scholarly journals Functional Implementation of the Posttranslational SecB-SecA Protein-Targeting Pathway in Bacillus subtilis

2011 ◽  
Vol 78 (3) ◽  
pp. 651-659 ◽  
Author(s):  
Liuyang Diao ◽  
Qilei Dong ◽  
Zhaohui Xu ◽  
Sheng Yang ◽  
Jiahai Zhou ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Targeting ◽  
Heterologous Proteins ◽  
Gram Positive ◽  
Content Type ◽  
E Coli ◽  
New Strategy ◽  
Secretory Production ◽  
Close Relatives

ABSTRACTBacillus subtilisand its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria,B. subtilisdoes not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein-targeting pathway intoB. subtilisby coexpressing SecB fromEscherichia colitogether with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of theB. subtilisSecA were replaced by the corresponding part of SecA fromE. coli.In vitropulldown experiments showed that, in contrast toB. subtilisSecA, the hybrid SecA protein gained the ability to efficiently bind toE. coliSecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that the secretion of both proteins byB. subtiliswas significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished the secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.

scholarly journals Organization of the Flagellar Switch Complex ofBacillus subtilis

2018 ◽  
Vol 201 (8) ◽  
Author(s):  
Elizabeth Ward ◽  
Eun A Kim ◽  
Joseph Panushka ◽  
Tayson Botelho ◽  
Trevor Meyer ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Interaction ◽  
Protein Complex ◽  
Cross Linking ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Middle Domain ◽  
Flagellar Rotation

ABSTRACTWhile the protein complex responsible for controlling the direction (clockwise [CW] or counterclockwise [CCW]) of flagellar rotation has been fairly well studied inEscherichia coliandSalmonella, less is known about the switch complex inBacillus subtilisor other Gram-positive species. Two component proteins (FliG and FliM) are shared betweenE. coliandB. subtilis, but in place of the protein FliN found inE. coli, theB. subtiliscomplex contains the larger protein FliY. Notably, inB. subtilisthe signaling protein CheY-phosphate induces a switch from CW to CCW rotation, opposite to its action inE. coli. Here, we have examined the architecture and function of the switch complex inB. subtilisusing targeted cross-linking, bacterial two-hybrid protein interaction experiments, and characterization of mutant phenotypes. In major respects, theB. subtilisswitch complex appears to be organized similarly to that inE. coli. The complex is organized around a ring built from the large middle domain of FliM; this ring supports an array of FliG subunits organized in a similar way to that ofE. coli, with the FliG C-terminal domain functioning in the generation of torque via conserved charged residues. Key differences fromE. coliinvolve the middle domain of FliY, which forms an additional, more outboard array, and the C-terminal domains of FliM and FliY, which are organized into both FliY homodimers and FliM heterodimers. Together, the results suggest that the CW and CCW conformational states are similar in the Gram-negative and Gram-positive switches but that CheY-phosphate drives oppositely directed movements in the two cases.IMPORTANCEFlagellar motility plays key roles in the survival of many bacteria and in the harmful action of many pathogens. Bacterial flagella rotate; the direction of flagellar rotation is controlled by a multisubunit protein complex termed the switch complex. This complex has been extensively studied in Gram-negative model species, but little is known about the complex inBacillus subtilisor other Gram-positive species. Notably, the switch complex in Gram-positive species responds to its effector CheY-phosphate (CheY-P) by switching to CCW rotation, whereas inE. coliorSalmonellaCheY-P acts in the opposite way, promoting CW rotation. In the work here, the architecture of theB. subtilisswitch complex has been probed using cross-linking, protein interaction measurements, and mutational approaches. The results cast light on the organization of the complex and provide a framework for understanding the mechanism of flagellar direction control inB. subtilisand other Gram-positive species.

scholarly journals Screening of Escherichia coli Species Biodiversity Reveals New Biofilm-Associated Antiadhesion Polysaccharides

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Olaya Rendueles ◽  
Laetitia Travier ◽  
Patricia Latour-Lambert ◽  
Thierry Fontaine ◽  
Julie Magnus ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Bacterial Adhesion ◽  
Bioactive Molecules ◽  
Bacterial Biofilms ◽  
Gram Positive ◽  
Content Type ◽  
E Coli ◽  
Species Biodiversity

ABSTRACTBacterial biofilms often form multispecies communities in which complex but ill-understood competition and cooperation interactions occur. In light of the profound physiological modifications associated with this lifestyle, we hypothesized that the biofilm environment might represent an untapped source of natural bioactive molecules interfering with bacterial adhesion or biofilm formation. We produced cell-free solutions extracted fromin vitromature biofilms formed by 122 naturalEscherichia coliisolates, and we screened these biofilm extracts for antiadhesion molecules active on a panel of Gram-positive and Gram-negative bacteria. Using this approach, we showed that 20% of the tested biofilm extracts contained molecules that antagonize bacterial growth or adhesion. We characterized a compound, produced by a commensal animalE. colistrain, for which activity is detected only in biofilm extract. Biochemical and genetic analyses showed that this compound corresponds to a new type of released high-molecular-weight polysaccharide whose biofilm-associated production is regulated by the RfaH protein. We demonstrated that the antiadhesion activity of this polysaccharide was restricted to Gram-positive bacteria and that its production reduced susceptibility to invasion and provided rapid exclusion ofStaphylococcus aureusfrom mixedE. coliandS. aureusbiofilms. Our results therefore demonstrate that biofilms contain molecules that contribute to the dynamics of mixed bacterial communities and that are not or only poorly detected in unconcentrated planktonic supernatants. Systematic identification of these compounds could lead to strategies that limit pathogen surface colonization and reduce the burden associated with the development of bacterial biofilms on medical devices.IMPORTANCEWe sought to demonstrate that bacterial biofilms are reservoirs for unknown molecules that antagonize bacterial adhesion. The use of natural strains representative ofEscherichia colispecies biodiversity showed that nonbiocidal antiadhesion polysaccharides are frequently found in mature biofilm extracts (bacterium-free suspensions which contain soluble molecules produced within the biofilm). Release of an antiadhesion polysaccharide confers a competitive advantage upon the producing strain against clinically relevant pathogens such asStaphylococcus aureus. Hence, exploring the biofilm environment provides a better understanding of bacterial interactions within complex communities and could lead to improved control of pathogen colonization.

scholarly journals Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis

2015 ◽  
Vol 197 (11) ◽  
pp. 1952-1962 ◽  
Author(s):  
Katherine A. Black ◽  
Patricia C. Dos Santos
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cysteine Desulfurase ◽  
Content Type ◽  
E Coli ◽  
Wobble Position ◽  
Lysine Trna ◽  
Domains Of Life

ABSTRACTThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U inEscherichia colirequires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). TheE. coliMnmA adenylates and subsequently thiolates tRNA to form the s2U modification.Bacillus subtilislacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene,yrvO, directly adjacent tomnmA. The genomic synteny ofyrvOandmnmAcombined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that theB. subtilisYrvO and MnmA are sufficient for s2U biosynthesis. A conditionalB. subtilisknockout strain showed that s2U abundance correlates with MnmA expression, andin vivocomplementation studies inE. coliIscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis.In vitroexperiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between theB. subtilisproteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype ofE. coliΔiscSand ΔmnmAstrains associated with s2U depletion is recovered byB. subtilis yrvOandmnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCEThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U inEscherichia colirequires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE).Bacillus subtilisand most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, themnmAcoding sequence is located adjacent toyrvO, encoding a cysteine desulfurase. In this work, we provide evidence that theB. subtilisYrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s2U biosynthesis in a pathway independent of the one used inE. coli.

scholarly journals High-Salinity Growth Conditions Promote Tat-Independent Secretion of Tat Substrates in Bacillus subtilis

2012 ◽  
Vol 78 (21) ◽  
pp. 7733-7744 ◽  
Author(s):  
René van der Ploeg ◽  
Carmine G. Monteferrante ◽  
Sjouke Piersma ◽  
James P. Barnett ◽  
Thijs R. H. M. Kouwen ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
High Salinity ◽  
Growth Conditions ◽  
Signal Peptides ◽  
Control Mechanisms ◽  
Heterologous Proteins ◽  
Content Type ◽  
E Coli

ABSTRACTThe Gram-positive bacteriumBacillus subtiliscontains two Tat translocases, which can facilitate transport of folded proteins across the plasma membrane. Previous research has shown that Tat-dependent protein secretion inB. subtilisis a highly selective process and that heterologous proteins, such as the green fluorescent protein (GFP), are poor Tat substrates in this organism. Nevertheless, when expressed inEscherichia coli, bothB. subtilisTat translocases facilitated exclusively Tat-dependent export of folded GFP when the twin-arginine (RR) signal peptides of theE. coliAmiA, DmsA, or MdoD proteins were attached. Therefore, the present studies were aimed at determining whether the same RR signal peptide-GFP precursors would also be exported Tat dependently inB. subtilis. In addition, we investigated the secretion of GFP fused to the full-length YwbN protein, a strict Tat substrate inB. subtilis. Several investigated GFP fusion proteins were indeed secreted inB. subtilis, but this secretion was shown to be completely Tat independent. At high-salinity growth conditions, the Tat-independent secretion of GFP as directed by the RR signal peptides from theE. coliAmiA, DmsA, or MdoD proteins was significantly enhanced, and this effect was strongest in strains lacking the TatAy-TatCy translocase. This implies that high environmental salinity has a negative influence on the avoidance of Tat-independent secretion of AmiA-GFP, DmsA-GFP, and MdoD-GFP. We conclude that as-yet-unidentified control mechanisms reject the investigated GFP fusion proteins for translocation by theB. subtilisTat machinery and, at the same time, set limits to their Tat-independent secretion, presumably via the Sec pathway.

scholarly journals A Novel Mobilizing Tool Based on the Conjugative Transfer System of the IncM Plasmid pCTX-M3

2020 ◽  
Vol 86 (17) ◽  
Author(s):  
Michał Dmowski ◽  
Izabela Kern-Zdanowicz
Keyword(s):  
Bacillus Subtilis ◽  
Lactococcus Lactis ◽  
Conjugative Transfer ◽  
Conjugation System ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Mobilizable Plasmid

ABSTRACT Conjugative plasmids are the main players in horizontal gene transfer in Gram-negative bacteria. DNA transfer tools constructed on the basis of such plasmids enable gene manipulation even in strains of clinical or environmental origin, which are often difficult to work with. The conjugation system of the IncM plasmid pCTX-M3 isolated from a clinical strain of Citrobacter freundii has been shown to enable efficient mobilization of oriTpCTX-M3-bearing plasmids into a broad range of hosts comprising Alpha-, Beta-, and Gammaproteobacteria. We constructed a helper plasmid, pMOBS, mediating such mobilization with an efficiency up to 1,000-fold higher than that achieved with native pCTX-M3. We also constructed Escherichia coli donor strains with chromosome-integrated conjugative transfer genes: S14 and S15, devoid of one putative regulator (orf35) of the pCTX-M3 tra genes, and S25 and S26, devoid of two putative regulators (orf35 and orf36) of the pCTX-M3 tra genes. Strains S14 and S15 and strains S25 and S26 are, respectively, up to 100 and 1,000 times more efficient in mobilization than pCTX-M3. Moreover, they also enable plasmid mobilization into the Gram-positive bacteria Bacillus subtilis and Lactococcus lactis. Additionally, the constructed E. coli strains carried no antibiotic resistance genes that are present in pCTX-M3 to facilitate manipulations with antibiotic-resistant recipient strains, such as those of clinical origin. To demonstrate possible application of the constructed tool, an antibacterial conjugation-based system was designed. Strain S26 was used for introduction of a mobilizable plasmid coding for a toxin, resulting in the elimination of over 90% of recipient E. coli cells. IMPORTANCE The conjugation of donor and recipient bacterial cells resulting in conjugative transfer of mobilizable plasmids is the preferred method enabling the introduction of DNA into strains for which other transfer methods are difficult to establish (e.g., clinical strains). We have constructed E. coli strains carrying the conjugation system of the IncM plasmid pCTX-M3 integrated into the chromosome. To increase the mobilization efficiency up to 1,000-fold, two putative regulators of this system, orf35 and orf36, were disabled. The constructed strains broaden the repertoire of tools for the introduction of DNA into the Gram-negative Alpha-, Beta-, and Gammaproteobacteria, as well as into Gram-positive bacteria such as Bacillus subtilis and Lactococcus lactis. The antibacterial procedure based on conjugation with the use of the orf35- and orf36-deficient strain lowered the recipient cell number by over 90% owing to the mobilizable plasmid-encoded toxin.

scholarly journals Control of Bacillus subtilis Replication Initiation during Physiological Transitions and Perturbations

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
John T. Sauls ◽  
Sarah E. Cox ◽  
Quynh Do ◽  
Victoria Castillo ◽  
Zulfar Ghulam-Jelani ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bacillus Subtilis ◽  
Steady State ◽  
Single Cell ◽  
Cell Size ◽  
Physiological Parameters ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
E Coli

ABSTRACT Bacillus subtilis and Escherichia coli are evolutionarily divergent model organisms whose analysis has enabled elucidation of fundamental differences between Gram-positive and Gram-negative bacteria, respectively. Despite their differences in cell cycle control at the molecular level, the two organisms follow the same phenomenological principle, known as the adder principle, for cell size homeostasis. We thus asked to what extent B. subtilis and E. coli share common physiological principles in coordinating growth and the cell cycle. We measured physiological parameters of B. subtilis under various steady-state growth conditions with and without translation inhibition at both the population and single-cell levels. These experiments revealed core physiological principles shared between B. subtilis and E. coli. Specifically, both organisms maintain an invariant cell size per replication origin at initiation, under all steady-state conditions, and even during nutrient shifts at the single-cell level. Furthermore, the two organisms also inherit the same “hierarchy” of physiological parameters. On the basis of these findings, we suggest that the basic principles of coordination between growth and the cell cycle in bacteria may have been established early in evolutionary history. IMPORTANCE High-throughput, quantitative approaches have enabled the discovery of fundamental principles describing bacterial physiology. These principles provide a foundation for predicting the behavior of biological systems, a widely held aspiration. However, these approaches are often exclusively applied to the best-known model organism, E. coli. In this report, we investigate to what extent quantitative principles discovered in Gram-negative E. coli are applicable to Gram-positive B. subtilis. We found that these two extremely divergent bacterial species employ deeply similar strategies in order to coordinate growth, cell size, and the cell cycle. These similarities mean that the quantitative physiological principles described here can likely provide a beachhead for others who wish to understand additional, less-studied prokaryotes.

scholarly journals Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli and In Vitro

2017 ◽  
Vol 199 (19) ◽  
Author(s):  
Daniel Parrell ◽  
Yang Zhang ◽  
Sandra Olenic ◽  
Lee Kroos
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Membrane Proteins ◽  
Direct Effects ◽  
Transmembrane Segment ◽  
Content Type ◽  
E Coli ◽  
Transmembrane Segments

ABSTRACT RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli, we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli. Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli, including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His6-MBP-RsiW(73–118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His6-MBP-FtsL(23–117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL. IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are typically inferred from genetic studies in the native bacterium. Evidence for direct effects has come sometimes from coexpression of the enzyme and potential substrate in a heterologous host and rarely from biochemical reconstitution of cleavage in vitro. We applied these two approaches to the B. subtilis enzyme RasP and its proposed substrates RsiW and FtsL. We discovered potential pitfalls and solutions in heterologous coexpression experiments in E. coli, providing evidence that both substrates are cleaved by RasP in vivo but, surprisingly, that only RsiW was cleaved in vitro, suggesting that FtsL has an additional requirement.

scholarly journals Gepotidacin (GSK2140944) In Vitro Activity against Gram-Positive and Gram-Negative Bacteria

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
R. K. Flamm ◽  
D. J. Farrell ◽  
P. R. Rhomberg ◽  
N. E. Scangarella-Oman ◽  
H. S. Sader
Keyword(s):  
Postantibiotic Effect ◽  
Gram Positive ◽  
Bacterial Dna ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Time Kill ◽  
Synergy Testing ◽  
Potential Use

ABSTRACT Gepotidacin is a first-in-class, novel triazaacenaphthylene antibiotic that inhibits bacterial DNA replication and has in vitro activity against susceptible and drug-resistant pathogens. Reference in vitro methods were used to investigate the MICs and minimum bactericidal concentrations (MBCs) of gepotidacin and comparator agents for Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli. Gepotidacin in vitro activity was also evaluated by using time-kill kinetics and broth microdilution checkerboard methods for synergy testing and for postantibiotic and subinhibitory effects. The MIC90 of gepotidacin for 50 S. aureus (including methicillin-resistant S. aureus [MRSA]) and 50 S. pneumoniae (including penicillin-nonsusceptible) isolates was 0.5 μg/ml, and for E. coli (n = 25 isolates), it was 4 μg/ml. Gepotidacin was bactericidal against S. aureus, S. pneumoniae, and E. coli, with MBC/MIC ratios of ≤4 against 98, 98, and 88% of the isolates tested, respectively. Time-kill curves indicated that the bactericidal activity of gepotidacin was observed at 4× or 10× MIC at 24 h for all of the isolates. S. aureus regrowth was observed in the presence of gepotidacin, and the resulting gepotidacin MICs were 2- to 128-fold higher than the baseline gepotidacin MICs. Checkerboard analysis of gepotidacin combined with other antimicrobials demonstrated no occurrences of antagonism with agents from multiple antimicrobial classes. The most common interaction when testing gepotidacin was indifference (fractional inhibitory concentration index of >0.5 to ≤4; 82.7% for Gram-positive isolates and 82.6% for Gram-negative isolates). The postantibiotic effect (PAE) of gepotidacin was short when it was tested against S. aureus (≤0.6 h against MRSA and MSSA), and the PAE–sub-MIC effect (SME) was extended (>8 h; three isolates at 0.5× MIC). The PAE of levofloxacin was modest (0.0 to 2.4 h), and the PAE-SME observed varied from 1.2 to >9 h at 0.5× MIC. These in vitro data indicate that gepotidacin is a bactericidal agent that exhibits a modest PAE and an extended PAE-SME against Gram-positive and -negative bacteria and merits further study for potential use in treating infections caused by these pathogens.

scholarly journals A Canonical Biotin Synthesis Enzyme, 8-Amino-7-Oxononanoate Synthase (BioF), Utilizes Different Acyl Chain Donors in Bacillus subtilis and Escherichia coli

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Miglena Manandhar ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Chemical Synthesis ◽  
Acyl Carrier Protein ◽  
Acyl Chain ◽  
Bacillus Sphaericus ◽  
Content Type ◽  
E Coli

ABSTRACTBioF (8-amino-7-oxononanoate synthase) is a strictly conserved enzyme that catalyzes the first step in assembly of the fused heterocyclic rings of biotin. The BioF acyl chain donor has long been thought to be pimeloyl-CoA. Indeed,in vitrotheEscherichia coliandBacillus sphaericusenzymes have been shown to condense pimeloyl-CoA withl-alanine in a pyridoxal 5′-phosphate-dependent reaction with concomitant CoA release and decarboxylation ofl-alanine. However, recentin vivostudies ofE. coliandBacillus subtilissuggested that the BioF proteins of the two bacteria could have different specificities for pimelate thioesters in thatE. coliBioF may utilize either pimeloyl coenzyme A (CoA) or the pimelate thioester of the acyl carrier protein (ACP) of fatty acid synthesis. In contrast,B. subtilisBioF seemed likely to be specific for pimeloyl-CoA and unable to utilize pimeloyl-ACP. We now report genetic andin vitrodata demonstrating thatB. subtilisBioF specifically utilizes pimeloyl-CoA.IMPORTANCEBiotin is an essential vitamin required by mammals and birds because, unlike bacteria, plants, and some fungi, these organisms cannot make biotin. Currently, the biotin included in vitamin tablets and animal feeds is made by chemical synthesis. This is partly because the biosynthetic pathways in bacteria are incompletely understood. This paper defines an enzyme of theBacillus subtilispathway and shows that it differs from that ofEscherichia coliin the ability to utilize specific precursors. These bacteria have been used in biotin production and these data may aid in making biotin produced by biotechnology commercially competitive with that produced by chemical synthesis.

scholarly journals Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Naomi Shimokawa-Chiba ◽  
Claudia Müller ◽  
Keigo Fujiwara ◽  
Bertrand Beckert ◽  
Koreaki Ito ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stop Codon ◽  
Release Factor ◽  
Positive Bacterium ◽  
Gram Positive ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Dead End ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus

AbstractRescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

Sign in / Sign up

Export Citation Format

Share Document