scholarly journals Engineering of membrane complex sphingolipids improves osmotic tolerance of Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Guoxing Zhu ◽  
Nannan Yin ◽  
Qiuling Luo ◽  
Jia Liu ◽  
Xiulai Chen ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Acyl Chain ◽  
Membrane Integrity ◽  
Wild Type ◽  
Adaptive Laboratory Evolution ◽  
Membrane Complex ◽  
Osmotic Tolerance ◽  
Novel Strategy ◽  
Complex Sphingolipids

ABSTRACTIn order to enhance the growth performance of S. cerevisiae under harsh environmental conditions, mutant XCG001, which tolerates up to 1.5M NaCl, was isolated via adaptive laboratory evolution (ALE). Comparisons made via transcriptome data of XCG001 and the wild-type strain identified ELO2 as being associated with osmotic tolerance. Overexpression of ELO2 increased the contents of inositol phosphorylceramide (IPC, t18:0/26:0), mannosylinositol phosphorylceramide (MIPC, t18:0/22:0(2OH)), MIPC (d18:0/22:0), MIPC (d20:0/24:0), mannosyldiinositol phosphorylceramide (M(IP)2C, d20:0/26:0), M(IP)2C (t18:0/26:0(2OH)) and M(IP)2C (d20:0/26:0(2OH)) by 88.3-, 166.9-, 63.3-, 23.9-, 27.9-, 113.8- and 208.1-fold at 1.0 M NaCl, respectively, compared those of strain XCG002. As a result, membrane integrity, cell growth and cell survival of the ELO2 overexpression strain (XCG010) increased by 24.4%, 29% and 22.1% at 1.0 M NaCl, respectively, compared those of strain XCG002. The findings provided a novel strategy for engineering complex sphingolipids to enhance osmotic tolerance.IMPORTANCEThis study demonstrated a novel strategy for manipulation membrane complex sphingolipids to enhance S. cerevisiae tolerance to osmotic stress. Osmotic tolerance was related to sphingolipid acyl chain elongase, Elo2, via transcriptome analysis of the wild-type strain and an osmotic tolerant strain generated from ALE. Overexpression of ELO2 increased complex sphingolipid with longer acyl chain, thus improved membrane integrity and osmotic tolerance.

scholarly journals Enhancement of Sphingolipid Synthesis Improves Osmotic Tolerance of Saccharomyces cerevisiae

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Guoxing Zhu ◽  
Nannan Yin ◽  
Qiuling Luo ◽  
Jia Liu ◽  
Xiulai Chen ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Acyl Chain ◽  
Membrane Integrity ◽  
Control Strain ◽  
Wild Type ◽  
Content Type ◽  
Osmotic Tolerance ◽  
Novel Strategy ◽  
Complex Sphingolipids

ABSTRACT To enhance the growth performance of Saccharomyces cerevisiae under osmotic stress, mutant XCG001, which tolerates up to 1.5 M NaCl, was isolated through adaptive laboratory evolution (ALE). Comparisons of the transcriptome data of mutant XCG001 and the wild-type strain identified ELO2 as being associated with osmotic tolerance. In the ELO2 overexpression strain (XCG010), the contents of inositol phosphorylceramide (IPC; t18:0/26:0), mannosylinositol phosphorylceramide [MIPC; t18:0/22:0(2OH)], MIPC (d18:0/22:0), MIPC (d20:0/24:0), mannosyldiinositol phosphorylceramide [M(IP)2C; d20:0/26:0], M(IP)2C [t18:0/26:0(2OH)], and M(IP)2C [d20:0/26:0(2OH)] increased by 88.3 times, 167 times, 63.3 times, 23.9 times, 27.9 times, 114 times, and 208 times at 1.0 M NaCl, respectively, compared with the corresponding values of the control strain XCG002. As a result, the membrane integrity, cell growth, and cell survival rate of strain XCG010 increased by 24.4% ± 1.0%, 21.9% ± 1.5%, and 22.1% ± 1.1% at 1.0 M NaCl, respectively, compared with the corresponding values of the control strain XCG002 (wild-type strain with a control plasmid). These findings provided a novel strategy for engineering complex sphingolipids to enhance osmotic tolerance. IMPORTANCE This study demonstrated a novel strategy for the manipulation of membrane complex sphingolipids to enhance S. cerevisiae tolerance to osmotic stress. Elo2, a sphingolipid acyl chain elongase, was related to osmotic tolerance through transcriptome analysis of the wild-type strain and an osmosis-tolerant strain generated from ALE. Overexpression of ELO2 increased the content of complex sphingolipid with longer acyl chain; thus, membrane integrity and osmotic tolerance improved.

scholarly journals Adaptive Laboratory Evolution of Cupriavidus necator H16 for Carbon Co-Utilization with Glycerol

2019 ◽  
Vol 20 (22) ◽  
pp. 5737 ◽  
Author(s):  
Miriam González-Villanueva ◽  
Hemanshi Galaiya ◽  
Paul Staniland ◽  
Jessica Staniland ◽  
Ian Savill ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Carbon Sources ◽  
Strain Engineering ◽  
Cupriavidus Necator ◽  
Superior Performance ◽  
Wild Type ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Cupriavidus Necator H16

Cupriavidus necator H16 is a non-pathogenic Gram-negative betaproteobacterium that can utilize a broad range of renewable heterotrophic resources to produce chemicals ranging from polyhydroxybutyrate (biopolymer) to alcohols, alkanes, and alkenes. However, C. necator H16 utilizes carbon sources to different efficiency, for example its growth in glycerol is 11.4 times slower than a favorable substrate like gluconate. This work used adaptive laboratory evolution to enhance the glycerol assimilation in C. necator H16 and identified a variant (v6C6) that can co-utilize gluconate and glycerol. The v6C6 variant has a specific growth rate in glycerol 9.5 times faster than the wild-type strain and grows faster in mixed gluconate–glycerol carbon sources compared to gluconate alone. It also accumulated more PHB when cultivated in glycerol medium compared to gluconate medium while the inverse is true for the wild-type strain. Through genome sequencing and expression studies, glycerol kinase was identified as the key enzyme for its improved glycerol utilization. The superior performance of v6C6 in assimilating pure glycerol was extended to crude glycerol (sweetwater) from an industrial fat splitting process. These results highlight the robustness of adaptive laboratory evolution for strain engineering and the versatility and potential of C. necator H16 for industrial waste glycerol valorization.

scholarly journals CgHog1-Mediated CgRds2 Phosphorylation Alters Glycerophospholipid Composition To Coordinate Osmotic Stress in Candida glabrata

2019 ◽  
Vol 85 (6) ◽  
Author(s):  
Chengjin Wu ◽  
Jiali Zhang ◽  
Guoxing Zhu ◽  
Rui Yao ◽  
Xiulai Chen ◽  
...  
Keyword(s):  
Cell Growth ◽  
Osmotic Stress ◽  
Cell Survival ◽  
Type Strain ◽  
Candida Glabrata ◽  
Wild Type Strain ◽  
Membrane Integrity ◽  
Wild Type ◽  
Content Type ◽  
Zinc Cluster

ABSTRACT Under stress conditions, Hog1 is required for cell survival through transiently phosphorylating downstream targets and reprogramming gene expression. Here, we report that Candida glabrata Hog1 (CgHog1) interacts with and phosphorylates CgRds2, a zinc cluster transcription factor, in response to osmotic stress. Additionally, we found that deletion of CgRDS2 led to decreases in cell growth and cell survival by 23.4% and 39.6%, respectively, at 1.5 M NaCl, compared with levels of the wild-type strain. This is attributed to significant downregulation of the expression levels of glycerophospholipid metabolism genes. As a result, the content of total glycerophospholipid decreased by 30.3%. Membrane integrity also decreased 47.6% in the Cgrds2Δ strain at 1.5 M NaCl. In contrast, overexpression of CgRDS2 increased the cell growth and cell survival by 10.2% and 6.3%, respectively, owing to a significant increase in the total glycerophospholipid content and increased membrane integrity by 27.2% and 12.1%, respectively, at 1.5 M NaCl, compared with levels for the wild-type strain. However, a strain in which the CgRDS2 gene encodes the replacement of Ser64 and Thr97 residues with alanines (Cgrds22A), harboring a CgRds2 protein that was not phosphorylated by CgHog1, failed to promote glycerophospholipid metabolism and membrane integrity at 1.5 M NaCl. Thus, the above results demonstrate that CgHog1-mediated CgRds2 phosphorylation enhanced glycerophospholipid composition and membrane integrity to resist osmotic stress in C. glabrata. IMPORTANCE This study explored the role of CgHog1-mediated CgRds2 phosphorylation in response to osmotic stress in Candida glabrata. CgHog1 interacts with and phosphorylates CgRds2, a zinc cluster transcription factor, under osmotic stress. Phosphorylated CgRds2 plays an important role in increasing glycerophospholipid composition and membrane integrity, thereby enhancing cell growth and survival.

A compensatory increase in trehalose synthesis in response to desiccation stress in Saccharomyces cerevisiae cells lacking the heat shock protein Hsp12p

2008 ◽  
Vol 54 (7) ◽  
pp. 559-568 ◽  
Author(s):  
Vanessa J. Shamrock ◽  
George G. Lindsey
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Flow Cytometry ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Integrity ◽  
Oxygen Species ◽  
Wild Type ◽  
Plasma Membrane Integrity ◽  
Trehalose Synthesis

The effect of HSP12 deletion on the response of yeast to desiccation was investigated. The Δhsp12 strain was found to be more desiccation tolerant than the wild-type strain. Furthermore, the increased intracellular trehalose levels in the Δhsp12 strain suggested that this strain compensated for the lack of Hsp12p synthesis by increasing trehalose synthesis, which facilitated increased desiccation tolerance. Results obtained from flow cytometry using the membrane exclusion dye propidium iodide suggested that Hsp12p helped maintain plasma membrane integrity during desiccation. Analysis of the oxidative loads experienced by the wild-type and Δhsp12 strains showed that during mid-exponential phase, the increased trehalose levels present in the Δhsp12 cells resulted in increased protection of these cells against reactive oxygen species compared with wild-type cells. During stationary phase, lower levels of reactive oxygen species reduction by reduced glutathione was enhanced in the wild-type strain, which displayed lower intracellular trehalose concentrations. Comparison of the tolerance of the wild-type and Δhsp12 strains with applied oxidative stress showed that the Δhsp12 strain was more tolerant to exogenously applied H2O2, which we attributed to the higher intracellular trehalose concentration. Flow cytometry demonstrated that Hsp12p played a role in maintaining plasma membrane integrity during applied oxidative stress.

scholarly journals The Structure of Neisseria meningitidis Lipid A Determines Outcome in Experimental Meningococcal Disease

2010 ◽  
Vol 78 (7) ◽  
pp. 3177-3186 ◽  
Author(s):  
Floris Fransen ◽  
Hendrik Jan Hamstra ◽  
Claire J. Boog ◽  
Jos P. van Putten ◽  
Germie P. J. M. van den Dobbelsteen ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Type Strain ◽  
Lipid A ◽  
Wild Type Strain ◽  
Large Fraction ◽  
Acyl Chain ◽  
Wild Type ◽  
Toll Like Receptor 4 ◽  
Lipid A Structure

ABSTRACTLipopolysaccharide (LPS), a major component of the meningococcal outer membrane, is sensed by the host through activation of Toll-like receptor 4 (TLR4). Recently, we demonstrated that a surprisingly large fraction ofNeisseria meningitidisdisease isolates are lipid A mutants, due to inactivating mutations in thelpxL1gene. ThelpxL1mutants activate human TLR4 much less efficiently than wild-type bacteria, which may be advantageous by allowing them to escape from the innate immune system. Here we investigated the influence of lipid A structure on virulence in a mouse model of meningococcal sepsis. One limitation, however, is that murine TLR4 recognizeslpxL1mutant bacteria much better than human TLR4. We show that anlpxL2mutant, another lipid A mutant lacking an acyl chain at a different position, activates murine TLR4 less efficiently than thelpxL1mutant. Therefore, thelpxL2mutant in mice might be a better model for infections withlpxL1mutants in humans. Interestingly, we found that thelpxL2mutant is more virulent in mice than the wild-type strain, whereas thelpxL1mutant is actually much less virulent than the wild-type strain. These results demonstrate the crucial role ofN. meningitidislipid A structure in virulence.

scholarly journals Candida glabrata Yap6 Recruits Med2 To Alter Glycerophospholipid Composition and Develop Acid pH Stress Resistance

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Pei Zhou ◽  
Xiaoke Yuan ◽  
Hui Liu ◽  
Yanli Qi ◽  
Xiulai Chen ◽  
...  
Keyword(s):  
Cell Growth ◽  
Type Strain ◽  
Candida Glabrata ◽  
Wild Type Strain ◽  
Membrane Integrity ◽  
Low Ph ◽  
Wild Type ◽  
Content Type ◽  
Acid Ph ◽  
Ph Stress

ABSTRACT Candida glabrata is a high-performance microbial cell factory for the production of organic acids. To elucidate the role of the C. glabrata Mediator tail subunit Med2 (CgMed2) at pH 2.0, we deleted or overexpressed CgMed2 and used transcriptome analysis to identify genes that are regulated by CgMed2. At pH 2.0, the deletion of CgMed2 resulted in a cell growth decrease of 26.1% and a survival decrease of 32.3%. Overexpression of CgMed2 increased cell growth by 12.4% and cell survival by 5.9% compared to the wild-type strain. Transcriptome and phenotypic analyses identified CgYap6 as a transcription factor involved in acid pH stress tolerance. Deletion of CgYap6 caused growth defects, whereas its overexpression enhanced cell growth at pH 2.0. Furthermore, total glycerophospholipid content and membrane integrity decreased by 33.4% and 21.8%, respectively, in the CgMed2Δ strain; however, overexpression of CgMed2 increased the total glycerophospholipid content and membrane integrity by 24.7% and 12.1%, respectively, compared with those of the wild-type strain at pH 2.0. These results demonstrated that under acid pH stress, CgMed2 physically interacts with CgYap6, which translocates from the cytoplasm to the nucleus after being phosphorylated by the protein kinase CgYak1. Once in the nucleus, CgYap6 recruits CgMed2 to express glycerophospholipid-related genes. Our study elucidated the function of CgMed2 under acid pH stress and provides a potential strategy to equip Candida glabrata with low-pH resistance during organic acid fermentation. IMPORTANCE This study investigated the function of the Mediator tail subunit CgMed2 in C. glabrata under low-pH stress. The protein kinase CgYak1 activates CgYap6 for the recruitment of CgMed2, which in turn increases glycerophospholipid content and membrane integrity to confer low-pH stress tolerance. This study establishes a new link between the Mediator tail subunit and transcription factors. Overall, these findings indicate that CgMed2 is a novel target to induce the low-pH stress response in C. glabrata.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nayeong Kim ◽  
Hyo Jeong Kim ◽  
Man Hwan Oh ◽  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Antimicrobial Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

scholarly journals FixJ family regulator AcfR of Azorhizobium caulinodans is involved in symbiosis with the host plant

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wei Liu ◽  
Xue Bai ◽  
Yan Li ◽  
Haikun Zhang ◽  
Xiaoke Hu
Keyword(s):  
Signal Transduction ◽  
Host Plant ◽  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Azorhizobium Caulinodans ◽  
Wild Type ◽  
Response Regulators ◽  
Free Living ◽  
Two Component

Abstract Background A wide variety of bacterial adaptative responses to environmental conditions are mediated by signal transduction pathways. Two-component signal transduction systems are one of the predominant means used by bacteria to sense the signals of the host plant and adjust their interaction behaviour. A total of seven open reading frames have been identified as putative two-component response regulators in the gram-negative nitrogen-fixing bacteria Azorhizobium caulinodans ORS571. However, the biological functions of these response regulators in the symbiotic interactions between A. caulinodans ORS571 and the host plant Sesbania rostrata have not been elucidated to date. Results In this study, we identified and investigated a two-component response regulator, AcfR, with a phosphorylatable N-terminal REC (receiver) domain and a C-terminal HTH (helix-turn-helix) LuxR DNA-binding domain in A. caulinodans ORS571. Phylogenetic analysis showed that AcfR possessed close evolutionary relationships with NarL/FixJ family regulators. In addition, six histidine kinases containing HATPase_c and HisKA domains were predicted to interact with AcfR. Furthermore, the biological function of AcfR in free-living and symbiotic conditions was elucidated by comparing the wild-type strain and the ΔacfR mutant strain. In the free-living state, the cell motility behaviour and exopolysaccharide production of the ΔacfR mutant were significantly reduced compared to those of the wild-type strain. In the symbiotic state, the ΔacfR mutant showed a competitive nodule defect on the stems and roots of the host plant, suggesting that AcfR can provide A. caulinodans with an effective competitive ability for symbiotic nodulation. Conclusions Our results showed that AcfR, as a response regulator, regulates numerous phenotypes of A. caulinodans under the free-living conditions and in symbiosis with the host plant. The results of this study help to elucidate the involvement of a REC + HTH_LuxR two-component response regulator in the Rhizobium-host plant interaction.

scholarly journals Mab_3083c Is a Homologue of RNase J and Plays a Role in Colony Morphotype, Aggregation, and Sliding Motility of Mycobacterium abscessus

2021 ◽  
Vol 9 (4) ◽  
pp. 676
Author(s):  
Ting-Yu Liu ◽  
Sheng-Hui Tsai ◽  
Jenn-Wei Chen ◽  
Yu-Ching Wang ◽  
Shiau-Ting Hu ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Opportunistic Pathogen ◽  
Mycobacterium Abscessus ◽  
Colony Morphology ◽  
Clinical Strain ◽  
Immunocompromised Patients ◽  
Clinical Infection ◽  
Wild Type ◽  
Sliding Motility

Mycobacterium abscessus is an opportunistic pathogen causing human diseases, especially in immunocompromised patients. M. abscessus strains with a rough morphotype are more virulent than those with a smooth morphotype. Morphotype switch may occur during a clinical infection. To investigate the genes involved in colony morphotype switching, we performed transposon mutagenesis in a rough clinical strain of M. abscessus. A morphotype switching mutant (smooth) named mab_3083c::Tn was obtained. This mutant was found to have a lower aggregative ability and a higher sliding motility than the wild type strain. However, its glycopeptidolipid (GPL) content remained the same as those of the wild type. Complementation of the mutant with a functional mab_3083c gene reverted its morphotype back to rough, indicating that mab_3083c is associated with colony morphology of M. abscessus. Bioinformatic analyses showed that mab_3083c has a 75.4% identity in amino acid sequence with the well-characterized ribonuclease J (RNase J) of M. smegmatis (RNase JMsmeg). Complementation of the mutant with the RNase J gene of M. smegmatis also switched its colony morphology from smooth back to rough. These results suggest that Mab_3083c is a homologue of RNase J and involved in regulating M. abscessus colony morphotype switching.

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