Anna Riccio

Plasminogen activator inhibitor type‐1 : reactive center and amino‐terminal heterogeneity determined by protein and cDNA sequencing

Both the urokinase‐type and tissue‐type plasminogen activator can convert their 5̃4 kDa type‐1 inhibitor (PAI‐1) to an inactive form with a lower apparent molecular mass. We have determined the amino‐terminal amino acid sequences of human native and converted PAI‐1, and isolated PAI‐1 cDNA and determined the nucleotide sequence in regions corresponding to the amino‐terminus and the cleavage site. The data show that the conversion of the inhibitor consists of cleavage of an Arg‐Met bond 33 residues from the carboxy‐terminus, thus localizing the reactive center of the inhibitor to that position. In addition, a heterogeneity was found at the amino‐terminus, with a Ser‐Ala‐Val‐His‐His form and a two‐residue shorter form (Val‐His‐His‐) occurring in approximately equal quantities.

Functional characterization of an ammonium transporter gene from Lotus japonicus

NH(4)(+) is the main product of symbiotic nitrogen fixation and the external concentration of combined nitrogen plays a key regulatory role in all the different step of plant-rhizobia interaction. We report the cloning and characterization of the first member of the ammonium transporter family, LjAMT1;1 from a leguminous plant, Lotus japonicus. Sequence analysis reveals a close relationship to plant transporters of the AMT1 family. The wild type and two mutated versions of LjAMT1;1 were expressed and functionally characterized in yeast. LjAMT1;1 is transcribed in roots, leaves and nodules of L. japonicus plants grown under low nitrogen conditions, consistent with a role in uptake of NH(4)(+) by the plant cells.

Nodule invasion and symbiosome differentiation during Rhizobium etli-Phaseolus vulgaris symbiosis

By means of a detailed ultrastructural analysis of nodules induced by Rhizobium etli on the roots of Phaseolus vulgaris, we observe that the development of host-invaded cells is not synchronous. An accumulation of mitochondria was found in freshly invaded host cells, containing only a few symbiosomes (SBs) that are released from highly branched intracellular ramification of the infection threads. Moreover, besides the fusion between the SB membrane with host secretory vesicles, we observe also a great number of fusions between the outer leaflets of adjoining SB membranes, thus resulting in structures that resemble the tight junction network (zona occludens with a five-layered structure) of epithelian cells. This process was found to be induced strongly and earlier both in the invaded host cells of ineffective nodules (elicited by Fix- mutant strains of R. etli) and in the older (senescence) invaded cells of effective nodules, whereas bacteroid division is seldom if ever observed. Our observations strongly suggest that multiple-occupancy SBs also arise by fusion of single-occupancy SBs and the physiological consequence of this process is discussed.

The ntrBC genes of Rhizobium leguminosarum are part of a complex operon subject to negative regulation

We report here that ntrB and ntrC genes of Rhizobium leguminosarum biovar phaseoli are cotranscribed with an open reading frame (called 0RF1) of unknown Unction. The promoter region of the 0RF1‐ntrB‐ntrC operon was mapped immediately upstream of ORF1 and two in vivo transcription initiation sites were identified, both preceded by −35/−10 promoter consensus sequences. Some major aspects differentiate ft leguminosarum from the enteric nitrogen regulatory system: the ntrBC genes are cotranscribed with 0RF1 which is homologous to an ORF located upstream of ntrBC of R. capsulatus and to the 0RF1 located upstream of the fis gene of Escherichia coir, ntrBC are not transcribed from a −24/−12 promoter and are only autogenously repressed. Moreover, the intracellular concentration of the NtrC protein increases when the bacterium is grown on ammonium salts, white under the same conditions the promoter of one of its target genes, glnII, is 12 times less active.

The Rhizobium etli metZ gene is essential for methionine biosynthesis and nodulation of Phaseolus vulgaris.

A mutant strain (CTNUX23) of Rhizobium etli carrying Tn5 unable to grow with sulfate as the sole sulfur source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a metZ (O-succinylhomoserine sulfhydrylase)-homologous gene. The CTNUX23 mutant strain had a growth dependency for methionine, although cystathionine or homocysteine, but not homoserine or O-succinylhomoserine, allowed growth of the mutant. RNase protection assays showed that the metZ-like gene had a basal level of expression in methionine- or cysteine-grown cells, which was induced when sulfate or thiosulfate was used. The metZ gene was cloned from the parent wild-type strain, CE3, and the resulting plasmid pAR204 relieved, after transformation, the methionine auxotrophy of both strains CTNUX23 of R. etli and PAO503(metZ) of Pseudomonas aeruginosa. Unlike strain CE3 or CTNUX23 (pAR204), strain CTNUX23 showed undetectable levels of O-succinylhomoserine sulfhydrylase activity. Strain CTNUX23 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) or to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris, unless methionine was added to the growth medium. These data and our previous results support the notion that cysteine or glutathione, but not methionine, is supplied by the root cells to bacteria growing inside the plant.

Regulation of nitrogen metabolism is altered in a glnB mutant strain of Rhizobium leguminosarum

We isolated a Rhizobium leguminosarum mutant strain altered in the glnB gene. This event, which has never been described in the Rhizobiaceae, is rare in comparison to mutants isolated in the contiguous gene, glnA. The glnB mutation removes the glnBA promoter but in vivo does not prevent glnA expression from its own promoter, which is not nitrogen regulated. The glnB mutant strain does not grow on nitrate as a sole nitrogen source and it is Nod+, Fix+. Two –24/–12 promoters, for the glnll and glnBA genes, are constitutively expressed in the glnB mutant, while two –35/–10‐like promoters for glnA and ntrBC are unaffected. We propose that the glnB gene product, the P11 protein, plays a negative role in the ability of NtrC to activate transcription from its target promoters and a positive role in the mechanism of nitrate utilization.

Ectopic expression of the Rhizobium etli amtB gene affects the symbiosome differentiation process and nodule development

Under conditions of nitrogen limitation, soil bacteria of the genus Rhizobium are able to induce the development of symbiotic nodules on the roots of leguminous plants. During nodule organogenesis, bacteria are released endocytotically inside the invaded plant cells where they differentiate into their endosymbiotic form called bacteroids. Bacteroids surrounded by a plant-derived peribacteroid membrane are nondividing, organelle-like structures, called symbiosomes, that use nitrogenase to reduce N2 to ammonia. Experiments performed in vitro with isolated symbiosomes have previously led to the suggestion that the NH3 produced by the bacteroids is released as NH4+ into the plant cytosol. Furthermore, it was observed that the bacterial amtB (ammonium/methylammonium transport B) gene is switched off very early during symbiosis, just when bacteria are released into the host cells. We report here that the ectopic expression of amtB in bacteroids alters the ability of bacteria to invade the host cells and the sym...

Glutamine utilization by Rhizobium etli

We undertook the study of the use of glutamine (Gln) as the source of carbon and energy by Rhizobium etli. Tn5-induced mutagenesis allowed us to identify several genes required for Gln utilization, including those coding for two broad-range amino acid transporters and a glutamate dehydrogenase. The isolated mutants were characterized by the analysis of their capacity i) to grow on different media, ii) to transport Gln (uptake assays), and iii) to utilize Gln as the C energy source (CO2 production from Gln). We show that Gln is degraded through the citric acid cycle and that its utilization as the sole C source is related to a change in the bacterial cell shape (from bacillary to coccoid form) and a high susceptibility to a thiol oxidative insult. Both these data and the analysis of ntr-dependent promoters suggested that Gln-grown bacteria are under a condition of C starvation and N sufficiency, and as expected, the addition of glucose counteracted the morphological change and increased both the bacterial growth rate and their resistance to oxidative stress. Finally, a nodulation analysis indicates that the genes involved in Gln transport and degradation are dispensable for the bacterial ability to induce and invade developing nodules, whereas those involved in gluconeogenesis and nucleotide biosynthesis are strictly required.

Sulphadimethoxine inhibits Phaseolus vulgaris root growth and development of N-fixing nodules

Sulphonamides contamination of cultivated lands occurs through the recurrent spreading of animal wastes from intensive farming. The aim of this study was to test the effect(s) of sulphadimethoxine on the beneficial N-fixing Rhizobium etli-Phaseolus vulgaris symbiosis under laboratory conditions. The consequence of increasing concentrations of sulphadimethoxine on the growth ability of free-living R. etli bacteria, as well as on seed germination, seedling development and growth of common bean plants was examined. We have established that sulphadimethoxine inhibited the growth of both symbiotic partners in a dose-dependent manner. Bacterial invasion occurring in developing root nodules was visualized by fluorescence microscopy generating EGFP-marked R. etli bacteria. Our results proved that the development of symbiotic N-fixing root nodules is hampered by sulphadimethoxine thus identifying sulphonamides as toxic compounds for the Rhizobium-legume symbiosis: a low-input sustainable agricultural practice.

Inhibition of glutamine synthetase II expression by the product of the gstI gene

We report the identification of a previously unrecognized gene that is involved in the regulation of the Rhizobium leguminosarum glnII (glutamine synthetase II) gene. This gene, which is situated immediately upstream of glnII, was identified by means of a deletion/complementation analysis performed in the heterologous background of Klebsiella pneumoniae. It has been designated gstI (glutamine synthetase translational Inhibitor) because, when a complete version of gstI is present, it is possible to detect glnII‐specific mRNA, but neither GSII activity nor GSII protein. The gstI gene encodes a small (63 amino acids) protein, which acts in cis or in trans with respect to glnII and is transcribed divergently with respect to glnII from a promoter that was found to be strongly repressed by the nitrogen transcriptional regulator NtrC. A mutated version of GstI lacking the last 14 amino acids completely lost its capacity to repress glnII expression. Our results indicate that gstI mediates the translation inhibition of glnII mRNA and, based on in silico analyses, a mechanism for GstI action is proposed.