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A Tn5-induced mutant strain (CTNUX5) of Rhizobium etli unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into an argC-homologous gene. Unlike its wild-type parent (strain CE3), the mutant strain CTNUX5 had an absolute dependency on arginine to grow. The argC gene was cloned from the wild-type strain CE3, and the resulting plasmid, pAR207, after transformation was shown to relieve the arginine auxotrophy of strain CTNUX5. Unlike strain CE3 or CTNUX5-pAR207, strain CTNUX5 showed undetectable levels of N-acetyl-gamma-glutamylphosphate reductase activity. Unless arginine was added to the growth medium, strain CTNUX5 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (nodulation factors) and to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris.

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.

A mutant strain (CTNUX4) of Rhizobium etli carrying Tn5 unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a trpB (tryptophan synthase)-homologous gene. When tested on the roots of Phaseolus vulgaris, strain CTNUX4 was able to induce only small, slightly pink, ineffective (Fix-) nodules. However, under free-living conditions, strain CTNUX4 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) unless tryptophan was added to the growth medium. These data and histological observations indicate that the lack of tryptophan biosynthesis affects the symbiotic behavior of R. etli.

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.

During development of root nodules, Rhizobium bacteria differentiate inside the invaded plant cells into N2-fixing bacteroids. Terminally differentiated bacteroids are unable to grow using the ammonia (NH3) produced therein by the nitrogenase complex. Therefore, the nitrogen assimilation activities of bacteroids, including the ammonium (NH4+) uptake activity, are expected to be repressed during symbiosis. By sequence homology the R. etli amtB (ammonium transport) gene was cloned and sequenced. As previously shown for its counterpart in other organisms, the R. etli amtB gene product mediates the transport of NH4+. The amtB gene is cotranscribed with the glnK gene (coding for a PII-like protein) from a nitrogen-regulated sigma 54-dependent promoter, which requires the transcriptional activator NtrC. Expression of the glnKamtB operon was found to be activated under nitrogen-limiting, free-living conditions, but down-regulated just when bacteria are released from the infection threads and before transcription of the nitrogenase genes. Our data suggest that the uncoupling between N2-fixation and NH3 assimilation observed in symbiosomes is generated by a transcriptional regulatory mechanism(s) beginning with the inactivation of NtrC in younger bacteroids.

We report here the isolation of a Rhizobium etli gene involved in lipoic acid metabolism, the lipA gene, which complements a lipA mutant strain of Escherichia coli. A promoter region (lipAp) was mapped immediately upstream of lipA and two in vivo transcription initiation sites were identified, preceded by sequences showing some homology to the -10/-35 promoter consensus sequences. The activity of the lipAp was found not to be regulated either by the carbon source or by the addition of lipoic acid. Moreover, quantitative analysis of the lipA transcript by RNase protection assays indicated its down-regulation during entry into stationary phase.

The DNA-binding activity of the NtrC protein can be demonstrated in gel retardation assays with concentrated protein extracts of Rhizobium etli. Using extracts from either the wild type or a ntrC mutant strain and an antiserum raised against the NtrC protein, we demonstrate specific binding of NtrC to the upstream regulatory region of the glnII gene, where two putative NtrC-binding sites are present. KNO3-grown bacteria contain less NtrC protein and more NtrC-binding activity than NH4Cl-grown bacteria, thus showing that with this protocol it is possible to detect changes in NtrC-binding activity. The advantages of this assay system in comparison with that using pure proteins is discussed.

Changes in temperature and a variety of other stimuli coordinately induce transcription of a specific set of heat-shock genes in all organisms. In the human fungal pathogen Histoplasma capsulatum, a temperature shift from 25 to 37 degrees C acts not only as a signal that causes transcription of heat-shock genes, but also triggers a morphological mycelium- to yeast-phase transition. The temperature-induced morphological transition may be viewed as a heat-shock response followed by cellular adaptation to a higher temperature. We have found that by inducing thermotolerance, i.e., an initial incubation at 34 degrees C, the thermosensitive attenuated Downs strain of H. capsulatum can be made to resemble those of the more temperature-tolerant G222B strain with respect to mitochondrial ATPase activity and electron transport efficiency at elevated temperatures. Furthermore, if the heat-shock response is first elicited by preincubation at milder temperatures or stress, transcription of heat-shock mRNA in mycelial cells of Downs strain that shifted to 37 degrees C proceeds at rates comparable to those of the virulent strains.

A Tn5 insertion mutant, strain CFN2012, of Rhizobium leguminosarum biovar phaseoli devoid of glutamine synthetase II (GSII) activity was analysed. It was shown to contain Tn5 within an 11-kb BamHI DNA fragment, which was isolated (pSM261) from the wild-type strain and, when introduced into strain CFN2012, was shown to complement the absence of GSII activity. The DNA sequence of the corresponding region from the wild-type allele revealed the presence of an ntrC regulatory gene, and restriction analysis indicated that the mutant allele carried the Tn5 insertion within it. Further analysis of strain CFN2012 indicated that this mutant has reduced levels of the PII regulatory protein and that, in contrast to ntrC mutants of other Rhizobiaceae, it grows on nitrate as the sole nitrogen source.

The complex molecular response of cells to sudden temperature changes is a well-characterized phenomenon. Although it is clear that the induction of heat shock proteins provides protection from heat in all of the organisms so far tested, very little is known about the role that this set of proteins plays in cellular homeostasis. Recently, putative roles for hsp60 and hsp70-like proteins have been proposed in Saccharomyces cerevisiae. hsp70-like proteins have been shown to be necessary for translocation of precursor polypeptides into mitochondria and endoplasmic reticulum, while hsp60 is required for the assembly of precursor polypeptides into oligomeric complexes following incorporation into the mitochondrial matrix. In this paper, we report that a brief temperature shock (44 degrees C) impairs coupling of oxidative phosphorylation in S. cerevisiae as measured indirectly by the Cl-CCP/oligomycin assay. Furthermore, at high temperature oligomycin stimulates rather than inhibits oxygen uptake under nonthermotolerant conditions. Pretreatment of cells for a short period of time at 37 degrees C, prior to exposure to higher temperatures rescues the capacity to maintain coupling between oxidative phosphorylation and electron transport. Inhibition of cytoplasmic RNA or protein synthesis during heat shock prevents the protection of this mitochondrial activity. We propose that one of the roles of the induction of heat shock proteins (or related activities) is to protect mitochondrial ATPase activity under conditions of further increase in temperature.

The indirect immunofluorescence procedure was used to localize prolactin (PRL)- and somatotrophin (STH)- producing cells in the pituitary distal lobe from Hyla arborea adult specimens. The following mammalian antisera were employed: rabbit anti-ovine PRL, antibovine PRL, anti-human PRL, anti-rat PRL, anti-ovine STH, anti-bovine STH, anti-human STH; monkey anti-rat STH. Immunocytochemical staining was suppressed by solid phase absorption of both anti-PRL and anti-STH with the specific antigen. Absorption of anti-PRL with STH and of anti-STH with PRL did not appreciably affect immunocytochemical staining. Treatment with the two antisera revealed two different reactive cell types, both acidophils. Using PRL antisera a strong fluorescence was found in the large acidophils located chiefly in the rostro-central and ventral areas of the distal lobe sagittal sections. A somewhat weaker fluorescence was observed using STH antisera in the fewer, small acidophils mostly concentrated in the dorso-caudal region and only sparsely scattered in the other areas of the pars distalis. Strikingly, the overall pattern of localization shown by the two cell types is similar to their already known distribution in the pituitary distal lobe of some other species of urodele and anuran amphibians.

Using an immunoperoxidase method the Authors have demonstrated carcinoembryonic antigen (CEA) in 4/7 patients with benign oral lesion and in 9/10 patients with oral cancer. All 4 oral leukoplakias were positive for CEA. The histologic presence of CEA in oral cancer was related mainly to the formation of well differentiated tissue. The anaplastic carcinoma was negative. These findings indicate that CEA is a product of differentiated cells and should not be considered exclusively an oncofetal antigen or a marker of undifferentiated cells.

The indirect immunofluorescence procedure was used to localize ACTH-producing cells in the pituitary distal lobe from Hyla arborea adult specimens; the antiserum employed was rabbit anti-synthetic ACTH (1-24) conjugated with BSA. Immunohistochemical staining was suppressed by solid-phase absorption of the anti-ACTH with the specific antigen. In the distal lobe one cell population is immunoreactive; these cells are predominantly localized in the anterior half, both ventrally and dorsally; they exclusively correspond to type-3 basophils. In the intermediate lobe the total cell population is immunoreactive. Parallel inspections carried out on the pituitary of Xenopus laevis specimens gave results fully consistent with those reported by other authors.