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.     

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     

Sucrose synthase and RuBisCo expression is similarly regulated by the nitrogen source in the nitrogen-fixing cyanobacterium Anabaena sp.

In higher plants and cyanobacteria, sucrose (Suc) metabolism is carried out by a similar set of enzymes. The function and regulation of Suc metabolism in cyanobacteria has begun to be elucidated. In strains of Anabaena sp., filamentous nitrogen-fixing cyanobacteria, Suc synthase (SuS, EC 2.4.1.13) controls Suc cell level through the cleavage of the disaccharide. The present work shows that there are two sus genes in Anabaena (Nostoc) sp. that are co-regulated regarding the nitrogen source; however, only susA accounts for the extractable SuS activity and for the control of the Suc level. Primer extension analysis has uncovered the sequence of the Anabaena susA and susB ammonium-activated putative promoters, which share a high sequence similarity with that of rbcLS encoding ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and other ammonium up-regulated genes. Moreover, susA and rbcLS expression is developmentally co-localized to the vegetative cells of the nitrogen-fixing cyanobacterial filaments. Our results strongly suggest the existence of a regulatory network that would coordinate the expression of key genes for Suc and nitrogen metabolism, carbon fixation, and development in Anabaena sp.     

Genome-wide analysis and phosphorylation sites identification of the 14-3-3 gene family and functional characterization of MeGRF3 in cassava

The biological functionality of many members of the 14-3-3 gene family is regulated via phosphorylation at multiple amino acid residues. The specific phosphorylation-mediated regulation of these proteins during cassava root tuberization, however, is not well understood. In this study, 15 different 14-3-3 genes (designated MeGRF1 – 15) were identified within the cassava genome. Based upon evolutionary conservation and structural analyses, these cassava 14-3-3 proteins were grouped into ε and non-ε clusters. We found these 15 MeGRF genes to be unevenly distributed across the eight cassava chromosomes. When comparing the expression of these genes during different developmental stages, we found that three of these genes (MeGRF9, 12 and 15) were overexpressed at all developmental stages at 75, 104, 135, 182 and 267 days post-planting relative to the fibrous root stage, whereas two (MeGRF5 and 7) were downregulated during these same points. In addition, the expression of most MeGRF genes changed significantly in the early and middle stages of root tuberization. This suggests that these different MeGRF genes likely play distinct regulatory roles during cassava root tuberization. Subsequently, 18 phosphorylated amino acid residues were detected on nine of these MeGRF proteins. A phosphomimetic mutation at serine-65 in MeGRF3 in Arabidopsis thaliana (Arabidopsis) slightly influenced starch metabolism in these plants, and significantly affected the role of MeGRF3 in salt stress responses. Together these results indicate that 14-3-3 genes play key roles in responses to abiotic stress and the regulation of starch metabolism, offering valuable insights into the functions of these genes in cassava.     

Essential role of the plasmid hik31 operon in regulating central metabolism in the dark in Synechocystis sp. PCC 6803

The plasmid hik31 operon (P3, slr6039‐slr6041) is located on the pSYSX plasmid in Synechocystis sp. PCC 6803. A P3 mutant (ΔP3) had a growth defect in the dark and a pigment defect that was worsened by the addition of glucose. The glucose defect was from incomplete metabolism of the substrate, was pH dependent, and completely overcome by the addition of bicarbonate. Addition of organic carbon and nitrogen sources partly alleviated the defects of the mutant in the dark. Electron micrographs of the mutant revealed larger cells with division defects, glycogen limitation, lack of carboxysomes, deteriorated thylakoids and accumulation of polyhydroxybutyrate and cyanophycin. A microarray experiment over two days of growth in light‐dark plus glucose revealed downregulation of several photosynthesis, amino acid biosynthesis, energy metabolism genes; and an upregulation of cell envelope and transport and binding genes in the mutant. ΔP3 had an imbalance in carbon and nitrogen levels and many sugar catabolic and cell division genes were negatively affected after the first dark period. The mutant suffered from oxidative and osmotic stress, macronutrient limitation, and an energy deficit. Therefore, the P3 operon is an important regulator of central metabolism and cell division in the dark.     

<Emphasis Type="Italic">para</Emphasis>-Nitrophenol 4-monooxygenase and hydroxyquinol 1,2-dioxygenase catalyze sequential transformation of 4-nitrocatechol in <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain WBC-3

Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen and energy. PnpA (PNP 4-monooxygenase) and PnpB (para-benzoquinone reductase) were shown to be involved in the initial steps of PNP catabolism via hydroquinone. We demonstrated here that PnpA also catalyzed monooxygenation of 4-nitrocatechol (4-NC) to hydroxyquinol, probably via hydroxyquinone. It was the first time that a single-component PNP monooxygenase has been shown to catalyze this conversion. PnpG encoded by a gene located in the PNP degradation cluster was purified as a His-tagged protein and identified as a hydroxyquinol dioxygenase catalyzing a ring-cleavage reaction of hydroxyquinol. Although all the genes necessary for 4-NC metabolism seemed to be present in the PNP degradation cluster in strain WBC-3, it was unable to grow on 4-NC as a sole source of carbon, nitrogen and energy. This was apparently due to the substrate’s inability to trigger the expression of genes involved in degradation. Nevertheless, strain WBC-3 could completely degrade both PNP and 4-NC when PNP was used as the inducer, demonstrating its potential in bioremediation of the environment polluted by both 4-NC and PNP.     

Enhanced curdlan production in <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749 by addition of low-polyphosphates

A large amount of adenosine triphosphate with high energy phosphate bonds is required for uridine triphosphate regeneration during curdlan biosynthesis by Agrobacterium sp. ATCC 31749. To supply high energy for curdlan synthesis, three low-polyphosphates (Na₄P₂O₇, Na₅P₃O₁₀, and (NaPO₃)₆) with higher energy phosphate bonds were employed to substitute for KH₂PO₄-K₂HPO₄ in fermentation medium. Two genes encoding the polyphosphate metabolizing enzymes, polyphosphate kinase and exopolyphosphatase, were amplified and showed 95% homology to those in Agrobacterium sp. C58 by sequence analysis. The curdlan yields were enhanced by 23 and 134% when phosphate concentrations 0.024 mol/L of Na₅P₃O₁₀ and 0.048 mol/L of (NaPO₃)₆ respectively, were added in the medium. The maximum curdlan yield of 30 ± 1.02 g/L was obtained with the addition of 0.048 mol/L of (NaPO₃)₆ with 5 g/L CaCO₃ in the medium. When CaCO₃ was removed from the culture and the three lowpolyphosphates were added, the pH and biomass yield dropped remarkably and little or no curdlan was produced. The culture containing 0.048 mol/L of (NaPO₃)₆ was mixed with KH₂PO₄-K₂HPO₄ and CaCO₃ in the medium, but showed no effect on curdlan production. However, curdlan yield was improved by 49 ∼ 60% when CaCO₃ was removed from the medium and KH₂PO₄-K₂HPO₄ acted as a buffer. It appears that the positive effect of (NaPO₃)₆ on curdlan production required the buffering capacity of CaCO₃ and the absence of KH₂PO₄-K₂HPO₄ competing as a phosphate supplier.