Osmotically sensitive mutants of Neurospora crassa are unable to grow on medium supplemented with 4% NaCl, have altered morphologies and cell-wall compositions, and are resistant to dicarboximide fungicides. Osmotic-1 (os-1) mutants have a unique characteristic of forming protoplasts that grow and divide in specialized liquid medium, suggesting that the os-1+ gene product is important for cell-wall assembly. A cosmid containing the os-1+ locus of N. crassa, isolated from a genomic cosmid library by chromosomal walk from a closely linked gene, was used to subclone the os-1+ gene by functional complementation of an os-1 mutant. Analysis of the sequence of complementing DNA predicts that os-1+ encodes a predicted protein similar to sensor-histidine kinases of bacteria and a yeast osmosensor-histidine kinase. Importantly, the predicted os-1+ protein is identical to the N. crassa nik-1 predicted protein that was identified by using polymerase chain reaction primers directed against histidine kinase consensus DNA sequences. Our results indicate that nik-1 and os-1 encode the same osmosensing histidine kinase that plays an important role in the regulation of cell-wall assembly and, probably, other cell responses to changes in external osmolarity.
The partners of the symbiotic association between Anthoceros punctatus L. and Nostoc spp. have been cultured separately in a pure state. The symbiotic association was reconstituted following dual culture in liquid Anthoceros growth medium with a variety of axenic Nostoc isolates and mutant strains. The heterocyst frequency of competent Nostoc strains increased four- to fivefold when in symbiotic association relative to free-living N2-grown cultures. Dinitrogen fixation by symbiotic Nostoc supported the growth of Anthoceros tissue, although this growth was nitrogen-limited relative to that supported by exogenous ammonium. When the association was reconstituted in the presence of two or three wild-type and mutant Nostoc strains some of these strains were found to compete in infection of Anthoceros tissue and a fraction of the symbiotic Nostoc colonies contained more than one strain. Exogenous ammonium did not affect infection, but repressed development of the symbiotic Nostoc colonies in Anthoceros tissue, and symbiotic Nostoc in N2-grown Anthoceros tissue appeared to regress from the symbiotic state in the presence of exogenous ammonium. The results show that the Anthoceros-Nostoc symbiotic association is amenable to specific experimental manipulations; their implications are discussed with respect to infection of Anthoceros tissue and control of the development of symbiotic Nostoc.
Yarrowia lipolytica DO613, carrying the xpr6-13 mutation, secretes an inactive precursor of alkaline extracellular protease that has not been cleaved after the Lys-Arg at the end of the pro-region. Compared to wild type, DO613 membrane preparations had significantly reduced ability to cleave after Lys-Arg of an artificial substrate. The XPR6 gene was cloned by complementation by screening for restoration of production of alkaline protease activity. Sequencing of a 3735 base pair SalI-SphI XPR6 fragment revealed a large open reading frame with a coding capacity of 976 amino acids (molecular weight, 110,016). The deduced amino acid sequence had significant homology to Saccharomyces cerevisiae Kex2p, a processing endoprotease that cleaves after pairs of basic amino acids. Disruption of the XPR6 gene was not lethal, but it resulted in several phenotypic changes. First, essentially no mature alkaline extracellular protease was produced indicating that the low levels produced by strains carrying previously isolated xpr6 alleles were due to leaky mutations. Second, mating type B strains carrying the disrupted XPR6 gene did not mate, but mating type A strains did. Third, the XPR6 disruption strains grew poorly on rich media at pH 5.5 and above. Cells remained physically attached after budding and continued to bud forming large dog balloon-like structures. In addition, these structures aggregated forming visible clumps in liquid culture. These growth aberrations were largely eliminated by growing cells in medium at pH 4. Fourth, no mycelial forms were observed regardless of the pH.
The initial product of fixation of [(13)N]N2 by pure cultures of the reconstituted symbiotic association between Anthoceros punctatus L. and Nostoc sp. strain ac 7801 was ammonium; it accounted for 75% of the total radioactivity recovered in methanolic extracts after 0.5 min and 14% after 10 min of incubation. Glutamine and glutamate were the primary organic products synthesized from [(13)N]N2 after incubation times of 0.5-10 min. The kinetics of labeling of these two amino acids were characteristic of a precursor (glutamine) and product (glutamate) relationship. Results of inhibition experiments with methionine sulfoximine (MSX) and diazo-oxonorleucine were also consistent with the assimilation of N2-derived NH 4 (+) by Anthoceros-Nostoc through the sequential activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1), with little or no assimilation by glutamate dehydrogenase (EC 1.3.1.3). Isolated symbiotic Nostoc assimilated exogenous (13)NH 4 (+) into glutamine and glutamate and their formation was inhibited by MSX, indicating operation of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway: However, relative to free-living cultures, isolated symbiotic Nostoc assimilated 80% less exogenous ammonium into glutamine and glutamate, implying that symbiotic Nostoc could assimilate only a fraction of N2-derived NH 4 (+) . This implication was tested by using Anthoceros associations reconstituted with wild-type or MSX-resistant strains of Nostoc incubated with [(13)N]N2 in the presence of MSX. The results of these experiments indicated that, in situ, symbiotic Nostoc assimilated about 10% of the N2-derived NH 4 (+) and that NH 4 (+) was made available to Anthoceros tissue where it was apparently assimilated by the GS-GOGAT pathway. Since less than 1% of the fixed N2 was lost to the suspension medium, it appears that transfer of NH 4 (+) from symbiont to host tissue was very efficient in this extracellular symbiotic association.
Anabaena sp. strain 7120 appeared more responsive to nitrogen control than A. cylindrica. Growth in the presence of nitrate strongly repressed the differentiation of heterocysts and fixation of dinitrogen in Anabaena sp. strain 7120, but only weakly in A. cylindrica. Nitrate assimilation by ammonium-grown cultures was strongly repressed in Anabaena sp. strain 7120, but less so in A. cylindrica. The repressive effect of nitrate on dinitrogen assimilation in Anabaena sp. strain 7120, compared to A. cylindnca, did not correlate with a greater rate of nitrate transport, reduction to ammonium, assimilation into amino acids, or growth. Although both species grew at similar rates with dinitrogen, A. cylindrica grew
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