scholarly journals σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

2018 ◽  
Author(s):  
Rémi Hocq ◽  
Maxime Bouilloux-Lafont ◽  
Nicolas Lopes Ferreira ◽  
François Wasels
Keyword(s):  
Point Mutations ◽  
Random Mutagenesis ◽  
Value Added ◽  
Clostridium Beijerinckii ◽  
Phenotypic Traits ◽  
Wild Type ◽  
Genomic Comparison ◽  
Alcohol Synthesis

Microbial production of butanol and isopropanol, two high value-added chemicals, is naturally occurring in the solventogenic Clostridium beijerinckii DSM 6423. Despite its ancient discovery, the precise mechanisms controlling alcohol synthesis in this microorganism are poorly understood. In this work, an allyl alcohol tolerant strain obtained by random mutagenesis was characterized. This strain, designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. Sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which encodes the σ54 sigma factor. Complementation with the wild-type sigL gene fully restored solvent production and sugar assimilation, demonstrating that σ54 plays a central role in regulating these pathways in C. beijerinckii DSM 6423. Genomic comparison with other strains further revealed that these functions are probably conserved among the C. beijerinckii strains. The importance of σ54 in C. beijerinckii was further assessed by the characterization of a sigL deletion mutant of the model strain NCIMB 8052 obtained with a CRISPR/Cas9 tool. The resulting mutant exhibited phenotypic traits similar to the AA strain, and was subsequently complemented with the sigL gene from either the wild type or the AA strains. The results of this experiment confirmed the crucial role of σ54 in the regulation of both solventogenesis and sugar consumption pathways in C. beijerinckii.

scholarly journals Genetic Analysis of Mutant Strains of Saccharomyces cerevisiae with Defects in Mannoprotein Synthesis

Proceedings ◽  
2020 ◽  
Vol 70 (1) ◽  
pp. 18
Author(s):  
Patricia Gil ◽  
Alberto Martínez ◽  
Elena Palencia ◽  
Rocío Velázquez ◽  
Manuel Ramírez ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Point Mutations ◽  
Random Mutagenesis ◽  
Selection Method ◽  
Wild Type ◽  
Truncated Protein ◽  
Mutant Strains

Mannan defective (mnn) mutants have constituted a fundamental tool in the study of the structure and biosynthesis of mannoproteins in Saccharomyces cerevisiae. They were isolated by the group of Dr. C.E. Ballou by random mutagenesis, and a selection method using specific antibodies obtained against the wild-type strain. Initially, the mutants were characterized biochemically, and in subsequent years the genes in which they were mutated were identified. All of them encode membrane proteins that catalyze the transfer of mannoses to N-oligosaccharides, sometimes isolated or as part of complexes made up of several proteins. However, the specific mutation of each of these mutants has only been identified in the case of mnn3. In this work, we have completed the characterization of the mutants by sequencing the mutated genes in each of them. As expected, they are point mutations that involve the change of one amino acid for another in the mutated protein, or for a stop signal, resulting in a truncated protein.

scholarly journals Enhanced production of Clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

2021 ◽  
Author(s):  
Chang-Hun Shin ◽  
Hang Soo Cho ◽  
Hyung-Jin Won ◽  
Ho Jeong Kwon ◽  
Chan-Wha Kim ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Mutant Selection ◽  
Enhanced Production ◽  
Industrial Strains ◽  
Glycerol Utilization

Abstract Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

Characterization of the Role of the FluG Protein in Asexual Development of Aspergillus nidulans

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 1027-1036 ◽  
Author(s):  
Cletus A D'Souza ◽  
Bee Na Lee ◽  
Thomas H Adams
Keyword(s):  
Aspergillus Nidulans ◽  
Negative Influence ◽  
Asexual Development ◽  
Wild Type ◽  
Significant Similarity ◽  
Developmental Defects ◽  
Asexual Sporulation ◽  
Type Strains

Abstract We showed previously that a ΔfluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG (∼400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of ΔfluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from ΔflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.

scholarly journals Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Michael J Prigge ◽  
Matthieu Platre ◽  
Nikita Kadakia ◽  
Yi Zhang ◽  
Kathleen Greenham ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Early Embryo ◽  
Wild Type ◽  
The Family ◽  
Dependent Inhibition ◽  
Root Gravitropism ◽  
Specialized Function ◽  
Mutant Lines

The TIR1/AFB auxin co-receptors mediate diverse responses to the plant hormone auxin. The Arabidopsis genome encodes six TIR1/AFB proteins representing three of the four clades that were established prior to angiosperm radiation. To determine the role of these proteins in plant development we performed an extensive genetic analysis involving the generation and characterization of all possible multiply-mutant lines. We find that loss of all six TIR1/AFB proteins results in early embryo defects and eventually seed abortion, and yet a single wild-type allele of TIR1 or AFB2 is sufficient to support growth throughout development. Our analysis reveals extensive functional overlap between even the most distantly related TIR1/AFB genes except for AFB1. Surprisingly, AFB1 has a specialized function in rapid auxin-dependent inhibition of root growth and early phase of root gravitropism. This activity may be related to a difference in subcellular localization compared to the other members of the family.

scholarly journals Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

2013 ◽  
Vol 57 (11) ◽  
pp. 5658-5664 ◽  
Author(s):  
Soo-Jin Yang ◽  
Nagendra N. Mishra ◽  
Aileen Rubio ◽  
Arnold S. Bayer
Keyword(s):  
Staphylococcus Aureus ◽  
Single Nucleotide Polymorphisms ◽  
Point Mutations ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Single Nucleotide ◽  
Content Type ◽  
Reading Frame ◽  
A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

Effect of temperature on transition and transversion mutagenesis: characterization of wild type and a mutator T4 DNA polymerase mutant

1984 ◽  
Vol 26 (3) ◽  
pp. 386-389 ◽  
Author(s):  
Linda J. Reha-Krantz ◽  
Sükran Parmaksizoglu
Keyword(s):  
Dna Polymerase ◽  
Low Temperatures ◽  
Bacteriophage T4 ◽  
Effect Of Temperature ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Mutational Site

The effect of temperature on genetically well-defined mutational pathways was examined in the bacteriophage T4. The mutational site was a T4 rII ochre mutant which could revert to rII+ via a transversion or to the amber convertant via a transition. Temperature did not strongly affect any of the pathways examined in a wild-type background; however, increased temperature reduced the mutational activity of a mutator DNA polymerase mutant. Possible models to explain the role of temperature in mutagenesis are discussed as well as the significance of low temperatures for in vitro mutagenesis reactions.Key words: bacteriophage T4, mutator, transition, transversion, temperature effects.

scholarly journals Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease

2015 ◽  
Vol 59 (8) ◽  
pp. 4669-4679 ◽  
Author(s):  
Nilmar Silvio Moretti ◽  
Leonardo da Silva Augusto ◽  
Tatiana Mordente Clemente ◽  
Raysa Paes Pinto Antunes ◽  
Nobuko Yoshida ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Drug Targets ◽  
Wild Type ◽  
Content Type ◽  
Damage Repair ◽  
Lysine Deacetylases

ABSTRACTAcetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD+-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation ofTrypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion byT. cruziat concentrations that did not affect host cell viability. In addition,in vivoinfection was partially controlled upon administration of salermide. There are two sirtuins inT. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed inEscherichia coliwith a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 μM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

scholarly journals Identification and Characterization of the Phage Gene sav, Involved in Sensitivity to the Lactococcal Abortive Infection Mechanism AbiV

2009 ◽  
Vol 75 (8) ◽  
pp. 2484-2494 ◽  
Author(s):  
Jakob Haaber ◽  
Geneviève M. Rousseau ◽  
Karin Hammer ◽  
Sylvain Moineau
Keyword(s):  
Gel Filtration ◽  
Point Mutations ◽  
Whole Genome ◽  
Wild Type ◽  
Abortive Infection ◽  
Native Form ◽  
Phage Gene ◽  
Infection Mechanism ◽  
Identification And Characterization

ABSTRACT Lactococcus lactis phage mutants that are insensitive to the recently characterized abortive infection mechanism AbiV were isolated and analyzed in an effort to elucidate factors involved in the sensitivity to AbiV. Whole-genome sequencing of the phage mutants p2.1 and p2.2 revealed mutations in an orf that is transcribed early, indicating that this orf was responsible for AbiV sensitivity. Sequencing of the homologous regions in the genomes of other AbiV-insensitive mutants derived from p2 and six other lactococcal wild-type phages revealed point mutations in the homologous orf sequences. The orf was named sav (for sensitivity to AbiV), and the encoded polypeptide was named SaV. The purification of a His-tagged SaV polypeptide by gel filtration suggested that the polypeptide formed a dimer in its native form. The overexpression of SaV in L. lactis and Escherichia coli led to a rapid toxic effect. Conserved, evolutionarily related regions in SaV polypeptides of different phage groups are likely to be responsible for the AbiV-sensitive phenotype and the toxicity.

scholarly journals Characterization of Biofilm Formation and the Role of BCR1 in Clinical Isolates of Candida parapsilosis

2013 ◽  
Vol 13 (4) ◽  
pp. 438-451 ◽  
Author(s):  
Srisuda Pannanusorn ◽  
Bernardo Ramírez-Zavala ◽  
Heinrich Lünsdorf ◽  
Birgitta Agerberth ◽  
Joachim Morschhäuser ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Candida Parapsilosis ◽  
Clinical Isolates ◽  
Wild Type ◽  
Content Type ◽  
Initial Adhesion ◽  
High Level ◽  
Increased Susceptibility

ABSTRACT In Candida parapsilosis , biofilm formation is considered to be a major virulence factor. Previously, we determined the ability of 33 clinical isolates causing bloodstream infection to form biofilms and identified three distinct groups of biofilm-forming strains (negative, low, and high). Here, we establish two different biofilm structures among strains forming large amounts of biofilm in which strains with complex spider-like structures formed robust biofilms on different surface materials with increased resistance to fluconazole. Surprisingly, the transcription factor Bcr1, required for biofilm formation in Candida albicans and C. parapsilosis , has an essential role only in strains with low capacity for biofilm formation. Although BCR1 leads to the formation of more and longer pseudohyphae, it was not required for initial adhesion and formation of mature biofilms in strains with a high level of biofilm formation. Furthermore, an additional phenotype affected by BCR1 was the switch in colony morphology from rough to crepe, but only in strains forming high levels of biofilm. All bcr1 Δ/Δ mutants showed increased proteolytic activity and increased susceptibility to the antimicrobial peptides protamine and RP-1 compared to corresponding wild-type and complemented strains. Taken together, our results demonstrate that biofilm formation in clinical isolates of C. parapsilosis is both dependent and independent of BCR1 , but even in strains which showed a BCR1 -independent biofilm phenotype, BCR1 has alternative physiological functions.

scholarly journals Characterization of the Opp Peptide Transporter ofCorynebacterium pseudotuberculosisand Its Role in Virulence and Pathogenicity

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Pablo M. R. O. Moraes ◽  
Nubia Seyffert ◽  
Wanderson M. Silva ◽  
Thiago L. P. Castro ◽  
Renata F. Silva ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Etiologic Agent ◽  
Peptide Transporter ◽  
Intercellular Signaling ◽  
Wild Type ◽  
Cell Nutrition ◽  
Oligopeptide Permease ◽  
Extracellular Environment

Despite the economic importance of caseous lymphadenitis (CLA), a chronic disease caused byCorynebacterium pseudotuberculosis, few genes related to the virulence of its etiologic agent have been characterized. The oligopeptide permease (Opp) transporters are located in the plasma membrane and have functions generally related to the uptake of peptides from the extracellular environment. These peptide transporters, in addition to having an important role in cell nutrition, also participate in the regulation of various processes involving intercellular signaling, including the control of the expression of virulence genes in pathogenic bacteria. To study the role of Opp inC. pseudotuberculosis, an OppD deficient strain was constructed via simple crossover with a nonreplicative plasmid carrying part of theoppDgene sequence. As occurred to the wild-type, the ΔoppDstrain showed impaired growth when exposed to the toxic glutathione peptide (GSH), indicating two possible scenarios: (i) that this component can be internalized by the bacterium through an Opp-independent pathway or (ii) that there is toxicity while the peptide is extracellular. Additionally, the ΔoppDmutant presented a reduced ability to adhere to and infect macrophages compared to the wild-type, although both strains exhibit the same potential to colonize spleens and cause injury and death to infected mice.

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