Sinorhizobium meliloti is a root-nodulating, nitrogen-fixing bacterium. An S. meliloti strain that is mutant for the rpoH(1) gene, which encodes a sigma(32)-like protein, elicits the formation of ineffective nodules on the host plant alfalfa. We characterized the rpoH(1) mutant for phenotypes related to symbiosis. Alfalfa nodules formed by the rpoH(1) mutant exhibited greatly reduced levels of acetylene reduction activity compared to the wild-type nodules. Whereas intracellular colonization by rhizobia was observed in a zone just below the apical meristem, we found ultrastructural abnormalities and signs of degeneration of bacteroids within many host cells in the proximally adjacent zone. In the proximal part of the nodule, only a few nodule cells contained bacteroids. In contrast, the rpoH(1) mutant showed normal induction of nitrogen fixation gene expression in microaerobic culture. These results suggest that the rpoH(1) mutation causes early senescence of bacteroids during the endosymbiotic process, but does not affect the invasion process or the synthesis of the nitrogenase machinery. The rpoH(1) mutant exhibited increased sensitivity to various agents and to acid pH, suggesting that RpoH(1) is required to protect the bacterial cell against environmental stresses encountered within the host. Since RpoH(1) was previously reported to be required for the synthesis of some heat shock proteins (Hsps), we examined the transcription of several genes for Hsp homologs. We found that transcription of groESL(5), lon, and clpB after heat shock was RpoH(1)-dependent, and conserved nucleotide sequences were found in the -35 and -10 regions upstream of the transcription start sites of these genes. Although groESL(5) expression is almost completely dependent on RpoH(1), we found that a groESL(5) mutant strain is still capable of normal symbiotic nitrogen fixation on alfalfa.
Graduate School ofAgricuttunalCaroteyoricin Er has been isolated as a phage-tail-like bacteriocin from the plant pathogen Erwinia carotovora Er [Kamimiya, S. et al., (197D, Agric. Biol. (]hem. 41,[911][912]. However, the fine morphology and str"ctural composition of carotoyoricin Er remained to be studied because a large ainount of contracted carotoyoricin Er were present in the bacteriocin preparations se far obtained.To obtain intact carotovoricin Er and its major parts, we deyeloped simple and eMcient purification methods inc]uding the use of sucrose density gradient ce"trifugation in the presence of 10-20% (vlv) ethanol. Electron microscopy for the purified carotoyoricin Er shewed the presence of a novel antenna-like structure at the proximal end of the phage-tai1-
Staphylococcal leukecidin consists of two separate proteins, LukS and LukF, which eooperatively lyse hllman a"d rabbit pelymorphenuclear leukocytes and rabbit erythrecytes. Here we studied the pore-forming properties of leukocidin alld the melecular architecture of the leukocidin pere. (1) Leukocidin caused an eMux of potassium iens from rabbit erythrocytes and swelling of the cells before hemolysis. However, ultimate lysis of the toxin-treated swollen erythrocytes did not occur when pelyethylene glycels with hydrodynanic dia!rteters of z2.1nm were present in the extracellular space. (2) Electron micrescopy shewed the presence of a ring-shaped structure with outer and inner diameters of 9 and 3 nm, respectiyely, en leukocidin-treated human polymorphonuclear leukocytes and rabbit erythrocytes. (3) Ring-shaped structures of the same dimensions were isolated from the target cells, and they contained LukS and LukF in a molar ratio ef 1:1. (4) A single ring-shaped toxin complex had a molecular size of 205 kDa. These results indicated that LukS and LukF ftssemble into a ring-shaped oligomer of approximately 200 kDa on the target cells, forming a membrane pore with a f"nctional diameter ef approximately 2 nm.
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