Xenogeneic modulation of the ClpCP protease of Bacillus subtilis by a phage-encoded adaptor-like protein

Mulvenna, Nancy; Hantke, Ingo; Burchell, Lynn; Nicod, Sophie; Bell, David; Turgay, Kürşad; Wigneshweraraj, Sivaramesh

doi:10.1074/jbc.ra119.010007

Cited by 11 publications

(12 citation statements)

References 37 publications

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“…How ReoM and ReoY exert their effect on ClpCP is currently unknown, but a fascinating possibility would be a function similar to that of an adaptor protein to target a subset of protein substrates, including MurA, to the ClpCP complex for degradation. Several ClpC adaptors are known in B. subtilis (52, 53), but an adaptor for Bs MurAA is not among them (26, 52). Interestingly, the ClpC adaptor protein McsB from B. subtilis is also subject to phosphorylation like ReoM but, unlike ReoM, it targets its substrate CtsR to the ClpCP machinery only when phosphorylated (54).…”

Section: Discussionmentioning

confidence: 99%

PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM

Wamp

Rutter

Rismondo

et al. 2019

Preprint

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30Peptidoglycan (PG) is the main component of bacterial cell walls and the target for many 31 antibiotics. PG biosynthesis is tightly coordinated with cell wall growth and turnover and many 32 of these control activities depend upon PASTA-domain containing eukaryotic-like 33 serine/threonine protein kinases (PASTA-eSTK) that sense PG fragments. However, only a few 34 PG biosynthetic enzymes are actually direct kinase substrates. Here, we identify the conserved 35 ReoM protein as a novel PASTA-eSTK substrate in the Gram-positive pathogen Listeria 36 monocytogenes. Our data show that the phosphorylation of ReoM is essential as it controls 37 ClpCP-dependent proteolytic degradation of the essential enzyme MurA, which catalyses the first 38 committed step in PG biosynthesis. We also identify ReoY as a second novel factor required for 39 degradation of ClpCP substrates. Collectively, our data imply that the first committed step of PG 40 biosynthesis is activated through control of ClpCP protease activity in response to signals of PG 41 homeostasis imbalance.42 43 44 45The cell wall of Gram-positive bacteria is a complicated three-dimensional structure that engulfs 46 the cell as a closed sacculus. The main component of bacterial cell walls is peptidoglycan (PG), a 47 network of glycan strands crosslinked together by short peptides (1). PG biosynthesis starts with 48 the conversion of UDP-GlcNAc into lipid II, a disaccharide pentapeptide that is ligated to a 49 100 positive bacteria, which explains how PG biosynthesis is adjusted in these bacteria to meet PG 101 production and repair needs. 102 103 6 RESULTS 104 105Novel gpsB suppressor mutations in the lmo1503 (reoM) and lmo1921 (reoY) genes 106 A L. monocytogenes ΔgpsB mutant is unable to replicate at 42°C but forms suppressors 107 correcting this defect with high frequency (28). In a preliminary selection of gpsB suppressors, 108 the clpC and murZ genes, important for the stability of the UDP-N-acetylglucosamine 1-109 carboxyvinyltransferase MurA, were found to be mutated (28). We have characterized three more 110 shg (suppression of heat sensitive growth) suppressor mutants (shg8, shg10 and shg12) isolated 111 from a ΔgpsB mutant incubated on a BHI agar plate at 42°C. These three shg strains grew as fast 112 as the wild type when cultivated at 37°C or 42°C, whereas the parental ΔgpsB mutant grew at a 113 reduced rate at 37°C and did not grow at 42°C (Fig. 1A-B), as shown previously (32). 114Illumina sequencing of the shg8, shg10 and shg12 genomes identified one SNP in each strain that 115 was not found in the parental ΔgpsB strain. Strain shg8 carried a mutation in the uncharacterized 116 lmo1921 gene (herein named reoY, see below) that exchanged H87 into tyrosine; the same gene 117 was affected by the introduction of a premature stop codon after the 73 rd codon of reoY to yield 118 strain shg10. Strain shg12 carried a mutation in the ribosomal binding site (RBS) of the lmo1503 119 gene (renamed reoM), encoding another protein of unknown function and conta...

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Section: Discussionmentioning

confidence: 99%

PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM

Wamp

Rutter

Rismondo

et al. 2019

Preprint

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“…E. coli was grown on LB medium (1% tryptone, 1% NaCl, 0.5 g/l yeast extract). Pseudomonads were grown either on M9 mineral salt medium (Miller 1972), supplemented with 0.4% glucose as carbon source and 100 μM FeCl 3 to avoid iron limitation (this medium is referred to as M9 medium throughout this study), or on complex media. King's B medium was used for P. putida (2% peptone, 0.15% K 2 HPO 4 , 0.15% MgSO 4 × 7H 2 O, 1% glycerol), and LB medium was used for P. fluorescens or P. aeruginosa.…”

Section: Methodsmentioning

confidence: 99%

“…The kan R resistance cassette from pK18mobSacB (Schäfer et al 1994 ) was amplified using primers KanR-SspI-SD-F (5′-GCA TAA TAT TAC AGG ATG AGG ATC GTT TCG C-3′) and KanR-BsaI-R (5′-GCT AAC CGC GAG ACC TCA GAA GAA CTC GTC AAG AAG-3′), and the amp R resistance cassette of pUCP20-ANT1 was substituted by this kan R resistance cassette using SspI/BsaI for cloning, resulting in pUCP20-ANT2. For promoter reporter studies, gfp was PCR-amplified from pHT01- gfp (Mulvenna et al 2019 ) using SD-GFP-SpeI-F (5′-GCA GAC TAG TAA AGG AGG AAG GAT CCA TGA G-3′) and GFP-SbfI-R (5′-GCA GCC TGC AGG TTA TTT GTA TAG TTC ATC CAT GCC-3′); the fragment was digested with SpeI/SbfI, and cloned into the corresponding sites of pUCP20-ANT2 to generate pUCP20-ANT2- gfp . The multiple cloning site was amplified from pUCP20 using primers MCS-F (5′-GCC GAC TAG TAG GAG ATA TAC ATA TGG AAT TCG AGC TCG GTA CCC GGG GAT CCT C-3′) and MCS-R (5′-TGC CAA GCT TGC ATG CCT GC-3′); the PCR-fragment was digested with SpeI/SbfI, and cloned into the corresponding sites of pUCP20-ANT2 to obtain pUCP20-ANT2-MCS.…”

Section: Methodsmentioning

confidence: 99%

A tunable anthranilate-inducible gene expression system for Pseudomonas species

Hoffmann

Sugue

Brüser

2020

Appl Microbiol Biotechnol

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Pseudomonads are among the most common bacteria in soils, limnic ecosystems, and human, animal, or plant host environments, including intensively studied species such as Pseudomonas aeruginosa, P. putida, or P. fluorescens. Various gene expression systems are established for some species, but there is still a need for a simple system that is suitable for a wide range of pseudomonads and that can be used for physiological applications, i.e., with a tuning capacity at lower expression levels. Here, we report the establishment of the anthranilate-dependent PantA promoter for tunable gene expression in pseudomonads. During studies on P. fluorescens, we constructed an anthranilate-inducible AntR/PantA-based expression system, named pUCP20-ANT, and used GFP as reporter to analyze gene expression. This system was compared with the rhamnose-inducible RhaSR/PrhaB-based expression system in an otherwise identical vector background. While the rhamnose-inducible system did not respond to lower inducer concentrations and always reached high levels over time when induced, expression levels of the pUCP20-ANT system could be adjusted to a range of distinct lower or higher levels by variation of anthranilate concentrations in the medium. Importantly, the anthranilate-inducible expression system worked also in strains of P. aeruginosa and P. putida and therefore will be most likely useful for physiological and biotechnological purposes in a wide range of pseudomonads. Key points • We established an anthranilate-inducible gene expression system for pseudomonads. • This system permits tuning of gene expression in a wide range of pseudomonads. • It will be very useful for physiological and biotechnological applications.

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“…This a non-specific mechanism that SPO1 employs to target different bacterial transcription apparatus, regardless of the structural variations of RNA polymerases. This opens up the possibility for the generation of genetically modified SPO1 for the purpose of targeted gene therapy [ 84 , 85 ].…”

Section: Scientific Biotechnology Environmental and Medical Potential—a Short Notementioning

confidence: 99%

Bacteriophages of Thermophilic ‘Bacillus Group’ Bacteria—A Review

et al. 2021

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Bacteriophages of thermophiles are of increasing interest owing to their important roles in many biogeochemical, ecological processes and in biotechnology applications, including emerging bionanotechnology. However, due to lack of in-depth investigation, they are underrepresented in the known prokaryotic virosphere. Therefore, there is a considerable potential for the discovery of novel bacteriophage-host systems in various environments: marine and terrestrial hot springs, compost piles, soil, industrial hot waters, among others. This review aims at providing a reference compendium of thermophages characterized thus far, which infect the species of thermophilic ‘Bacillus group’ bacteria, mostly from Geobacillus sp. We have listed 56 thermophages, out of which the majority belong to the Siphoviridae family, others belong to the Myoviridae and Podoviridae families and, apparently, a few belong to the Sphaerolipoviridae, Tectiviridae or Corticoviridae families. All of their genomes are composed of dsDNA, either linear, circular or circularly permuted. Fourteen genomes have been sequenced; their sizes vary greatly from 35,055 bp to an exceptionally large genome of 160,590 bp. We have also included our unpublished data on TP-84, which infects Geobacillus stearothermophilus (G. stearothermophilus). Since the TP-84 genome sequence shows essentially no similarity to any previously characterized bacteriophage, we have defined TP-84 as a new species in the newly proposed genus Tp84virus within the Siphoviridae family. The information summary presented here may be helpful in comparative deciphering of the molecular basis of the thermophages’ biology, biotechnology and in analyzing the environmental aspects of the thermophages’ effect on the thermophile community.

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Xenogeneic modulation of the ClpCP protease of Bacillus subtilis by a phage-encoded adaptor-like protein

Cited by 11 publications

References 37 publications

PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM

PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM

A tunable anthranilate-inducible gene expression system for Pseudomonas species

Bacteriophages of Thermophilic ‘Bacillus Group’ Bacteria—A Review

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