Signal peptides for recombinant protein secretion in bacterial expression systems

Freudl, Roland

doi:10.1186/s12934-018-0901-3

Cited by 200 publications

(184 citation statements)

References 81 publications

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“…7B, lane 4). The expression of the C-terminal truncation mutant, dsbA(ss)-ExbD(44–58)-His6X was undetectable (data not shown). Because dsbA(ss)-ExbD(49-63) was expressed similarly to the parent peptide but was significantly less inhibitory, it suggested that the first five residues of the dsbA(ss)-ExbD(44-63) peptide were important to inhibit TonB system activity.…”

Section: Resultsmentioning

confidence: 94%

An ExbD Disordered Domain Peptide Inhibits TonB System Activity

Postle

2020

Preprint

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The TonB system energizes transport of essential nutrients, such as iron siderophores, across unenergized outer membranes of Gram-negative bacteria. The integral cytoplasmic membrane proteins of the TonB system--ExbB, ExbD, and TonB--transduce the protonmotive force of the cytoplasmic membrane to TonB-dependent outer membrane transporters for active transport. ExbD protein is anchored in the cytoplasmic membrane, with the majority of it occupying the periplasm. We previously identified a conserved motif within a periplasmic disordered domain that is essential for TonB system function. Here we demonstrated that export of a peptide derived from that motif into the periplasm prevented TonB system function and inhibited all known ExbD interactions in vivo. Formaldehyde crosslinking captured the ExbD peptide in multiple ExbD and TonB complexes. Furthermore, peptides with mutations in the conserved motif not only had significantly reduced ability to inhibit TonB system activity, but they also altered interactions with ExbD and TonB, indicating the specificity of the interaction. Conserved motif peptide interactions with ExbD and TonB mostly occurred between Stage II and Stage III of the TonB energy transduction cycle, a transition that is characterized by the use of protonmotive force. Taken together, the data suggest that the ExbD disordered domain motif has multiple interactions with TonB and ExbD during between Stage II and III of the TonB energization cycle. Because of the essentiality of the motif, it may be a potential template for design of novel antibiotics that target the TonB system.IMPORTANCEGram-negative bacteria are intrinsically antibiotic-resistant due to the diffusion barrier posed by their outer membranes. The TonB system allows them to circumvent this barrier for their own nutritional needs, including iron. The ability of bacteria to acquire iron is a virulence factor for many Gram-negative pathogens. However, no antibiotics currently target the TonB system. Because TonB and ExbD must interact productively in the periplasm for transport across the outer membrane, they constitute attractive targets for potential antibiotic development where chemical characteristics need not accommodate the need to cross the hydrophobic cytoplasmic membrane. Here we show that a small ExbD-derived peptide can interfere with the TonB-ExbD interaction to inhibit the TonB system in vivo.

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Section: Resultsmentioning

confidence: 94%

An ExbD Disordered Domain Peptide Inhibits TonB System Activity

Postle

2020

Preprint

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“…Another important finding here is the observation that translocation is one of the mechanisms for gene compatibility. To date, a general trend in the MBL signal peptide sequence has been identified for each translocation system (e.g., the Sec protein translocation pathway) (46)(47)(48)(49)(50), however, we have little understanding of the relationship between the signal peptide sequence and its translocation efficacy, let alone the incompatibility between the translocation system of a particular organism and the signal peptide sequence of a particular gene. Indeed, the signal peptide is the most variable region in the MBL sequence.…”

Section: Discussionmentioning

confidence: 99%

The molecular mechanisms underlying hidden phenotypic variation among metallo-ß-lactamases

Tokuriki

Socha

2018

Preprint

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Genetic variation among orthologous genes has been largely formed through neutral genetic drift to maintain the same functional role. In some circumstances, however, this genetic variation can create critical phenotypic variation, particularly when genes are transferred to a new host by horizontal gene transfer (HGT). Unveiling "hidden phenotypic variation" through HGT is especially important for genes that confer resistance to antibiotics, which continue to disseminate to new organisms through HGT.Despite this biomedical importance, our understanding of the molecular mechanisms that underlie hidden phenotypic variation remains limited. Here we sought to determine the extent of hidden phenotypic variation in the B1 metallo-β-lactamase (MBL) family, as well as to determine its molecular basis by systematically characterizing eight MBL orthologs when they are expressed in three different organisms (E. coli, P. aeruginosa, and K. pneumoniae). We found that these MBLs confer diverse levels of resistance in each organism, which cannot be explained by variation in catalytic efficiency alone; rather, it is the combination of the catalytic efficiency and abundance of functional periplasmic enzyme that best predicts the observed variation in resistance. The level of functional periplasmic expression varied dramatically between MBL orthologs and between hosts.This was the result changes at multiple levels of each enzyme's functional: 1) the quantity of mRNA; 2) the amount of MBL expressed; and 3) the efficacy of functional enzyme translocation to the periplasm. Overall, we see that it is the interaction between each gene and the host's underlying cellular processes (transcription, translation, and translocation) that determines MBL genetic incompatibility thorough HGT. These hostspecific processes may constrain the effective spread and deployment of MBLs to certain host species, and could explain the current observed distribution bias. Author SummaryOrthologous genes spread among different organisms, typically maintaining the same functional role within the cell while accumulating some, presumably functionally-inert, genetic variation over time. However, these seemingly neutral gene sequence changes among orthologs can be revealed to have substantial difference in protein phenotypes, and thus, organismal fitness, when they are transferred to other host species. This socalled "hidden phenotypic variation" through horizontal gene transfer may play an important role in dissemination of antibiotic resistance genes, in particular. In this work, we systematically investigated the extent of phenotypic variation in eight orthologous antibiotic resistant genes from the metallo-β-lactamases family (MBLs), and identified the molecular causes underlying the observed phenotypic variation. We found that functional protein expression varied substantially among MBLs (causing significant variation in the level of antibiotic resistance conferred), and that this could not be explained by variation in catalytic efficiency alone. Instead, we s...

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“…The first 26 residues of VIM-2 has been identified as the signal peptide 14,15,52 , which is a sequence used to translocate the enzyme to the periplasm, then cleaved after transport [53][54][55][56] . Our DMS data supports the known length of the signal peptide as mutations to Cys are much less deleterious before residue 26, suggesting these positions are not incorporated into the mature enzyme in the periplasm.…”

Section: Codon and Amino Acid Optimization In The Signal Peptidementioning

confidence: 99%

“…with 1 or more positive residues, a hydrophobic (H) region (residues 8-21) and a carboxy terminal (C) region (residues 22-26) that precedes the cleavage site containing a PXAXS motif ( Fig. 5a) [53][54][55][56] . The signal peptide is conserved at 17 of 25 positions across all VIM variants, and the remaining are binary differences between the conserved sequences of the VIM-1 and VIM-2 clades ( Fig.…”

Section: Codon and Amino Acid Optimization In The Signal Peptidementioning

confidence: 99%

Comprehensive exploration of the translocation, stability and substrate recognition requirements in VIM-2 lactamase

Chen

Fowler

Tokuriki

2020

Preprint

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Metallo-β-lactamases (MBLs) degrade a broad spectrum of β-lactam antibiotics, and are a major disseminating source for multidrug resistant bacteria. Despite many biochemical studies in diverse MBLs, molecular understanding of the roles of residues in the enzyme's stability and function, and especially substrate specificity, is lacking. Here, we employ deep mutational scanning (DMS) to generate comprehensive single amino acid variant data on a major clinical MBL, VIM-2, by measuring the effect of thousands of VIM-2 mutants on the degradation of three representative classes of β-lactams (ampicillin, cefotaxime, and meropenem) and at two different temperatures (25°C and 37°C). We revealed residues responsible for expression and translocation, and mutations that increase resistance and/or alter substrate specificity. The distribution of specificityaltering mutations unveiled distinct molecular recognition of the three substrates. Moreover, these function-altering mutations are frequently observed among naturally occurring variants, suggesting that the enzymes has continuously evolved to become more potent resistance genes.

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Signal peptides for recombinant protein secretion in bacterial expression systems

Cited by 200 publications

References 81 publications

An ExbD Disordered Domain Peptide Inhibits TonB System Activity

An ExbD Disordered Domain Peptide Inhibits TonB System Activity

The molecular mechanisms underlying hidden phenotypic variation among metallo-ß-lactamases

Comprehensive exploration of the translocation, stability and substrate recognition requirements in VIM-2 lactamase

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