Second-site Suppressor Mutations for the Serine 202 to Phenylalanine Substitution within the Interdomain Loop of the Tetracycline Efflux Protein Tet(C)

Sapunaric, Frédéric; Levy, Stuart B.

doi:10.1074/jbc.m302658200

Cited by 8 publications

(6 citation statements)

References 39 publications

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“…Hydrophilic residues within the interdomain loop of LacY are required to permit a temporal delay for the insertion of both domains. In TetA(B), the cytoplasmic interdomain loop is more than a linker between the a and b domains since mutations in it affect resistance (Sapunaric & Levy, 2003;Saraceni-Richards & Levy, 2000a). We have shown here that this loop also possesses at least three residues that appear to be involved in interacting with the tetracycline/ Mg 2+ substrate.…”

Section: Resultsmentioning

confidence: 89%

“…Since the inactivating Ser201Cys substitution of class B is at a position similar to that of the inactivating Ser202Phe mutation of class C (Saraceni-Richards & Levy, 2000a), we examined whether the former would be suppressed by Leu9Phe, which corresponds to the Leu11Phe in TM1 which suppresses the class C mutation (Sapunaric & Levy, 2003). The Leu9Phe replacement indeed suppressed the Ser201Cys mutation, restoring a nearly wild-type level of tetracycline resistance; the Leu9Phe replacement by itself had little effect on TetA(B) function (Table 2).…”

Section: Resultsmentioning

confidence: 99%

“…The insertion of five residues (between Arg190 and Pro191) which lengthened the TetA(C) interdomain loop increased tetracycline susceptibility (Jewell et al, 1999). In TetA(C), the interdomain mutation Ser202Phe led to a 12-fold decrease in activity (Saraceni-Richards & Levy, 2000a); second-site suppressor mutations which restored resistance were found in TM1, in the interdomain loop and in the cytoplasmic loop connecting TM8 and TM9 (Sapunaric & Levy, 2003). To examine further the role of the interdomain loop of TetA(B) in substrate specificity, we performed site-directed mutagenesis at Asp190, Glu192 and Ser201, and assayed resistance to tetracycline and tetracycline analogues.…”

Section: Introductionmentioning

confidence: 99%

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Substitutions in the interdomain loop of the Tn10 TetA efflux transporter alter tetracycline resistance and substrate specificity

Sapunaric

Levy

2005

Microbiology

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Cysteine replacement of Asp190, Glu192 and Ser201 residues in the cytoplasmic interdomain loop of the TetA(B) tetracycline efflux antiporter from Tn10 reduces tetracycline resistance [Tamura, N., Konishi, S., Iwaki, S., Kimura-Someya, T., Nada, S. & Yamaguchi, A. (2001). J Biol Chem 276, 20330–20339]. It was found that these Cys substitutions altered the substrate specificity of TetA(B), increasing the relative resistance to doxycycline and minocycline over that to tetracycline by three- to sixfold. Substitutions of Asp190 and Glu192 by Ala, Asn and Gln also impaired the ability of TetA(B) to mediate tetracycline resistance while Ser201Ala and Ser201Thr substitutions did not. A Leu9Phe substitution in the first transmembrane helix of TetA(B) suppressed the Ser201Cys mutation, undoing the alterations in resistance and specificity. That the interdomain loop might contact substrate during transport, as is suggested from its role in substrate specificity, is unexpected considering that the primary sequence in the loop is not conserved among a group of otherwise homologous TetA proteins. However, in the interdomain loop of 11 of 14 homologous TetA efflux proteins, computational analysis revealed a short α-helix, which includes some residues affecting activity and substrate specificity. Perhaps this conserved secondary structure accounts for the role of the non-conserved interdomain loop in TetA function.

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Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Substitutions in the interdomain loop of the Tn10 TetA efflux transporter alter tetracycline resistance and substrate specificity

Sapunaric

Levy

2005

Microbiology

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“…However, for the large majority of compensatory effects detected, the mechanistic explanation remains unclear. In addition to intrinsically advantageous second-site mutations acting merely in an additive way with the disadvantageous primary mutation, intrinsically near-neutral compensatory mutations have been observed in some cases (46,48). Mutually compensatory effects of individually disadvantageous mutations leading to a neutral phenotype, or even providing a selective advantage relative to the situation where no mutation is present, may also occur in proteins (3,25,36).…”

mentioning

confidence: 99%

Deterministic, Compensatory Mutational Events in the Capsid of Foot-and-Mouth Disease Virus in Response to the Introduction of Mutations Found in Viruses from Persistent Infections

Mateo

Mateu

2007

J Virol

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The evolution of foot-and-mouth disease virus (FMDV) (biological clone C-S8c1) in persistently infected cells led to the emergence of a variant (R100) that displayed increased virulence, reduced stability, and other modified phenotypic traits. Some mutations fixed in the R100 genome involved a cluster of highly conserved residues around the capsid pores that participate in interactions with each other and/or between capsid protomers. We have investigated phenotypic and genotypic changes that occurred when these replacements were introduced into the C-S8c1 capsid. The C3007V and M3014L mutations exerted no effect on plaque size or viral yield during lytic infections, or on virion stability, but led to a reduction in biological fitness; the D3009A mutation caused drastic reductions in plaque size and viability. Remarkably, competition of the C3007V mutant with the nonmutated virus invariably resulted in the fixation of the D3009A mutation in the C3007V capsid. In turn, the presence of the D3009A mutation invariably led to the fixation of the M3014L mutation. In both cases, two individually disadvantageous mutations led, together, to an increase in fitness, as the double mutants outcompeted the nonmutated genotype. The higher fitness of C3007V/D3009A was related to a faster multiplication rate. These observations provide evidence for a chain of linked, compensatory mutational events in a defined region of the FMDV capsid. Furthermore, they indicate that the clustering of unique amino acid replacements in viruses from persistent infections may also occur in cytolytic infections in response to changes caused by previous mutations without an involvement of the new mutations in the adaptation to a different environment.

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“…As a starting point for their studies, Sapunaric et al [67] used a Phe substitution for Ser 202 (S202F) in the large cytoplasmic loop, the so-called interdomain loop, between TM helices 6 and 7 of the Tet(C) protein. Similar to earlier studies, point mutations in this cytoplasmic loop resulted in a significant reduction in pump activity [48].…”

Section: Effluxmentioning

confidence: 99%

Genetic Determinants of Tetracycline Resistance and their Effect on Tetracycline and Glycylcycline Antibiotics

Ca¹,

Murphy²,

Bradford³

2008

AIAMC

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Tetracycline class antibiotics have been a hallmark in terms of safety profile and spectrum of activity for nearly 60 years. Multiple resistance mechanisms have evolved to counter the tetracyclines with both of the chief mechanismsefflux and ribosomal protection -widely spread in Gram-positive and Gram-negative organisms. The utility of the tetracyclines as growth promoters in domesticated food animals as well as for pest control in aquaculture and horticulture has resulted in the dissemination of tetracycline resistance determinants in the environment, which may act as a reservoir for resistance genes. Focused chemistry efforts to develop tetracycline derivatives not subject to the efflux and ribosomal protection mechanism resulted in the discovery of tigecycline, the first in class glycylcycline antibiotic. Tigecycline has successfully reclaimed the spectrum, safety and efficacy of the tetracyclines.

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Second-site Suppressor Mutations for the Serine 202 to Phenylalanine Substitution within the Interdomain Loop of the Tetracycline Efflux Protein Tet(C)

Cited by 8 publications

References 39 publications

Substitutions in the interdomain loop of the Tn10 TetA efflux transporter alter tetracycline resistance and substrate specificity

Substitutions in the interdomain loop of the Tn10 TetA efflux transporter alter tetracycline resistance and substrate specificity

Deterministic, Compensatory Mutational Events in the Capsid of Foot-and-Mouth Disease Virus in Response to the Introduction of Mutations Found in Viruses from Persistent Infections

Genetic Determinants of Tetracycline Resistance and their Effect on Tetracycline and Glycylcycline Antibiotics

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