scholarly journals Development and Characterization of Acidic-pH Tolerant Mutants of Zymomonas mobilis through Adaptation and Next Generation Sequencing based Genome Resequencing and RNA-Seq

2020 ◽  
Author(s):  
Qing Yang ◽  
Yongfu Yang ◽  
Yin Tang ◽  
Xia Wang ◽  
Yunhao Chen ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Acidic Ph ◽  
Rna Seq ◽  
Genome Resequencing ◽  
Ph Tolerance ◽  
Mutant Strains ◽  
Rnd Efflux Pump ◽  
Ph Conditions ◽  
Generation Sequencing ◽  
Better Than

Abstract Background: Acid pretreatment is a common strategy used to break down the hemicellulose component of the lignocellulosic biomass to release pentoses, and a subsequent enzymatic hydrolysis step is usually applied to release hexoses from the cellulose. The hydrolysate after pretreatment and enzymatic hydrolysis containing both hexoses and pentoses can then be used as substrates for biochemical production. However, the acid-pretreated liquor can also be directly used as the substrate for microbial fermentation, which has an acidic pH and contains inhibitory compounds generated during pretreatment. Although the natural ethanologenic bacterium Zymomonas mobilis can grow in a broad range of pH 3.5~7.5, cell growth and ethanol fermentation are still affected under acidic-pH conditions below pH 4.0. Results: In this study, adaptive laboratory evolution (ALE) strategy was applied to adapt Z. mobilis under acidic-pH conditions. Two mutant strains named 3.6M and 3.5M with enhanced acidic-pH tolerance were selected and confirmed, of which 3.5M grew better than ZM4 but worse than 3.6M in acidic-pH conditions that is served as a reference strain between 3.6M and ZM4 to help unravel the acidic-pH tolerance mechanism. Mutant strains 3.5M and 3.6M exhibited 50~130% enhancement on growth rate, 4~9 h reduction on fermentation time to consume glucose, and 20~63% improvement on ethanol productivity than wild-type ZM4 at pH 3.8. Next-generation sequencing (NGS)-based whole genome resequencing (WGR) and RNA-Seq technologies were applied to unravel the acidic-pH tolerance mechanism of mutant strains. WGR result indicated that compared to wild-type ZM4, 3.5M and 3.6M have seven and five single nucleotide polymorphisms (SNPs) respectively, among which four are shared in common. Additionally, RNA-Seq result showed that the upregulation of genes involved in glycolysis and the downregulation of flagellar and mobility related genes would help generate and redistribute cellular energy to resist acidic pH while keeping normal biological processes in Z. mobilis. Moreover, genes involved in RND efflux pump, ATP-binding cassette (ABC) transporter, proton consumption, and alkaline metabolite production were significantly upregulated in mutants under the acidic-pH condition compared with ZM4, which could help maintain the pH homeostasis in mutant strains for acidic-pH resistance. Furthermore, our results demonstrated that in mutant 3.6M, genes encoding F1F0 ATPase to pump excess protons out of cells were upregulated under pH 3.8 compared to pH 6.2. This difference might help mutant 3.6M manage acidic conditions better than ZM4 and 3.5M. A few gene targets were then selected for genetics study to explore their role on acidic-pH tolerance, and our results demonstrated that the expression of two operons in the shuttle plasmids, ZMO0956-ZMO0958 encoding cytochrome bc1 complex and ZMO1428-ZMO1432 encoding RND efflux pump, could help Z. mobilis tolerate acidic-pH conditions. Conclusion: An acidic-pH tolerant mutant 3.6M obtained through this study can be used for commercial bioethanol production under acidic fermentation conditions. In addition, the molecular mechanism of acidic-pH tolerance of Z. mobilis was further proposed, which can facilitate future research on rational design of synthetic microorganisms with enhanced tolerance against acidic-pH conditions. Moreover, the strategy developed in this study combining approaches of ALE, genome resequencing, RNA-Seq, and classical genetics study for mutant evolution and characterization can be applied in other industrial microorganisms.

scholarly journals Development and Characterization of Acidic-pH Tolerant Mutants of Zymomonas mobilis through Adaptation and Next Generation Sequencing based Genome Resequencing and RNA-Seq

2020 ◽  
Author(s):  
Qing Yang ◽  
Yongfu Yang ◽  
Xia Wang ◽  
Yunhao Chen ◽  
Wei Shen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Zymomonas Mobilis ◽  
Acidic Condition ◽  
Acidic Ph ◽  
Rna Seq ◽  
Genome Resequencing ◽  
Ph Tolerance ◽  
Mutant Strains ◽  
Ph Conditions ◽  
Generation Sequencing

Abstract Background: Acid pretreatment is a common strategy used to break down lignocellulosic biomass as substrate for biochemical production, which however generates inhibitory compounds and results in acidic pH condition. Although the natural ethanologenic bacterium Zymomonas mobilis can grow in a broad pH range, cell growth and ethanol fermentation are still affected at acidic pH conditions below pH 4.0.Results: In this study, adaptive laboratory evolution (ALE) strategy was applied to adapt Z. mobilis under acidic pH condition. Two mutant strains named 3.6M and 3.5M with enhanced acidic-pH tolerance were selected and confirmed. Mutant strains 3.6M and 3.5M exhibited 50~130% enhancement on growth rate, 4~9 h reduction on fermentation time, and 20~63% improvement on ethanol productivity than wild-type ZM4 at pH 3.8. Next-generation sequencing (NGS)-based whole genome resequencing (WGR) and RNA-Seq technologies were applied to unravel the acidic pH tolerance mechanism of mutant strains. WGR result indicated that mutations in four genes ZMO0421 (Ala67Thr), ZMO0712 (Gly539Asp), ZMO1432 (Pro480Leu), and ZMO1733 (Thr7Lys) with non-synonymous amino acid changes might be related with the acidic-pH tolerance. Additionally, RNA-Seq result showed that the upregulation of genes involved in glycolysis and the downregulation of mobility related genes would help generate and redistribute cellular energy to help resist acidic pH while keep normal biological processes in Z. mobilis . Moreover, genes involved in RND efflux pump, ATP-binding cassette (ABC) transporter, proton consumption, and alkaline metabolite production were significantly upregulated in mutants under acidic condition compared with ZM4, which could help maintain the pH homeostasis in mutant strains for low acidic-pH resistance. Furthermore, our results also demonstrated that genes related to branch amino acid biosynthesis from threonine to isoleucine were significantly upregulated in mutant 3.6M under acidic condition compared with ZM4, and genes encoding F 1 F 0 ATPase to pump excess protons out of cells were upregulated in mutant 3.6M under pH 3.8 compared with pH 6.2. These differences could help mutant 3.6M manage acidic condition better than ZM4. A few gene targets were then selected for genetics study to confirm their role on acidic pH tolerance, and our results demonstrated that the expression of two operons in the shuttle plasmids could help Z. mobilis tolerate acidic pH condition, which are ZMO0956-ZMO0958 encoding cytochrome bc1 complex and ZMO1428-ZMO1432 encoding RND efflux pump.Conclusion: Two acidic-pH tolerant mutants 3.6M and 3.5M obtained through this study especially 3.6M can be used as candidate strains for commercial bioethanol production under acidic fermentation conditions, and molecular mechanism of acidic pH tolerance of Z. mobilis \was further proposed, which can facilitate future research on rational design of synthetic microorganisms with enhanced tolerance against acidic pH conditions. In addition, the strategy developed in this study combining approaches of ALE, genome resequencing, RNA-Seq and classical genetics study for mutant evolution and characterization can be applied in other industrial microorganisms.

Screening of Natural Products and Derivatives for the Identification of RND Efflux Pump Inhibitors

2016 ◽  
Vol 19 (9) ◽  
pp. 705-713 ◽  
Author(s):  
Debarati Choudhury ◽  
Anupam Talukdar ◽  
Pankaj Chetia ◽  
Amitabha Bhattacharjee ◽  
Manabendra Choudhury
Keyword(s):  
Natural Products ◽  
Efflux Pump ◽  
Rnd Efflux Pump ◽  
Efflux Pump Inhibitors

scholarly journals Transcriptomic Profiling of Skeletal Muscle Reveals Candidate Genes Influencing Muscle Growth and Associated Lipid Composition in Portuguese Local Pig Breeds

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1423
Author(s):  
André Albuquerque ◽  
Cristina Óvilo ◽  
Yolanda Núñez ◽  
Rita Benítez ◽  
Adrián López-Garcia ◽  
...  
Keyword(s):  
Candidate Genes ◽  
Muscle Growth ◽  
Rna Seq ◽  
Next Generation Sequencing Technology ◽  
Pig Breeds ◽  
Nfkb Activation ◽  
Main Gene ◽  
Slow Type ◽  
Longissimus Lumborum ◽  
Generation Sequencing

Gene expression is one of the main factors to influence meat quality by modulating fatty acid metabolism, composition, and deposition rates in muscle tissue. This study aimed to explore the transcriptomics of the Longissimus lumborum muscle in two local pig breeds with distinct genetic background using next-generation sequencing technology and Real-Time qPCR. RNA-seq yielded 49 differentially expressed genes between breeds, 34 overexpressed in the Alentejano (AL) and 15 in the Bísaro (BI) breed. Specific slow type myosin heavy chain components were associated with AL (MYH7) and BI (MYH3) pigs, while an overexpression of MAP3K14 in AL may be associated with their lower loin proportion, induced insulin resistance, and increased inflammatory response via NFkB activation. Overexpression of RUFY1 in AL pigs may explain the higher intramuscular (IMF) content via higher GLUT4 recruitment and consequently higher glucose uptake that can be stored as fat. Several candidate genes for lipid metabolism, excluded in the RNA-seq analysis due to low counts, such as ACLY, ADIPOQ, ELOVL6, LEP and ME1 were identified by qPCR as main gene factors defining the processes that influence meat composition and quality. These results agree with the fatter profile of the AL pig breed and adiponectin resistance can be postulated as responsible for the overexpression of MAP3K14′s coding product NIK, failing to restore insulin sensitivity.

scholarly journals A mutation in LacDWARF1 results in a GA-deficient dwarf phenotype in sponge gourd (Luffa acutangula)

2021 ◽  
Author(s):  
Gangjun Zhao ◽  
Caixia Luo ◽  
Jianning Luo ◽  
Junxing Li ◽  
Hao Gong ◽  
...  
Keyword(s):  
Gene Annotation ◽  
Recessive Gene ◽  
Genomic Region ◽  
Dwarf Mutant ◽  
Rna Seq ◽  
Dwarf Phenotype ◽  
Sponge Gourd ◽  
Response To Stress ◽  
Luffa Acutangula ◽  
Generation Sequencing

Abstract Key message A dwarfism gene LacDWARF1 was mapped by combined BSA-Seq and comparative genomics analyses to a 65.4 kb physical genomic region on chromosome 05. Abstract Dwarf architecture is one of the most important traits utilized in Cucurbitaceae breeding because it saves labor and increases the harvest index. To our knowledge, there has been no prior research about dwarfism in the sponge gourd. This study reports the first dwarf mutant WJ209 with a decrease in cell size and internodes. A genetic analysis revealed that the mutant phenotype was controlled by a single recessive gene, which is designated Lacdwarf1 (Lacd1). Combined with bulked segregate analysis and next-generation sequencing, we quickly mapped a 65.4 kb region on chromosome 5 using F2 segregation population with InDel and SNP polymorphism markers. Gene annotation revealed that Lac05g019500 encodes a gibberellin 3β-hydroxylase (GA3ox) that functions as the most likely candidate gene for Lacd1. DNA sequence analysis showed that there is an approximately 4 kb insertion in the first intron of Lac05g019500 in WJ209. Lac05g019500 is transcribed incorrectly in the dwarf mutant owing to the presence of the insertion. Moreover, the bioactive GAs decreased significantly in WJ209, and the dwarf phenotype could be restored by exogenous GA3 treatment, indicating that WJ209 is a GA-deficient mutant. All these results support the conclusion that Lac05g019500 is the Lacd1 gene. In addition, RNA-Seq revealed that many genes, including those related to plant hormones, cellular process, cell wall, membrane and response to stress, were significantly altered in WJ209 compared with the wild type. This study will aid in the use of molecular marker-assisted breeding in the dwarf sponge gourd.

scholarly journals Heterocyclic boronic acids display sialic acid selective binding in a hypoxic tumor relevant acidic environment

2017 ◽  
Vol 8 (9) ◽  
pp. 6165-6170 ◽  
Author(s):  
A. Matsumoto ◽  
A. J. Stephenson-Brown ◽  
T. Khan ◽  
T. Miyazawa ◽  
H. Cabral ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Boronic Acids ◽  
Sialic Acids ◽  
Strong Interactions ◽  
Acidic Ph ◽  
Acidic Environment ◽  
Selective Binding ◽  
High Affinity ◽  
Ph Conditions ◽  
Hypoxic Tumor

A group of heterocyclic boronic acids demonstrating unusually high affinity and selectivity for sialic acids are described, with strong interactions under the weakly acidic pH conditions associated with a hypoxic tumoral microenvironment.

scholarly journals Erratum: Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study

2014 ◽  
Vol 32 (11) ◽  
pp. 1166-1166 ◽  
Author(s):  
Sheng Li ◽  
Scott W Tighe ◽  
Charles M Nicolet ◽  
Deborah Grove ◽  
Shawn Levy ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Transcriptome Profiling ◽  
Rna Seq ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals Target (MexB)- and Efflux-Based Mechanisms Decreasing the Effectiveness of the Efflux Pump Inhibitor D13-9001 in Pseudomonas aeruginosa PAO1: Uncovering a New Role for MexMN-OprM in Efflux of β-Lactams and a Novel Regulatory Circuit (MmnRS) Controlling MexMN Expression

2018 ◽  
Vol 63 (2) ◽  
pp. e01718-18 ◽  
Author(s):  
Srijan Ranjitkar ◽  
Adriana K. Jones ◽  
Mina Mostafavi ◽  
Zachary Zwirko ◽  
Oleg Iartchouk ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Response Regulator ◽  
Heavy Metal Resistance ◽  
Metal Resistance ◽  
Rna Seq ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
Regulatory Circuit ◽  
Outer Membrane Channel

ABSTRACT Efflux pumps contribute to antibiotic resistance in Gram-negative pathogens. Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13-9001 specifically inhibits MexAB-OprM in Pseudomonas aeruginosa. Mutants with decreased susceptibility to MexAB-OprM inhibition by D13-9001 were identified, and these fell into two categories: those with alterations in the target MexB (F628L and ΔV177) and those with an alteration in a putative sensor kinase of unknown function, PA1438 (L172P). The alterations in MexB were consistent with reported structural studies of the D13-9001 interaction with MexB. The PA1438L172P alteration mediated a >150-fold upregulation of MexMN pump gene expression and a >50-fold upregulation of PA1438 and the neighboring response regulator gene, PA1437. We propose that these be renamed mmnR and mmnS for MexMN regulator and MexMN sensor, respectively. MexMN was shown to partner with the outer membrane channel protein OprM and to pump several β-lactams, monobactams, and tazobactam. Upregulated MexMN functionally replaced MexAB-OprM to efflux these compounds but was insusceptible to inhibition by D13-9001. MmnSL172P also mediated a decrease in susceptibility to imipenem and biapenem that was independent of MexMN-OprM. Expression of oprD, encoding the uptake channel for these compounds, was downregulated, suggesting that this channel is also part of the MmnSR regulon. Transcriptome sequencing (RNA-seq) of cells encoding MmnSL172P revealed, among other things, an interrelationship between the regulation of mexMN and genes involved in heavy metal resistance.

scholarly journals Cloning, Sequencing, and Characterization of the SdeAB Multidrug Efflux Pump of Serratia marcescens

2005 ◽  
Vol 49 (4) ◽  
pp. 1495-1501 ◽  
Author(s):  
Ayush Kumar ◽  
Elizabeth A. Worobec
Keyword(s):  
Serratia Marcescens ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Library ◽  
Efflux Pump ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Diverse Range ◽  
Mutant Strains ◽  
Encoding Genes

ABSTRACT Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.

scholarly journals Overproduction of Inactive Variants of the Murein Synthase PBP1B Causes Lysis in Escherichia coli

2003 ◽  
Vol 185 (18) ◽  
pp. 5342-5348 ◽  
Author(s):  
Ute Meisel ◽  
Joachim-Volker Höltje ◽  
Waldemar Vollmer
Keyword(s):  
Escherichia Coli ◽  
Single Amino Acid ◽  
Penicillin Binding Protein ◽  
Amino Acid Exchange ◽  
Lytic Transglycosylases ◽  
Mutant Strains ◽  
Ph Conditions ◽  
Expression Plasmids ◽  
Acid Exchange

ABSTRACT Penicillin-binding protein 1B (PBP1B) of Escherichia coli is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms (α, β, and γ) which differ in the length of their N-terminal cytoplasmic region. Expression plasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lytic transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a multienzyme complex is discussed.

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