglycan, such as exoR mutants, are defective in symbiosis with host plants, leading to the hypothesis that high levels of succinoglycan production might be detrimental to symbiotic development. This study demonstrates that increased succinoglycan production itself is not detrimental to symbiotic development and, in fact, enhances the symbiotic productivity of S. meliloti 1021 with the host plant Medicago truncatula cv. Jemalong A17. Increased succinoglycan production was engineered by overexpression of the exoY gene, which encodes the enzyme responsible for the first step in succinoglycan biosynthesis. These results suggest that the level of symbiotic exopolysaccharide produced by a rhizobial species is one of the factors involved in optimizing the interaction with plant hosts.     

Talin and vinculin play distinct roles in filopodial motility in the neuronal growth cone

Filopodial motility is critical for many biological processes, particularly for axon guidance. This motility is based on altering the F-actin-based cytoskeleton, but the mechanisms of how this occurs and the actin-associated proteins that function in this process remain unclear. We investigated two of these proteins found in filopodia, talin and vinculin, by inactivating them in subregions of chick dorsal root ganglia neuronal growth cones and by observing subsequent behavior by video-enhanced microscopy and quantitative morphometry. Microscale chromophore-assisted laser inactivation of talin resulted in the temporary cessation of filopodial extension and retraction. Inactivation of vinculin caused an increased incidence of filopodial bending and buckling within the laser spot but had no effect on extension or retraction. These findings show that talin acts in filopodial motility and may couple both extension and retraction to actin dynamics. They also suggest that vinculin is not required for filopodial extension and retraction but plays a role in the structural integrity of filopodia.     

Nitrate-induced ethylene biosynthesis and the control of nodulation in alfalfa

We previously reported that inhibition of ethylene biosynthesis with aminoethoxyvinylglycine (AVG) eliminated the inhibitory effect of NO₃^– on nodulation of alfalfa (Medicago sativa L. cv. Aragon) plants grown aeroponically. In this work, the effect of Ag⁺, as an inhibitor of ethylene action, has been studied in plants growing aeroponically or in darkened tubes with vermiculite, and low-nitrate or high-nitrate solution. Vermiculite-grown plants developed up to 3 times as many nodules as did those growing aeroponically. Nodule formation was mirrored by dry-matter accumulation. High (10 mol m^–3) NO₃^– applied from planting inhibited nodulation to an equal extent (c. 50%) in the two growth conditions. In contrast, Ag⁺ treatment increased nodule formation at all NO₃^– concentrations assayed under the two growth conditions, with the stimulation being higher in plants grown aeroponically. Finally, no effect of Ag⁺ (10 mmol m^–3) on plant growth was observed in either of the growth conditions. The effectiveness of NO₃^– as a nodulation inhibitor and enhancer of ethylene biosynthesis in roots of alfalfa was also studied. Within 24 h after inoculation, 10 mol m^–3 NO₃^– exerted most of its inhibitory effect on nodulation. At the same time, both 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity and ethylene evolution rates markedly increased in inoculated and uninoculated alfalfa roots treated with NO₃^–. Support for a role of endogenous ethylene in the control of nodule formation in legumes is discussed.     

Ras-related proteins (Rab) play significant roles in sperm motility and capacitation status

Rab proteins are widely known for their involvement in establishing Golgi apparatus and controlling Golgi trafficking in eukaryotic cells. Specifically, Rab proteins play significant roles in acrosome formation and exocytosis. Furthermore, mechanisms involved in the regulation of Rab proteins during capacitation have been identified. However, there has been no direct evaluation to assess the correlation between Rab proteins and sperm function. Consequently, this study was designed to analyze the correlation between Rab proteins and sperm functions. Individually, we analyzed the sperm motility patterns, motion kinematics, capacitation status, and Rab protein expression levels of sperm samples from 31 boars before and after capacitation. As a result, we discovered that Rab3A, Rab5, Rab11, Rab14, and Rab27A correlated with various sperm motility patterns, motion kinematics before capacitation. Rab3A, Rab5, Rab11, Rab14, and Rab34 correlated with various sperm motility patterns, motion kinematics after capacitation. Moreover, Rab4 and Rab34 were associated with capacitation status before capacitation, and Rab3A, 25, and 27A correlated with capacitation status after capacitation. This is the first study to analyze the correlation between Rab proteins and sperm functions. Collectively, our results indicate that specific sperm motility and kinematics, as well as the structural condition of the sperm head and capacitation status, regulate individual Rab protein. Therefore, we expect that the current findings will be used to identify the etiology of idiopathic male infertility patients and to diagnose male fertility and that Rab proteins will be employed as biomarkers to predict and analyze male fertility.     

茎瘤固氮根瘤菌ORS571 GGDEF和EAL结构域蛋白的预测及功能研究

[目的]环二鸟苷酸c-di-GMP是细菌中广泛存在的第二信使,能够调控多种细胞功能。c-di-GMP的合成与水解分别由含有GGDEF结构域和EAL结构域的蛋白催化。本研究针对茎瘤固氮根瘤菌ORS571的GGDEF和EAL结构域相关蛋白进行基因组学分析,并对三个同时含有GGDEF和EAL结构域的蛋白（AZC_3085、AZC_3226和AZC_4658）进行功能研究。[方法]利用SMART数据库对含有GGDEF和EAL结构域的蛋白进行结构域预测。利用CLUSTALW程序对蛋白序列进行比较分析。通过同源重组的方法构建突变株,并对突变株的细胞运动能力、胞外多糖合成、生物膜形成及与豆科宿主的结瘤等表型进行测定。[结果]茎瘤固氮根瘤菌ORS571中一共存在37个GGDEF和EAL结构域蛋白。突变株△4658的运动能力较野生型有下降,但是其胞外多糖合成能力、生物膜形成能力和竞争性结瘤能力较野生型有提高。此外,实验结果表明突变株△4658的胞内c-di-GMP水平高于野生型。突变株△3085和△3226的各种表型与野生型相比没有明显差异。[结论]茎瘤固氮根瘤菌ORS571编码如此大数量的GGDEF和EAL结构域蛋白,表明c-di-GMP可能在其信号转导过程中起到非常重要的作用。同时具有GGDEF和EAL结构域的蛋白AZC_4658对茎瘤固氮根瘤菌ORS571的运动能力、胞外多糖合成、生物膜形成及与宿主的结瘤起到一定的调节作用。     

Two StAR-related lipid transfer proteins play specific roles in endocytosis,exocytosis, and motility in the parasitic protist Entamoeba histolytica

Lipid transfer proteins (LTPs) are the key contributor of organelle-specific lipid distribution and cellular lipid homeostasis. Here, we report a novel implication of LTPs in phagocytosis, trogocytosis, pinocytosis, biosynthetic secretion, recycling of pinosomes, and motility of the parasitic protist E. histolytica, the etiological agent of human amoebiasis. We show that two StAR-related lipid transfer (START) domain-containing LTPs (named as EhLTP1 and 3) are involved in these biological pathways in an LTP-specific manner. Our findings provide novel implications of LTPs, which are relevant to the elucidation of pathophysiology of the diseases caused by parasitic protists.     

Quorum sensing regulates exopolysaccharide production, motility, and virulence in Pseudomonas syringae

The N-acyl homoserine lactone (AHL)-mediated quorum-sensing system in the phytopathogen Pseudomonas syringae pv. syringae requires the AHL synthase AhlI and the regulator AhlR, and is additionally subject to regulation by AefR. The contribution of quorum sensing to the expression of a variety of traits expected to be involved in epiphytic fitness and virulence of P syringae were examined. Both an aefR- mutant and an ahlI- ahlR- double mutant, deficient in AHL production, were significantly impaired in alginate production and had an increased susceptibility to hydrogen peroxide compared with the wild-type strain. These mutants were hypermotile in culture, invaded leaves more rapidly, and caused an increased incidence of brown spot lesions on bean leaves after a 48-h moist incubation. Interestingly, an aefR- mutant was both the most motile and virulent. Like the wild-type strain, the AHL-deficient mutant strains incited water-soaked lesions on bean pods. However, lesions caused by an ahlI- ahlR- double mutant were larger, whereas those incited by an aefR- mutant were smaller. In contrast, tissue maceration of pods, which occurs at a later stage of infection, was completely abolished in the AHL-deficient mutants. Both the incidence of disease and in planta growth of P syringae pv. tabaci were greatly reduced in transgenic tobacco plants that produced AHL compared with wild-type plants. These results demonstrate that quorum sensing in E syringae regulates traits that contribute to epiphytic fitness as well as to distinct stages of disease development during plant infection.     

'Ca. Liberibacter asiaticus' proteins orthologous with pSymA-encoded proteins of Sinorhizobium meliloti: hypothetical roles in plant host interaction

Sinorhizobium meliloti strain 1021, a nitrogen-fixing, root-nodulating bacterial microsymbiont of alfalfa, has a 3.5 Mbp circular chromosome and two megaplasmids including 1.3 Mbp pSymA carrying nonessential 'accessory' genes for nitrogen fixation (nif), nodulation and host specificity (nod). A related bacterium, psyllid-vectored 'Ca. Liberibacter asiaticus,' is an obligate phytopathogen with a reduced genome that was previously analyzed for genes orthologous to genes on the S. meliloti circular chromosome. In general, proteins encoded by pSymA genes are more similar in sequence alignment to those encoded by S. meliloti chromosomal orthologs than to orthologous proteins encoded by genes carried on the 'Ca. Liberibacter asiaticus' genome. Only two 'Ca. Liberibacter asiaticus' proteins were identified as having orthologous proteins encoded on pSymA but not also encoded on the chromosome of S. meliloti. These two orthologous gene pairs encode a Na(+)/K+ antiporter (shared with intracellular pathogens of the family Bartonellacea) and a Co++, Zn++ and Cd++ cation efflux protein that is shared with the phytopathogen Agrobacterium. Another shared protein, a redox-regulated K+ efflux pump may regulate cytoplasmic pH and homeostasis. The pSymA and 'Ca. Liberibacter asiaticus' orthologs of the latter protein are more highly similar in amino acid alignment compared with the alignment of the pSymA-encoded protein with its S. meliloti chromosomal homolog. About 182 pSymA encoded proteins have sequence similarity (≤ E-10) with 'Ca. Liberibacter asiaticus' proteins, often present as multiple orthologs of single 'Ca. Liberibacter asiaticus' proteins. These proteins are involved with amino acid uptake, cell surface structure, chaperonins, electron transport, export of bioactive molecules, cellular homeostasis, regulation of gene expression, signal transduction and synthesis of amino acids and metabolic cofactors. The presence of multiple orthologs defies mutational analysis and is consistent with the hypothesis that these proteins may be of particular importance in host/microbe interaction and their duplication likely facilitates their ongoing evolution.     

GGDEF proteins YeaI, YedQ, and YfiN reduce early biofilm formation and swimming motility in Escherichia coli

The second messenger 3′–5′-cyclic diguanylic acid (c-di-GMP) promotes biofilm formation, and c-di-GMP is synthesized by diguanylate cyclases (characterized by a GGDEF domain) and degraded by phosphodiesterases. Here, we evaluated the effect of the 12 E. coli GGDEF-only proteins on biofilm formation and motility. Deletions of the genes encoding the GGDEF proteins YeaI, YedQ, YfiN, YeaJ, and YneF increased swimming motility as expected for strains with reduced c-di-GMP. Alanine substitution in the EGEVF motif of YeaI abolished its impact on swimming motility. In addition, extracellular DNA (eDNA) was increased as expected for the deletions of yeaI (tenfold), yedQ (1.8-fold), and yfiN (3.2-fold). As a result of the significantly enhanced motility, but contrary to current models of decreased biofilm formation with decreased diguanylate cyclase activity, early biofilm formation increased dramatically for the deletions of yeaI (30-fold), yedQ (12-fold), and yfiN (18-fold). Our results indicate that YeaI, YedQ, and YfiN are active diguanylate cyclases that reduce motility, eDNA, and early biofilm formation and contrary to the current paradigm, the results indicate that c-di-GMP levels should be reduced, not increased, for initial biofilm formation so c-di-GMP levels must be regulated in a temporal fashion in biofilms.     

Expression of a Serratia marcescens chitinase gene in Sinorhizobium fredii USDA191 and Sinorhizobium meliloti RCR2011 impedes soybean and alfalfa nodulation.

A gene encoding chitinase from Serratia marcescens BJL200 was cloned into a broad-host-range vector (pRK415) and mobilized into Sinorhizobium fredii USDA191. Chitinolytic activity was detected in S. fredii USDA191 transconjugants that carried the S. marcescens chiB gene. Chitinase-producing S. fredii USDA191 formed nodules on soybean cultivar McCall. However, there was a delay in nodule formation and a marked decrease in the total number of nodules formed by the chitinase-producing S. fredii in comparison with the wild-type strain. Expression of chitinase in S. meliloti RCR2011 also impeded alfalfa nodulation. Thin-layer chromatography of 14C-labeled Nod factors from chitinase-producing S. fredii USDA191 revealed hydrolysis of lipochitooligosaccharides.     

A fadD mutant of Sinorhizobium meliloti shows multicellular swarming migration and is impaired in nodulation efficiency on alfalfa roots

Swarming is a form of bacterial translocation that involves cell differentiation and is characterized by a rapid and co-ordinated population migration across solid surfaces. We have isolated a Tn5 mutant of Sinorhizobium meliloti GR4 showing conditional swarming. Swarm cells from the mutant strain QS77 induced on semi-solid minimal medium in response to different signals are hyperflagellated and about twice as long as wild-type cells. Genetic and physiological characterization of the mutant strain indicates that QS77 is altered in a gene encoding a homologue of the FadD protein (long-chain fatty acyl-CoA ligase) of several microorganisms. Interestingly and similar to a less virulent Xanthomonas campestris fadD(rpfB) mutant, QS77 is impaired in establishing an association with its host plant. In trans expression of multicopy fadD restored growth on oleate, control of motility and the symbiotic phenotype of QS77, as well as acyl-CoA synthetase activity of an Escherichia coli fadD mutant. The S. meliloti QS77 strain shows a reduction in nod gene expression as well as a differential regulation of motility genes in response to environmental conditions. These data suggest that, in S. meliloti, fatty acid derivatives may act as intracellular signals controlling motility and symbiotic performance through gene expression.