Most microalgae accumulate neutral lipids, including triacylglycerol (TAG), into spherical structures called lipid bodies (LBs) under environmental stress conditions such as nutrient depletion. In green algae, starch accumulation precedes TAG accumulation, and the starch is thought to be a substrate for TAG synthesis. However, the relationship between TAG synthesis and the starch content in red algae, as well as how TAG accumulation is regulated, is unclear. In this study, we cultured the primitive red alga Cyanidioschyzon merolae under nitrogen-depleted conditions, and monitored the formation of starch granules (SGs) and LBs using microscopy. SGs stained with potassium iodide were observed at 24 h; however, LBs stained specifically with BODIPY 493/503 were observed after 48 h. Quantitative analysis of neutral sugar and cytomorphological semi-quantitative analysis of TAG accumulation also supported these results. Thus, the accumulation of starch occurred and preceded the accumulation of TAG in cells of C. merolae. However, TAG accumulation was not accompanied by a decrease in the starch content, suggesting that the starch is a major carbon storage sink, at least under nitrogen-depleted conditions. Quantitative real-time PCR revealed that the mRNA levels of genes involved in starch and TAG synthesis rarely changed during the culture period, suggesting that starch and TAG synthesis in C. merolae are not controlled through gene transcription but at other stages, such as translation and/or enzymatic activity.
Bacteria capable of degrading cis-dichloroethene (cDCE) were screened from cDCE-contaminated soil, and YKD221, a bacterial strain that exhibited a higher growth on minimal salt agar plates in the presence of cDCE than in the absence of cDCE, were isolated. Phylogenetic studies of the 16S rRNA as well as gyrB, rpoD, and recA in YKD221 indicated that this strain is closely related to the type strains of Pseudomonas plecoglossicida, monteilii, and putida. An average nucleotide identity analysis indicated that YKD221 is most closely related to P. putida strains, including the type strain, which suggests that YKD221 belongs to P. putida. Although the genome of YKD221 was very similar to that of P. putida F1, a toluene-degrading strain, the YKD221 genome has 15 single-nucleotide polymorphisms and 4 insertions compared with the F1 genome. YKD221 caused the release of sufficient chloride ions from cDCE to suggest that the strain is able to completely dechlorinate and degrade cDCE. YKD221 also degraded trichloroethene but was unable to degrade trans-dichloroethene and tetrachloroethene. The degradation activity of YKD221 was elevated after growth on toluene. Inactivation of todC1, which encodes for a large subunit of the catalytic terminal component in toluene dioxygenase, resulted in a complete loss of growth on toluene and cDCE degradation activity. This is the first evidence of the involvement of todC1C2BA-coded toluene dioxygenase in cDCE degradation. YKD221 did not appear to grow on cDCE in a minimal salt liquid medium. However, YKD221 did exhibit an enhanced increase in cell concentration and volume of cells during growth on minimal salt agar plates with cDCE when first grown in LB medium. This behavior appears to have led us to misinterpret our initial results on YKD221 as an indication of improved growth in the presence of cDCE.
The pH of a microbiological culture is important for both cell growth and chitinase accumulation, but the optimal pH is not normally the same for both. The objective of this study was to investigate the effect of pH on chitinase production by Chitinolyticbacter meiyuanensis strain SYBC-H1 (ATCC BAA-2140) in a mineral medium. The results of batch culture at different pH values showed that the optimum pH for cell growth and chitinase production varied with time, although KOH produced the best results for cell growth and chitinase production, NaOH was chosen because of cost considerations. We designed a three-stage pH control strategy using NaOH as the neutralizing agent. Maximum cell growth (1.07 g dry cell weight/l) and maximum chitinase activity (13.6 U/ml) were observed after culture at 26°C for 72 h in a mineral medium. These values were greater by 129% and 162%, respectively, and the length of time to attain maximum chitinase activity was decreased by 12 h, compared with results from an earlier study (Hao et al., 2011b).
In the budding yeast Saccharomyces cerevisiae, the AVT genes (AVT1–7), which encode vacuolar amino acid transporters belonging to the amino acid vacuolar transport (AVT)-family, were significantly upregulated in response to exogenous proline. To reveal a novel role of the Avt proteins in proline homeostasis, we analyzed the effects of deletion or overexpression of the AVT genes on the subcellular distribution of amino acids after the addition of proline to the cells grown in minimal medium. Among seven AVT gene disruptants, avt1Δ and avt7Δ showed the lowest ratios of vacuolar proline. Consistently, overexpression of the AVT1 gene specifically enhanced the vacuolar localization of proline. Since double disruption of the AVT1 and AVT7 genes did not completely abrogate vacuolar accumulation of proline, it is presumed that Avt1 has a dominant role, and Avt7 and other Avt proteins have redundant functions, in the localization of proline into the vacuolar lumen. In contrast, deletion of the AVT3 gene increased vacuolar proline, although the highly expressed AVT3 gene interfered with the accumulation of proline in the vacuole. Based on these results, it appears that Avt3 is the major protein involved in the export of proline from the vacuole. We also observed vacuolar membrane localization of GFP-fused Avt1, Avt3, and Avt7 proteins. Taken together, our data suggest that the AVT genes induced by exogenous proline are involved in the bidirectional transport of proline across the vacuolar membrane.
Two-component signal transduction systems (TCS) are involved in widespread cellular responses to diverse signals from bacteria to plants. Cyanobacteria have evolved photoperception systems for efficient photosynthesis, and some histidine kinases are known to function as photosensors. In this study, we attempt to reconstruct the photoperception system in Escherichia coli to make an easily controllable ON/OFF switch for gene expressions. For this purpose, a CcaS-CcaR two-component system from Nostoc punctiforme was expressed with phycocyanobilin (PCB) producing enzymes in E. coli which carries a G-box-controlled reporter gene. We succeeded to endow E. coli with a gene activation switch that is regulated in a light-color dependent manner. The possibility of such a switch for the development of synthetic biology is pointed out.
Mycothiol (MSH) plays a major role in protecting cells against oxidative stress and detoxification from a broad range of exogenous toxic agents. In the present study, we reveal that intracellular MSH contributes significantly to the adaptation to acidic conditions in the model organism Corynebacterium glutamicum. We present evidence that MSH confers C. glutamicum with the ability to adapt to acidic conditions by maintaining pHi homeostasis, scavenging reactive oxygen species (ROS), and protecting methionine synthesis by the S-mycothiolation modification of methionine synthase (MetE). The role of MSH in acid adaptation was further confirmed by improving the acid tolerance of C. glutamicum by overexpressing the key MSH synthesis gene mshA. Hence, our work provides insights into a previously unknown, but important, aspect of the C. glutamicum cellular response to acid stress. The results reported here may help to understand acid tolerance mechanisms in acid sensitive bacteria and may open a new avenue for improving acid resistance in industry strains for the production of bio-based chemicals from renewable biomass.
Cyanobacteria are photosynthetic microorganisms that serve as experimental model organisms for the study of photosynthesis, environmental stress responses, and the production of biofuels. Genetic tools for bioengineering have been developed as a result of such studies. However, there is still room for improvement for the tight control of experimental protein expression in these microorganisms. Here, we describe an expression system controlled by a theophylline-responsive riboswitch that we have constructed in the cyanobacterium Synechocystis sp. PCC 6803. We demonstrate that, in response to different theophylline concentrations, this riboswitch can tightly control green fluorescence protein expression in Synechocystis. Thus, this system is useful as a tool for genetic engineering and the synthetic biology of cyanobacteria.