NtrC-dependent regulatory network for curdlan biosynthesis in response to nitrogen limitation in Agrobacterium sp. ATCC 31749

The mechanism of nitrogen signal regulating curdlan biosynthesis in Agrobacterium sp. ATCC 31749 was investigated. Under nitrogen limitation, more carbon flux is directed to curdlan synthesis with low specific growth rate. When ntrB and ntrC genes in Agrobacterium sp. were inactivated, NH₄Cl utilization ability was significantly impaired in the ntrB and ntrC mutants and curdlan production was significantly reduced. Through proteomic analysis, nearly 40 proteins did not express in ntrC mutant compared with wild type strain. The levels of 22 proteins were significantly increased and 21 proteins were repressed after nitrogen exhaustion. Phosphoglucomutase activity in Agrobacterium sp. was also decreased. However, phosphoglucomutase activity in the ntrC mutant did not change. On that basis, an NtrC-dependent regulatory network for curdlan biosynthesis in response to nitrogen limitation in Agrobacterium sp. ATCC 31749 is proposed.     

ntrB基因对Agrobacterium sp.ATCC31749合成胞外多糖的调控研究

利用三亲本接合方法将含有ntrB基因两端同源序列的自杀载体pJQ-ntrB-cat导入土壤杆菌(Agrobacterium sp.ATCC 31749)中,获得了ntrB基因突变株.结果表明,ntrB突变株对NH4Cl和KNO3的利用能力有所下降.当分别以谷氨酸和谷氨酰胺为氮源时,ntrB突变株生长状况与野生菌相同.n...     

Proteomic analysis of curdlan-producing Agrobacterium sp. in response to pH downshift

During batch cultivation of Agrobacterium sp. ATCC 31750, proteome analysis in response to a pH downshift from 7.0 to 5.5 was carried out using two-dimensional electrophoresis and matrix-assisted laser desorption-ionization-time of flight mass spectrometry. When the pH of the exponentially growing Agrobacterium sp. culture was downshifted to pH 5.5, the synthesis level of 27 intracellular proteins showed significant changes in level over a prolonged period of time compared with the batch culture controlled at pH 7.0. In particular, the intracellular protein level of the beta-1,3-glucan synthase catalytic subunit, UTP-glucose-1-phosphate uridylyltransferase, and phosphoglucomutase, which are key metabolic enzymes in the curdlan biosynthesis pathway, were more than 10-, 3- and 17-times higher in the low pH culture. On the other hand, the level of orotidine5-phosphate decarboxylase (conversion of OMP to UMP) was significantly up-regulated after pH downshift. The accumulation of UMP may direct the metabolic flow towards the biosynthetic route of UTP, which is a key metabolic precursor for UDP-glucose. Therefore, it is possible that increase of cellular metabolic enzymes during pH downshift culture can enhance the metabolic flux of the biosynthesis of key precursor, such as UTP- and UDP-glucose, resulting in an increase in curdlan biosynthesis.     

A pair of iron-responsive genes encoding protein kinases with a Ser/Thr kinase domain and a His kinase domain are regulated by NtcA in the Cyanobacterium Anabaena sp. strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 can fix N(2) when combined nitrogen is not available in the growth medium. It has a family of 13 genes encoding proteins with both a Ser/Thr kinase domain and a His kinase domain. The function of these enzymes is unknown. Two of them are encoded by pkn41 (alr0709) and pkn42 (alr0710). These two genes are separated by only 72 bp on the chromosome, and our results indicate that they are cotranscribed. The expression of pkn41 and pkn42 is induced by iron deprivation irrespective of the nature of the nitrogen source. Mutants inactivating either pkn41, pkn42, or both grow similarly to the wild type under normal conditions, but their growth is impaired either in the presence of an iron chelator or under conditions of nitrogen fixation and iron limitation, two situations where the demand for iron is particularly strong. Consistent with these results, these mutants display lower iron content than the wild type and a higher level of expression for nifJ1 and nifJ2, which encode pyruvate:ferredoxin oxidoreductases. Both nifJ1 and nifJ2 are known to be induced by iron limitation. NtcA, a global regulatory factor for different metabolic pathways, binds to the putative promoter region of pkn41, and the induction of pkn41 in response to iron limitation no longer occurs in an ntcA mutant. Our results suggest that ntcA not only regulates the expression of genes involved in nitrogen and carbon metabolism but also coordinates iron acquisition and nitrogen metabolism by activating the expression of pkn41 and pkn42.     

Cloning and characterization of the phosphatidylserine synthase gene of Agrobacterium sp. strain ATCC 31749 and effect of its inactivation on production of high-molecular-mass (1-->3)-beta-D-glucan (curdlan)

Genes involved in the production of the extracellular (1-->3)-beta-glucan, curdlan, by Agrobacterium sp. strain ATCC 31749 were described previously (Stasinopoulos et al., Glycobiology 9:31-41, 1999). To identify additional curdlan-related genes whose protein products occur in the cell envelope, the transposon TnphoA was used as a specific genetic probe. One mutant was unable to produce high-molecular-mass curdlan when a previously uncharacterized gene, pss(AG), encoding a 30-kDa, membrane-associated phosphatidylserine synthase was disrupted. The membranes of the mutant lacked phosphatidylethanolamine (PE), whereas the phosphatidylcholine (PC) content was unchanged and that of both phosphatidylglycerol and cardiolipin was increased. In the mutant, the continued appearance of PC revealed that its production by this Agrobacterium strain is not solely dependent on PE in a pathway controlled by the Pss(AG) protein at its first step. Moreover, PC can be produced in a medium lacking choline. When the pss(AG)::TnphoA mutation was complemented by the intact pss(AG) gene, both the curdlan deficiency and the phospholipid profile were restored to wild-type, demonstrating a functional relationship between these two characteristics. The effect of the changed phospholipid profile could occur through an alteration in the overall charge distribution on the membrane or a specific requirement for PE for the folding into or maintenance of an active conformation of any or all of the structural proteins involved in curdlan production or transport.     

Changes in gene expression during hairy root formation by<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> infection in ginseng

Researchers have widely adopted the hairy root culture system as a means for producing secondary metabolites, including ginsenosides from ginseng. Although bacterial genes are involved, the aspects of plant gene expression are unclear. Using a cDNA microarray approach, we identified genes that are differentially expressed in ginseng hairy roots afterAgrobacterium rhizogenes infection. Our goal was to gain an initial understanding of the correlation between hairy root morphology and ginsenoside production. Among the 250 genes analyzed here, 63 (including 14 that are unclassified) were differentially expressed in a hairy root line containing a high level of ginsenosides. Of the genes that had been functionally categorized, 29% and 17% were active in metabolism and stress responses, respectively. Most were primarily associated with ribosomal proteins, thereby functioning in protein synthesis and destination. Their expression was down-regulated in hairy roots having less lateral branching. This phenotype may have resulted from the manipulation of metabolic activities by the translational machinery.     

Production of curdlan using sucrose or sugar cane molasses by two-step fed-batch cultivation of Agrobacterium species

Maltose and sucrose were efficient carbon sources for the production of curdlan by a strain of Agrobacterium sp. A two-step, fed-batch operation was designed in which biomass was first produced, followed by curdlan production which was stimulated by nitrogen limitation. There exists an optimal timing for nitrogen limitation for curdlan production in the two-step, fed-batch operation. Maximum curdlan production (60 g L⁻¹) was obtained from sucrose with a productivity of 0.2 g L⁻¹ h⁻¹ when nitrogen was limited at a cell concentration of 16.0 g L⁻¹. It was also noted that the curdlan yield from sucrose was as high as 0.45 g curdlan g⁻¹ sucrose, and the highest specific production rate was 1.0 g curdlan g⁻¹ cells h⁻¹ right after nitrogen limitation. Of particular importance was the use of molasses as a cheap carbon source to produce curdlan in the two-step, fed-batch cultivation. As high as 42 g L⁻¹ of curdlan with a yield of 0.35 g curdlan g⁻¹ total sugar was obtained after 120 h of fed-batch cultivation. Received 20 August 1996/ Accepted in revised form 26 November 1996