ABC transporter content diversity in
            <i>Streptococcus pneumoniae</i>
            impacts competence regulation and bacteriocin production

Wang, Charles Y.; Patel, Nisha; Wholey, Wei-Yun; Dawid, Suzanne

doi:10.1073/pnas.1804668115

Cited by 31 publications

(63 citation statements)

References 61 publications

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“…Monitoring protein secretion through the plasma membrane using NanoBiT has already been described in mammalian cells 33 and in Gram-positive bacteria 34 . However, employing the same approach to Gram-negative bacteria is considerably challenging because the outer membrane in their cell envelope provides a low permeability barrier.…”

Section: Discussionmentioning

confidence: 99%

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation Across Biological Membranes

Pereira

Allen

Watkins

et al. 2018

Preprint

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Abbreviations:AA -antimycin A; IMM -inner mitochondrial membrane; mt-11S -mitochondrial-targeted 11S; H6 11S -Nterminal 6xHis tagged version of 11S; OMM -outer mitochondrial membrane; OVA -oligomycin, valinomycin and antimycin A (full depolarisation cocktail); MTS -mitochondrial targeting sequence, NanoBiT -NanoLuc Binary Technology; PAM -presequence-translocase-associated import-motor; pep86high-affinity version of NanoBiT small fragment; pep114 -small fragment of NanoBiT; PMF -proton motive force; TIM -translocase of the inner membrane; TOM -translocase of the outer membrane; 11Slarge fragment of the NanoBiT; Δ Ψ -membrane potential. 2 ABSTRACTProtein translocation is a fundamental process in biology. Major gaps in our understanding of this process arises due the poor sensitivity, low time-resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes, and allows continuous acquisition with a time-resolution in the order of seconds -yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system, and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigour unattainable through classical methods.

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Section: Discussionmentioning

confidence: 99%

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation Across Biological Membranes

Pereira

Allen

Watkins

et al. 2018

Preprint

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“…To elucidate whether production and export of CSP are affected in chaining cells, we employed the HiBiT tag detection system (Aggarwal et al, 2018;Wang et al, 2018). The HiBiT tag was placed under the control of the comCDE promoter, either with (strain ADP308) or without (strain ADP312) the leader peptide sequence of comC.…”

Section: Cell Chaining Reduces the Shared Csp Poolmentioning

confidence: 99%

“…Combined, these results strongly suggest that comC transcription and ComAB activity is not affected by cell chaining and CSP is exported at similar rates in chains of cells. (Wang et al, 2018). Right, HiBiT lacks the ComC leader peptide and accumulates in the cytoplasm since it cannot be recognized and exported by ComAB.…”

Section: Cell Chaining Reduces the Shared Csp Poolmentioning

confidence: 99%

Antibiotic-induced cell chaining triggers pneumococcal competence by reshaping quorum sensing to autocrine signaling

Domenech

Slager

Veening

2018

Preprint

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Streptococcus pneumoniae can acquire antibiotic resistance by activation of competence and subsequent DNA uptake. Here, we demonstrate that aztreonam (ATM) and clavulanic acid (CLA) promote competence. We show that both compounds induce cell chain formation by targeting the D,D-carboxypeptidase PBP3. In support of the hypothesis that chain formation promotes competence, we demonstrate that an autolysin mutant (ΔlytB) is hypercompetent. Since competence is initiated by the binding of a small extracellular peptide (CSP) to a membrane-anchored receptor (ComD), we wondered if chain formation alters CSP diffusion kinetics. Indeed, the presence of ATM or CLA affects competence synchronization by shifting from global to local quorum sensing, as CSP is primarily retained to chained cells, rather than shared in a common pool. Importantly, autocrine-like signaling prolongs the time-window in which the population is able to transform. Together, these insights demonstrate the versatility of quorum sensing and highlight the importance of an accurate antibiotic prescription.

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“…In the gram-positive opportunistic pathogen Streptococcus pneumoniae (pneumococcus), the PCATs ComAB and BlpAB secrete quorum-sensing pheromones that control two important cellular pathways: genetic competence (the ability to take up and incorporate extracellular DNA into the genome) and production of the major family of pneumococcal bacteriocins (Blp bacteriocins, or pneumocins) (6, 7, 21, 22). ComAB and BlpAB secrete the same GG peptides, including the competence- and pneumocin-inducing pheromones and the pneumocins (23–25). Substrate sharing between ComAB and BlpAB affects competence and pneumocin regulation and influences when and with what effectiveness naturally occurring BlpAB + and BlpAB − strains can employ pneumocin-mediated killing (25, 26).…”

Section: Introductionmentioning

confidence: 99%

“…ComAB and BlpAB secrete the same GG peptides, including the competence- and pneumocin-inducing pheromones and the pneumocins (23–25). Substrate sharing between ComAB and BlpAB affects competence and pneumocin regulation and influences when and with what effectiveness naturally occurring BlpAB + and BlpAB − strains can employ pneumocin-mediated killing (25, 26).…”

Section: Introductionmentioning

confidence: 99%

Molecular Determinants of Substrate Selectivity of a Pneumococcal Rgg-regulated Peptidase-Containing ABC Transporter

Wang¹,

Medlin

Nguyen

et al. 2019

Preprint

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word count: (abstract/importance: 235/145) 14 Text word count: 4911 15 32 Abstract 33 34 Peptidase-containing ABC transporters (PCATs) are a widely distributed family of 35 transporters which secrete double-glycine (GG) peptides. In the opportunistic pathogen 36 Streptococcus pneumoniae (pneumococcus), the PCATs ComAB and BlpAB have been 37 shown to secrete quorum-sensing pheromones and bacteriocins related to the 38 competence and pneumocin pathways. Here, we describe another pneumococcal 39 PCAT, RtgAB, encoded by the rtg locus and found intact in 17% of strains. The 40 Rgg/SHP-like quorum sensing system RtgR/S, which uses a peptide pheromone with a 41 distinctive Trp-X-Trp motif, regulates expression of the rtg locus and provides a 42 competitive fitness advantage in a mouse model of nasopharyngeal colonization. RtgAB 43 secretes a set of co-regulated rtg GG peptides. ComAB and BlpAB, which share a 44 substrate pool with each other, do not secrete the rtg GG peptides. Similarly, RtgAB 45 does not efficiently secrete ComAB/BlpAB substrates. We examined the molecular 46 determinants of substrate selectivity between ComAB, BlpAB, and RtgAB and found 47 that the GG peptide signal sequences contain all the information necessary to direct 48 secretion through specific transporters. Secretion through ComAB and BlpAB depends 49 largely on the identity of four conserved hydrophobic signal sequence residues 50 previously implicated in substrate recognition by PCATs. In contrast, a motif situated at 51 the N-terminal end of the signal sequence, found only in rtg GG peptides, directs 52 secretion through RtgAB. These findings illustrate the complexity in predicting 53 substrate-PCAT pairings by demonstrating specificity that is not dictated solely by signal 54 sequence residues previously implicated in substrate recognition. 55 56 Importance 57 58The export of peptides from the cell is a fundamental process carried out by all 59 bacteria. One method of bacterial peptide export relies on a family of transporters called 60 peptidase-containing ABC transporters (PCATs). PCATs export so-called GG peptides 61 which carry out diverse functions, including cell-to-cell communication and inter-62 bacterial competition. In this work, we describe a PCAT-encoding genetic locus, rtg, in 63 the pathogen Streptococcus pneumoniae (pneumococcus). The rtg locus is linked to 64 increased competitive fitness advantage in a mouse model of nasopharyngeal 65 colonization. We also describe how the rtg PCAT preferentially secretes a set of co-66 regulated GG peptides but not GG peptides secreted by other pneumococcal PCATs. 67These findings illuminate a relatively understudied part of PCAT biology: how these 68 transporters discriminate between different subsets of GG peptides.

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ABC transporter content diversity in Streptococcus pneumoniae impacts competence regulation and bacteriocin production

Cited by 31 publications

References 61 publications

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation Across Biological Membranes

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation Across Biological Membranes

Antibiotic-induced cell chaining triggers pneumococcal competence by reshaping quorum sensing to autocrine signaling

Molecular Determinants of Substrate Selectivity of a Pneumococcal Rgg-regulated Peptidase-Containing ABC Transporter

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