Functional and evolutionary relatedness of genes for exopolysaccharide synthesis in Rhizobium meliloti and Rhizobium sp. strain NGR234.

Rhizobium meliloti SU47 and Rhizobium sp. strain NGR234 produce distinct exopolysaccharides that have some similarities in structure. R. meliloti has a narrow host range, whereas Rhizobium strain NGR234 has a very broad host range. In cross-species complementation and hybridization experiments, we found that several of the genes required for the production of the two polysaccharides were functionally interchangeable and similar in evolutionary origin. NGR234 exoC and exoY corresponded to R. meliloti exoB and exoF, respectively. NGR234 exoD was found to be an operon that included genes equivalent to exoM, exoA, and exoL in R. meliloti. Complementation of R. meliloti exoP, -N, and -G by NGR234 R'3222 indicated that additional equivalent genes remain to be found on the R-prime. We were not able to complement NGR234 exoB with R. meliloti DNA. In addition to functional and evolutionary equivalence of individual genes, the general organization of the exo regions was similar between the two species. It is likely that the same ancestral genes were used in the evolution of both exopolysaccharide biosynthetic pathways and probably of pathways in other species as well.     

Oxidation-induced Structural Changes of Ceruloplasmin Foster NGR Motif Deamidation That Promotes Integrin Binding and Signaling

Asparagine deamidation occurs spontaneously in proteins during aging; deamidation of Asn-Gly-Arg (NGR) sites can lead to the formation of isoAsp-Gly-Arg (isoDGR), a motif that can recognize the RGD-binding site of integrins. Ceruloplasmin (Cp), a ferroxidase present in the cerebrospinal fluid (CSF), contains two NGR sites in its sequence: one exposed on the protein surface (⁵⁶⁸NGR) and the other buried in the tertiary structure (⁹⁶²NGR). Considering that Cp can undergo oxidative modifications in the CSF of neurodegenerative diseases, we investigated the effect of oxidation on the deamidation of both NGR motifs and, consequently, on the acquisition of integrin binding properties. We observed that the exposed ⁵⁶⁸NGR site can deamidate under conditions mimicking accelerated Asn aging. In contrast, the hidden ⁹⁶²NGR site can deamidate exclusively when aging occurs under oxidative conditions, suggesting that oxidation-induced structural changes foster deamidation at this site. NGR deamidation in Cp was associated with gain of integrin-binding function, intracellular signaling, and cell pro-adhesive activity. Finally, Cp aging in the CSF from Alzheimer disease patients, but not in control CSF, causes Cp deamidation with gain of integrin-binding function, suggesting that this transition might also occur in pathological conditions. In conclusion, both Cp NGR sites can deamidate during aging under oxidative conditions, likely as a consequence of oxidative-induced structural changes, thereby promoting a gain of function in integrin binding, signaling, and cell adhesion.     

A Unified Mechanism for Aminopeptidase N-based Tumor Cell Motility and Tumor-homing Therapy

Tumor cell surface aminopeptidase N (APN or CD13) has two puzzling functions unrelated to its enzymatic activity: mediating tumor cell motility and serving as a receptor for tumor-homing peptides (peptides that bring anti-cancer drugs to tumor cells). To investigate APN-based tumor-homing therapy, we determined the crystal structure of APN complexed with a tumor-homing peptide containing a representative Asn-Gly-Arg (NGR) motif. The tumor-homing peptide binds to the APN enzymatic active site, but it resists APN degradation due to a distorted scissile peptide bond. To explore APN-based tumor cell motility, we examined the interactions between APN and extracellular matrix (ECM) proteins. APN binds to, but does not degrade, NGR motifs in ECM proteins that share similar conformations with the NGR motif in the APN-bound tumor-homing peptide. Therefore, APN-based tumor cell motility and tumor-homing therapy rely on a unified mechanism in which both functions are driven by the specific and stable interactions between APN and the NGR motifs in ECM proteins and tumor-homing peptides. This study further implicates APN as an integrin-like molecule that functions broadly in cell motility and adhesion by interacting with its signature NGR motifs in the extracellular environment.     

自然生草制苹果园土壤微生物、酶活性和养分含量对行间草耕翻还田的响应

果园生草是提高果园生产力、促进果园可持续生产的有效措施。为探究自然生草制苹果园行间草耕翻还田对土壤生物学性状和养分含量的影响,本试验以清耕(CK)为对照,设置草被刈割还田(NG)和草被刈割耕翻还田处理(NGR),研究不同草被还田方式对行间0~20 cm土层土壤微生物数量、酶活性以及不同形态氮、钾含量的影响。结果表明: 苹果园土壤中微生物以细菌占绝对优势,放线菌次之,真菌数量最少;与CK相比,NG和NGR处理均显著提高了土壤细菌和真菌数量,以NGR处理提升效果最显著,显著提高了土壤脲酶、蔗糖酶和过氧化氢酶活性,增幅分别为59.0%、20.7%、38.3%和73.5%、45.9%、67.8%。NGR处理显著提高了土壤氮、钾养分含量,铵态氮、硝态氮、颗粒有机态氮、微生物生物量氮及速效钾和水溶性钾含量分别为CK的1.5、1.8、1.6、2.0、1.3和1.4倍,显著提高了果实可溶性糖含量和糖酸比,进而提高了果实品质。综上,NGR可提高苹果园土壤微生物数量、酶活性、氮钾养分含量和果实品质,是生草制苹果园一种可行的行间草被还田方式。     

Molecular cloning of cDNAs encoding ribonuclease-related proteins in Nicotiana glutinosa leaves, as induced in response to wounding or to TMV-infection

We earlier isolated a cDNA clone (NGR1) encoding a wound-inducible ribonuclease (RNase NW) from leaves of Nicotiana glutinosa [Kariu et al. Biosci. Biotechnol. Biochem., 62, 1144-1151 (1998)]. In this study, two distinct cDNA clones, NGR2 and NGR3, encoding proteins with a ribonuclease-related sequence in the N. glutinosa leaves, were amplified and sequenced. The nucleotide sequences of NGR2 and NGR3 consist of 1244 bp and 1069 bp, and have open reading frames encoding 277 (RNase NGR2) and 236 (RNase NGR3) amino acid residues, respectively. The deduced amino acid sequences of the putative RNases NGR2 and NGR3 showed 33% and 58% amino acid sequence identity, respectively, with that of RNase NW and 32% identity with each other. Sequence comparison showed that NGR2 is similar to RNase RNS2 (61%) from Arabidopsis thaliana, while NGR3 is related to RNase LX (84%) from tomato (Lycopersicon esculentum). RNA gel blot analysis showed that the RNase NGR2 gene is constitutively expressed to measurable levels; it is not increased by either wounding or TMV infection. In contrast, the expression of the NGR3 gene is induced after 48 h upon TMV infection.     

Rhizobia utilize pathogen-like effector proteins during symbiosis

A type III protein secretion system (T3SS) is an important host range determinant for the infection of legumes by Rhizobium sp. NGR234. Although a functional T3SS can have either beneficial or detrimental effects on nodule formation, only the rhizobial-specific positively acting effector proteins, NopL and NopP, have been characterized. NGR234 possesses three open reading frames potentially encoding homologues of effector proteins from pathogenic bacteria. NopJ, NopM and NopT are secreted by the T3SS of NGR234. All three can have negative effects on the interaction with legumes, but NopM and NopT also stimulate nodulation on certain plants. NopT belongs to a family of pathogenic effector proteases, typified by the avirulence protein, AvrPphB. The protease domain of NopT is required for its recognition and a subsequent strong inhibition in infection of Crotalaria juncea . In contrast, the negative effects of NopJ are relatively minor when compared with those induced by its Avr homologues. Thus NGR234 uses a mixture of rhizobial-specific and pathogen-derived effector proteins. Whereas some legumes recognize an effector as potentially pathogen-derived, leading to a block in the infection process, others perceive both the negative- and positive-acting effectors concomitantly. It is this equilibrium of effector action that leads to modulation of symbiotic development.     

Involvement of multiple loci in quorum quenching of autoinducer I molecules in the nitrogen-fixing symbiont Rhizobium (Sinorhizobium) sp. strain NGR234

Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. Since we have previously described the complete genome sequence of NGR234, we now report on a genome-wide functional analysis of the genes and enzymes involved in autoinducer I hydrolysis in this microbe. Altogether we identified five cosmid clones that repeatedly gave a positive result in our function-based approach for the detection of autoinducer I hydrolase genes. Of these five cosmid clones, two were located on pNGR234b and three were on cNGR234. Subcloning and in vitro mutagenesis in combination with BLAST analyses identified the corresponding open reading frames (ORFs) of all cosmid clones: dlhR, qsdR1, qsdR2, aldR, and hitR-hydR. Analyses of recombinant DlhR and QsdR1 proteins by using high-performance liquid chromatography-mass spectrometry (HPLC-MS) demonstrate that these enzymes function as acyl homoserine lactone (AHL) lactonases. Furthermore, we showed that these enzymes inhibited biofilm formation and other quorum-sensing-dependent processes in Pseudomonas aeruginosa, Chromobacterium violaceum, and Agrobacterium tumefaciens. Finally, our experimental data suggest that competitive colonization of roots in the rhizospheres of cowpea plants is affected by DlhR and QsdR1.     

Rhizobium sp. strain NGR234 and R. fredii USDA257 share exceptionally broad, nested host ranges

Genetically, Rhizobium sp. strain NGR234 and R. fredii USDA257 are closely related. Small differences in their nodulation genes result in NGR234 secreting larger amounts of more diverse lipo-oligosaccharidic Nod factors than USDA257. What effects these differences have on nodulation were analyzed by inoculating 452 species of legumes, representing all three subfamilies of the Leguminosae, as well as the nonlegume Parasponia andersonii, with both strains. The two bacteria nodulated P. andersonii, induced ineffective outgrowths on Delonix regia, and nodulated Chamaecrista fasciculata, a member of the only nodulating genus of the Caesalpinieae tested. Both strains nodulated a range of mimosoid legumes, especially the Australian species of Acacia, and the tribe Ingeae. Highest compatibilities were found with the papilionoid tribes Phaseoleae and Desmodieae. On Vigna spp. (Phaseoleae), both bacteria formed more effective symbioses than rhizobia of the "cowpea" (V. unguiculata) miscellany. USDA257 nodulated an exact subset (79 genera) of the NGR234 hosts (112 genera). If only one of the bacteria formed effective, nitrogen-fixing nodules it was usually NGR234. The only exceptions were with Apios americana, Glycine max, and G. soja. Few correlations can be drawn between Nod-factor substituents and the ability to nodulate specific legumes. Relationships between the ability to nodulate and the origin of the host were not apparent. As both P. andersonii and NGR234 originate from Indonesia/Malaysia/Papua New Guinea, and NGR234's preferred hosts (Desmodiinae/Phaseoleae) are largely Asian, we suggest that broad host range originated in Southeast Asia and spread outward.     

The Rhizobium sp. strain NGR234 systemically suppresses arbuscular mycorrhizal root colonization in a split‐root system of barley (Hordeum vulgare)

Nitrogen‐fixing bacteria (rhizobia) form a nodule symbiosis with legumes, but also induce certain effects on non‐host plants. Here, we used a split‐root system of barley to examine whether inoculation with Rhizobium sp. strain NGR234 on one side of a split‐root system systemically affects arbuscular mycorrhizal (AM) root colonization on the other side. Mutant strains of NGR234 deficient in Nod factor production (strain NGRΔnodABC), perception of flavonoids (strain NGRΔnodD1) and secretion of type 3 effector proteins (strain NGRΩrhcN) were included in this study. Inoculation resulted in a systemic reduction of AM root colonization with all tested strains. However, the suppressive effect of strain NGRΩrhcN was less pronounced. Moreover, levels of salicylic acid, an endogenous molecule related to plant defense, were increased in roots challenged with rhizobia. These data indicate that barley roots perceived NGR234 and that a systemic regulatory mechanism of AM root colonization was activated. The suppressive effect appears to be Nod factor independent, but enhanced by type 3 effector proteins of NGR234.     

Disulfide bond formation in NGR fiber-modified adenovirus is essential for retargeting to aminopeptidase N

The peptide motif NGR (asparagine-glycine-arginine) is known to bind to aminopeptidase N (APN). We have constructed five adenoviruses (Ads) bearing NGR in the HI loop of the adenoviral fiber protein. We compared the targeting properties of the NGR peptide within different amino acid environments and showed that their cellular receptor(s) were not identical. Ads containing NGR within potentially cyclic sequences flanked by cysteines retargeted viruses mainly to APN, while Ads containing NGR within linear sequences not containing cysteines retargeted Ads mainly to alpha(v)beta(3) integrin, albeit with a lower affinity. Finally, we show evidence that disulfide bond formation within an Ad bearing the CDCNGRCFC sequence is essential for retargeting to APN, suggesting that this sequence does indeed assume a cyclic structure which facilitates NGR binding to APN. Therefore, our study underscores the importance of cysteine residues flanking targeting peptides for not only affinity but also specificity of the retargeted Ad.     

Structural basis for the interaction of isoDGR with the RGD-binding site of alphavbeta3 integrin

Asparagine deamidation at the NGR sequence in the 5th type I repeat of fibronectin (FN-I5) generates isoDGR, an alphavbeta3 integrin-binding motif regulating endothelial cell adhesion and proliferation. By NMR and molecular dynamics studies, we analyzed the structure of CisoDGRC (isoDGR-2C), a cyclic beta-peptide mimicking the FN-I5 site, and compared it with NGR, RGD, or DGR-containing cyclopeptides. Docking experiments show that isoDGR, exploiting an inverted orientation as compared with RGD, favorably interacts with the RGD-binding site of alphavbeta3, both recapitulating canonical RGD-alphavbeta3 contacts and establishing additional polar interactions. Conversely, NGR and DGR motifs lack the fundamental pharmacophoric requirements for high receptor affinity. Therefore, unlike NGR and DGR, isoDGR is a new natural recognition motif of the RGD-binding pocket of alphavbeta3. These findings contribute to explain the different functional properties of FN-I5 before and after deamidation, and provide support for the hypothesis that NGR --> isoDGR transition can work as a molecular timer for activating latent integrin-binding sites in proteins, thus regulating protein function.     

iso-DGR Sequences Do Not Mediate Binding of Fibronectin N-terminal Modules to Adherent Fibronectin-null Fibroblasts

Fibronectin (FN) without an RGD sequence (FN-RGE), and thus lacking the principal binding site for α5β1 integrin, is deposited into the extracellular matrix of mouse embryos. Spontaneous conversion of ²⁶³NGR and/or ⁵⁰¹NGR to iso-DGR possibly explains this enigma, i.e. ligation of iso-DGR by αvβ3 integrin may allow cells to assemble FN. Partial modification of ²⁶³NGR to DGR or iso-DGR was detected in purified plasma FN by mass spectrometry. To test functions of the conversion, one or both NGR sequences were mutated to QGR in recombinant N-terminal 70-kDa construct of FN (70K), full-length FN, or FN-RGE. The mutations did not affect the binding of soluble 70K to already adherent fibroblasts or the ability of soluble 70K to compete with non-mutant FN or FN-RGE for binding to FN assembly sites. Non-mutant FN and FN-N263Q/N501Q with both NGRs mutated to QGRs were assembled equally well by adherent fibroblasts. FN-RGE and FN-RGE-N263Q/N501Q were also assembled equally well. Although substrate-bound 70K mediated cell adhesion in the presence of 1 mm Mn²⁺ by a mechanism that was inhibited by cyclic RGD peptide, the peptide did not inhibit 70K binding to cell surface. Mutations of the NGR sequences had no effect on Mn²⁺-enhanced cell adhesion to adsorbed 70K but caused a decrease in cell adhesion to reduced and alkylated 70K. These results demonstrate that iso-DGR sequences spontaneously converted from NGR are cryptic and do not mediate the interaction of the 70K region of FN with the cell surface during FN assembly.     

NopB, a type III secreted protein of Rhizobium sp. strain NGR234, is associated with pilus-like surface appendages

Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGROmegarhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB.     

豇豆根瘤菌NGR234和紫云英根瘤菌109感染紫云英的比较研究

利用光学和电子显微镜对紫云英根瘤菌菌株109和广宿主的快生型根瘤菌菌株NGR234感染温带型豆科植物紫云英进行了研究,结果表明根瘤菌感染紫云英是通过在根毛中形成侵染线的途径。电子显微镜研究揭示了固氮根瘤中细胞内侵染线的存在。接种二天后,首先可观察到根毛的卷曲或分枝。接种四至五天后,在每株植物卷曲的根毛中可看到侵染线。接种八至十天后的植株出现肉眼可见的根瘤。菌株NGR234能够在紫云英上诱导根毛的卷曲,侵染线和根瘤的形成,但所形成的根瘤却未能固氮,根瘤中无明显的类菌体区,但有少数包有细菌的侵染线。NGR234抗抗菌素的衍生菌均未能使紫云英结瘤。将NGR234的共生质粒转移至三叶草、苜蓿、豌豆、快生型大豆根瘤菌和农杆菌,亦未能使这些细菌获得紫云英上结瘤的能力。     

Role of BacA in lipopolysaccharide synthesis, peptide transport, and nodulation by Rhizobium sp. strain NGR234

BacA of Sinorhizobium meliloti plays an essential role in the establishment of nitrogen-fixing symbioses with Medicago plants, where it is involved in peptide import and in the addition of very-long-chain fatty acids (VLCFA) to lipid A of lipopolysaccharide (LPS). We investigated the role of BacA in Rhizobium species strain NGR234 by mutating the bacA gene. In the NGR234 bacA mutant, peptide import was impaired, but no effect on VLCFA addition was observed. More importantly, the symbiotic ability of the mutant was comparable to that of the wild type for a variety of legume species. Concurrently, an acpXL mutant of NGR234 was created and assayed. In rhizobia, AcpXL is a dedicated acyl carrier protein necessary for the addition of VLCFA to lipid A. LPS extracted from the NGR234 mutant lacked VLCFA, and this mutant was severely impaired in the ability to form functional nodules with the majority of legumes tested. Our work demonstrates the importance of VLCFA in the NGR234-legume symbiosis and also shows that the necessity of BacA for bacteroid differentiation is restricted to specific legume-Rhizobium interactions.     

Molecular cloning of thentrA gene of the broad host-rangeRhizobium sp. NGR234, and phenotypes of a site-directed mutant

Summary The clonedntrA (rpoN) gene andntrA mutants ofRhizobium meliloti were used to isolate the homologous gene from the broad-host rangeRhizobium sp. NGR234 by hybridization and interspecies complementation. The NGR234 locus was analyzed by deletion and insertional mutagenesis. A site-directedntrA mutant, NGR234rn1, was made with an interposon, GmI, and its phenotype was examined ex planta and in symbiosis. NGR234rn1 formed Fix⁻ nodules on six genera tested from among its legume hosts, including both indeterminate and determinate nodule-type plants. Formation of nodules onMacroptilium was delayed, and expression of anR. meliloti nodABC-lacZ fusion was reduced by the mutant allele.     

Structure-activity relationships of linear and cyclic peptides containing the NGR tumor-homing motif

Cyclic and linear peptides containing the Asn-Gly-Arg (NGR) motif have proven useful for delivering various anti-tumor compounds and viral particles to tumor vessels. We have investigated the role of cyclic constraints on the structure and tumor-homing properties of NGR peptides using tumor necrosis factor-alpha (TNF) derivatives containing disulfide-bridged (CNGRC-TNF) and linear (GNGRG-TNF) NGR domains. Experiments carried out in animal models showed that both GNGRG and CNGRC can target TNF to tumors. However, the anti-tumor activity of CNGRC-TNF was >10-fold higher than that of GNGRG-TNF. Molecular dynamic simulation of cyclic CNGRC showed the presence of a bend geometry involving residues Gly(3)-Arg(4). Molecular dynamic simulation of the same peptide without disulfide constraints showed that the most populated and thermodynamically favored configuration is characterized by the presence of a beta-turn involving residues Gly(3)-Arg(4) and hydrogen bonding interactions between the backbone atoms of Asn(2) and Cys(5). These results suggest that the NGR motif has a strong propensity to form beta-turn in linear peptides and may explain the finding that GNGRG peptide can target TNF to tumors, albeit to a lower extent than CNGRC. The disulfide bridge constraint is critical for stabilizing the bent conformation and for increasing the tumor targeting efficiency.