Dataset Information

Mutation network-based understanding of pleiotropic and epistatic mutational behavior of Enterococcus faecalis FMN-dependent azoreductase.

ABSTRACT: We previously identified a highly active homodimeric FMN-dependent NADH-preferred azoreductase (AzoA) from Enterococcus faecalis, which cleaves the azo bonds (R-N?N-R) of diverse azo dyes, and determined its crystal structure. The preliminary network-based mutational analysis suggested that the two residues, Arg-21 and Asn-121, have an apparent mutational potential for fine-tuning of AzoA, based on their beneficial pleiotropic feedbacks. However, epistasis between the two promising mutational spots in AzoA has not been obtained in terms of substrate binding and azoreductase activity. In this study, we further quantified, visualized, and described the pleiotropic and/or epistatic behavior of six single or double mutations at the positions, Arg-21 and Asn-121, as a further research endeavor for beneficial fine-tuning of AzoA. Based on this network-based mutational analysis, we showed that pleiotropy and epistasis are common, sensitive, and complex mutational behaviors, depending mainly on the structural and functional responsibility and the physicochemical properties of the residue(s) in AzoA.

SUBMITTER: Sun J

PROVIDER: S-EPMC5704035 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Mutation network-based understanding of pleiotropic and epistatic mutational behavior of <i>Enterococcus faecalis</i> FMN-dependent azoreductase.

Sun Jinyan J Kweon Ohgew O Jin Jinshan J He Gui-Xin GX Li Xiyu X Cerniglia Carl E CE Chen Huizhong H

Biochemistry and biophysics reports 20171106

We previously identified a highly active homodimeric FMN-dependent NADH-preferred azoreductase (AzoA) from <i>Enterococcus faecalis</i>, which cleaves the azo bonds (R-N˭N-R) of diverse azo dyes, and determined its crystal structure. The preliminary network-based mutational analysis suggested that the two residues, Arg-21 and Asn-121, have an apparent mutational potential for fine-tuning of AzoA, based on their beneficial pleiotropic feedbacks. However, epistasis between the two promising mutati ...[more]

PMID: 29214224

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Project description:Enterococci are Gram-positive bacteria present in the healthy human microbiota, but they are also a leading cause of nosocomial infections. Maltodextrin utilization by Enterococcus faecalis has been identified as an important factor for colonization of mammalians hosts. Here, we show that the LacI/GalR transcriptional regulator MalR, the maltose gene regulator, is also the main regulator of the operons encoding an ABC transporter (mdxEFG) and three metabolic enzymes (mmdH-gmdH-mmgT) required for the uptake and catabolism of maltotetraose and longer maltodextrins. The utilization of maltose and maltodextrins is consequently coordinated and induced by the disaccharide maltose, which binds to MalR. Carbon catabolite repression of the mdxEFG and mmdH-gmdH-mmgT operons is mediated by both P-Ser-HPr/MalR and P-Ser-HPr/CcpA. The latter complex exerts only moderate catabolite repression, which became visible when comparing maltodextrin operon expression levels of a malR- mutant (with a mutant allele for the malR gene) and a malR- ΔccpA double mutant grown in the presence of maltose, which is transported via a phosphotransferase system and, thus, favors the formation of P-Ser-HPr. Moreover, maltodextrin transport via MdxEFG slows rapidly when glucose is added, suggesting an additional regulation via inducer exclusion. This complex regulation of metabolic operons likely allows E. faecalis to fine-tune gene expression in response to changing environmental conditions.IMPORTANCEEnterococcus faecalis represents a leading cause of hospital-acquired infections worldwide. Several studies highlighted the importance of carbohydrate metabolism in the infection process of this bacterium. The genes required for maltodextrin metabolism are particularly induced during mouse infection and, therefore, should play an important role for pathogenesis. Since no data were hitherto available concerning the regulation of expression of the maltodextrin operons, we have conducted experiments to study the underlying mechanisms.

Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Dataset Information

Mutation network-based understanding of pleiotropic and epistatic mutational behavior of Enterococcus faecalis FMN-dependent azoreductase.

Publications

Mutation network-based understanding of pleiotropic and epistatic mutational behavior of <i>Enterococcus faecalis</i> FMN-dependent azoreductase.

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