Photorespiratory 2-phosphoglycolate metabolism and photoreduction of O<Subscript>2</Subscript> cooperate in high-light acclimation of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. strain PCC 6803

In cyanobacteria, photorespiratory 2-phosphoglycolate (2PG) metabolism is mediated by three different routes, including one route involving the glycine decarboxylase complex (Gcv). It has been suggested that, in addition to conversion of 2PG into non-toxic intermediates, this pathway is important for acclimation to high-light. The photoreduction of O₂ (Mehler reaction), which is mediated by two flavoproteins Flv1 and Flv3 in cyanobacteria, dissipates excess reductants under high-light by the four electron-reduction of oxygen to water. Single and double mutants defective in these processes were constructed to investigate the relation between photorespiratory 2PG-metabolism and the photoreduction of O₂ in the cyanobacterium Synechocystis sp. PCC 6803. The single mutants Δflv1, Δflv3, and ΔgcvT, as well as the double mutant Δflv1/ΔgcvT, were completely segregated but not the double mutant Δflv3/ΔgcvT, suggesting that the T-protein subunit of the Gcv (GcvT) and Flv3 proteins cooperate in an essential process. This assumption is supported by the following results: (1) The mutant Δflv3/ΔgcvT showed a considerable longer lag phase and sometimes bleached after shifts from slow (low light, air CO₂) to rapid (standard light, 5% CO₂) growing conditions. (2) Photoinhibition experiments indicated a decreased ability of the mutant Δflv3/ΔgcvT to cope with high-light. (3) Fluorescence measurements showed that the photosynthetic electron chain is reduced in this mutant. Our data suggest that the photorespiratory 2PG-metabolism and the photoreduction of O₂, particularly that catalyzed by Flv3, cooperate during acclimation to high-light stress in cyanobacteria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Identification and characterization of a novel enzyme related to the synthesis of PUFAs derived from <Emphasis Type="Italic">Thraustochytrium aureum</Emphasis> ATCC 34304

In Thraustochytrids, Thraustochytrium aureum ATCC 34304 was able to produce high levels of several polyunsaturated fatty acids. In the present study, a novel gene encoding protein was cloned from the DHA rich microbe, T. aureum ATCC 34304. The functional analysis of a novel gene was demonstrated by its heterologous expression in Pichia pastoris. The gene was able to synthesize C20 and C22 PUFAs, as well as, to mediate different elongations (Δ9, Δ6, and Δ5) and one Δ5 desaturation activities. The conversion rates of the Δ9 elongation (n-3) and Δ5 desaturation products were found to be higher in response to the novel enzyme than the controls (TaElo and Tad5, respectively). The other Δ9 elongation (n-6) and Δ5 elongation products were slightly lower than those of the control (TaElo). The full length of the 1,374 bp gene contained 458 amino acids that showed very limited homology with desaturases and elongases from various organisms. In addition, the rate of synthesis of PUFAs was evaluated at temperatures ranging from 10 to 30°C. The elongation products were found to decrease dramatically and the desaturation products were found to increase dramatically at 10°C. TaNE was confirmed to be a multifunctional enzyme with higher activity towards Δ6 elongations than Δ9, Δ5 elongations, and Δ5 desaturation.     

Deletion of the <Emphasis Type="Italic">CgTPI</Emphasis> Gene Encoding Triose Phosphate Isomerase of <Emphasis Type="Italic">Candida glycerinogenes</Emphasis> Inhibits the Biosynthesis of Glycerol

The yeast Candida glycerinogenes produces a high yield of glycerol only in response to a medium-osmotic stress, but little is known about the relationship between osmoadaptation and glycerol metabolism. The CgTPI gene encoding triose phosphate isomerase of C. glycerinogenes was cloned and sequenced, and its functionality was confirmed by complementation of Saccharomyces cerevisiae tpi1 Δ. The roles of CgTpip in the glycerol biosynthesis and the osmoadaptation were investigated. Unlike S. cerevisiae tpi1 Δ and Klyuveromyces lactis tpi1 Δ, the mutant lacking CgTPI significantly decreased the rate of glucose consumption and the glycerol yield. Furthermore, the mutants decreased osmotolerance to glucose and NaCl. The results suggest that CgTPI might be crucial for a high yield of glycerol by C. glycerinogenes. The inhibition of glycerol biosynthesis might be related to the reduced ability of osmoadaptation to high external osmolarity. To our knowledge, this is the first report that inactivation of a yeast TPI gene inhibits the biosynthesis of glycerol.     

Effect of the chitin binding domain deletion from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> chitinase Chi255 on its stability in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus thuringiensis subsp. kurstaki BUPM255 secretes a chitobiosidase Chi255 having an expected molecular weight of 70.665 kDa. When the corresponding gene, chi255, was expressed in E. coli, the active form, extracted from the periplasmic fraction of E. coli/pBADchi255, was of about 54 kDa, which suggested that Chi255 was excessively degraded by the action of E. coli proteases. Therefore, in vitro progressive C-terminal Chi255 deleted derivatives were constructed in order to study their stability and their activity in E. coli. Interestingly, when the chitin binding domain (CBD) was deleted from Chi255, an active form (Chi2555Δ5) of expected size of about 60 kDa was extracted from the E. coli periplasmic fraction, without the observation of any proteolytic degradation. Compared to Chi255, Chi255Δ5 exhibited a higher chitinase activity on colloidal chitin. Both of the enzymes exhibit activities at broad pH and temperature ranges with maximal enzyme activities at pH 5 and pH 6 and at temperatures 50°C and 40°C, respectively for Chi255 and Chi255Δ5. Thus, it was concluded that the C-terminal deletion of Chi255 CBD might be a nice tool for avoiding the excessive chitinase degradation, observed in the native chitinase, and for improving its activity.     

Regulation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Plasma membrane H<Superscript>+</Superscript>-ATPase (Pma1) by Dextrose and Hsp30 during Exposure to Thermal Stress

Pma1p is an essential plasma membrane H⁺-pump in Saccharomyces cerevisiae that pumps out H⁺ at the expense of cellular ATP. Its activity is induced by glucose at 30°C and is inhibited by Hsp30 during exposure to heat shock conditions. To further investigate the regulation of Pma1 function by glucose and Hsp30 during exposure to thermal stress, we estimated Pma1 activity, its protein levels and ser-phosphorylation status in membrane fractions isolated from BY4741 and hsp30Δ cells grown in dextrose and sorbitol at 30°C, and following exposure at 40°C for 30 min. Our results demonstrate that Pma1 activity and protein levels were reduced in Hsp30⁺ cells following exposure to thermal stress in dextrose media. The above was not observed in hsp30Δ cells wherein Pma1 activity did not decrease following exposure to similar conditions. Although Pma1p levels decreased in heat-shocked hsp30Δ cells, it was lower compared to that observed in Hsp30⁺ cells. Total ser-phosphorylation of Pma1 also showed a decrease following exposure to heat shock condition in dextrose media in both BY4741 and hsp30Δ cells. Its levels were also reduced in BY4741 cells upon heat shock treatment in sorbitol unlike that observed in hsp30Δ cells wherein it was increased. Taken together the above indicate that heat shock induced reduction in Pma1 activity and protein levels in dextrose media required Hsp30. To examine functional interactions between dextrose utilization, Hsp30 and the regulation of various aspects of Pma1, we determined if dextrose regulated other functions attributed to Hsp30. Results demonstrate that the deletion of HSP30 rendered cells dependent on dextrose utilization for survival during exposure to lethal heat stress. Our study has hence been able to establish a functional relationship between glucose utilization, Hsp30 function and the regulation of Pma1 activity. Finally, since the deletion of HSP30 renders Pma1p levels and its activity unresponsive to thermal stress in dextrose media, we concluded that Hsp30 is necessary to maintain Pma1 in a regulation competent conformation. Hsp30 may thus act as a chaperone in the S. cerevisiae plasma membrane.     

Deletion analysis of the C-terminal region of a molecular chaperone DnaK from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Bacillus licheniformis DnaK (BlDnaK) is predicted to consist of a 45-kDa N-terminal ATPase domain and a 25-kDa C-terminal substrate-binding domain. In this study, the full-length BlDnaK and its T86W and three C-terminally truncated mutants were constructed to evaluate the role of up to C-terminal 255 amino acids of the protein. The steady-state ATPase activity for BlDnaK, T86W, T86W/ΔC120, T86W/ΔC249, and T86W/ΔC255 was 65.68, 53.21, 116.04, 321.38, and 90.59 nmol Pi/min per mg, respectively. In vivo, BldnaK, T86W and T86W/ΔC120 genes allowed an E. coli dnaK756-ts mutant to grow at 44°C. Except for T86W/ΔC255, simultaneous addition of B. licheniformis DnaJ and GrpE, and NR-peptide synergistically stimulated the ATPase activity of BlDnaK, T86W, T86W/ΔC120, and T86W/ΔC249 by 16.9-, 13.9-, 33.9-, 9.9-fold, respectively. Measurement of intrinsic tryptophan fluorescence revealed significant alterations of microenvironment of aromatic amino acids in the C-terminally truncated mutants. The temperature-dependent signal in the far-UV region for T86W was consistent with that of BlDnaK, but the C-terminally truncated mutant proteins showed a higher sensitivity toward temperature-induced denaturation. These results suggest that C-terminal truncations alter the ATPase activity and thermal stability of BlDnaK and induce the conformation change of the ATPase domain. Wan-Chi Liang and Min-Guan Lin contributed equally to this work.     

Biochemical properties and physiological roles of NADP-dependent malic enzyme in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Malic enzymes catalyze the reversible oxidative decarboxylation of L-malate using NAD(P)⁺ as a cofactor. NADP-dependent malic enzyme (MaeB) from Escherichia coli MG1655 was expressed and purified as a fusion protein. The molecular weight of MaeB was about 83 kDa, as determined by SDS-PAGE. The recombinant MaeB showed a maximum activity at pH 7.8 and 46°C. MaeB activity was dependent on the presence of Mn²⁺ but was strongly inhibited by Zn²⁺. In order to understand the physiological roles, recombinant E. coli strains (icd ^NADP/ΔmaeB and icd ^NAD/ΔmaeB) containing NADP-dependent isocitrate dehydrogenase (IDH), or engineered NAD-dependent IDH with the deletion of the maeB gene, were constructed using homologous recombination. During growth on acetate, icd ^NAD/ΔmaeB grew poorly, having a growth rate only 60% that of the wild-type strain (icd ^NADP). Furthermore, icd ^NADP/ΔmaeB exhibited a 2-fold greater adaptability to acetate than icd ^NAD/ΔmaeB, which may be explained by more NADPH production for biosynthesis in icd ^NADP/ΔmaeB due to its NADP-dependent IDH. These results indicated that MaeB was important for NADPH production for bacterial growth on acetate. We also observed that MaeB activity was significantly enhanced (7.83-fold) in icd ^NAD, which was about 3-fold higher than that in icd ^NADP, when switching from glucose to acetate. The marked increase of MaeB activity was probably induced by the shortage of NADPH in icd ^NAD. Evidently, MaeB contributed to the NADPH generation needed for bacterial growth on two carbon compounds.     

Roles of <Emphasis Type="Italic">Saccharomyces cerevisiae RAD17</Emphasis> and <Emphasis Type="Italic">CHK1</Emphasis> checkpoint genes in the repair of double-strand breaks in cycling cells

Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Δ/chk1Δ and rad17Δ/rad17Δ, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Δ/rad17Δ cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Δ/rad17Δ cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Δ/chk1Δ cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Δ/chk1Δ mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background. Nelson Bracesco and Ema C. Candreva have contributed equally to this article.     

Isolation,Functional Characterization,and Expression Pattern of a Vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Gene <Emphasis Type="Italic">TrNHX1</Emphasis> from <Emphasis Type="Italic">Trifolium repens</Emphasis> L.

Plant vacuolar Na⁺/H⁺ antiporter plays an important role in salt tolerance. A vacuolar Na⁺/H⁺ antiporter gene TrNHX1 was cloned from Trifolium repens L., a forage legume, by RT-PCR and RACE methods using degenerate oligonucleotide primers. The TrNHX1 sequence contains 2,394 nucleotides and an open-reading frame of 1,626 nucleotides that encodes a protein of 541 amino acids with a deduced molecular mass of 59.5 kDa. The deduced amino acid sequence of TrNHX1 is 78% identical to that of a vacuolar Na⁺/H⁺ antiporter of Arabidopsis thaliana, AtNHX1, and contains the consensus amiloride-binding domain. TrNHX1 could partially complement the NaCl-sensitive phenotypes of yeast mutants Δnhx1 and Δena1-4Δnhx1, and a similar complementation was also observed in the presence of LiCl and KCl. In addition, it was found that TrNHX1 suppressed the hygromycin-sensitive phenotype of yeast mutant Δena1-4Δnhx1. The expression of TrNHX1 in T. repens increased in the presence of 150 mM NaCl, and this result accords with that of Na⁺ contents determination under the same treatment. These results suggest that TrNHX1 functions as a vacuolar Na⁺/H⁺ antiporter and plays an important role in salt tolerance and ion homeostasis in T. repens.