scholarly journals Degradation of Extracytoplasmic Catalysts for Protein Folding in Bacillus subtilis

2013 ◽  
Vol 80 (4) ◽  
pp. 1463-1468 ◽  
Author(s):  
Laxmi Krishnappa ◽  
Carmine G. Monteferrante ◽  
Jolanda Neef ◽  
Annette Dreisbach ◽  
Jan Maarten van Dijl
Keyword(s):  
Quality Control ◽  
Protein Folding ◽  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Protein Secretion ◽  
Secreted Proteins ◽  
Biotechnological Production ◽  
Content Type ◽  
Wide Range ◽  
Mutant Strains

ABSTRACTThe general protein secretion pathway ofBacillus subtilishas a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold into their active and protease-resistant conformations once membrane passage is completed. The lipoprotein PrsA and the membrane proteins HtrA and HtrB facilitate the extracytoplasmic folding and quality control of exported proteins. Among the native exported proteins ofB. subtilisare at least 10 proteases that have previously been implicated in the degradation of heterologous secreted proteins. Recently, we have shown that these proteases also degrade many native membrane proteins, lipoproteins, and secreted proteins. The present studies were therefore aimed at assessing to what extent these proteases also degrade extracytoplasmic catalysts for protein folding. To this end, we employed a collection of markerless protease mutant strains that lack up to 10 different extracytoplasmic proteases. The results show that PrsA, HtrA, and HtrB are indeed substrates of multiple extracytoplasmic proteases. Thus, improved protein secretion by multiple-protease-mutant strains may be related to both reduced proteolysis and improved posttranslocational protein folding and quality control.

scholarly journals The Redox Cofactor F420Protects Mycobacteria from Diverse Antimicrobial Compounds and Mediates a Reductive Detoxification System

2016 ◽  
Vol 82 (23) ◽  
pp. 6810-6818 ◽  
Author(s):  
Thanavit Jirapanjawat ◽  
Blair Ney ◽  
Matthew C. Taylor ◽  
Andrew C. Warden ◽  
Shahana Afroze ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Mycobacterium Smegmatis ◽  
Antimicrobial Compounds ◽  
Loss Of Function ◽  
Detoxifying Enzymes ◽  
Content Type ◽  
Wide Range ◽  
Detoxification System ◽  
Mutant Strains

ABSTRACTA defining feature of mycobacterial redox metabolism is the use of an unusual deazaflavin cofactor, F420. This cofactor enhances the persistence of environmental and pathogenic mycobacteria, including after antimicrobial treatment, although the molecular basis for this remains to be understood. In this work, we explored our hypothesis that F420enhances persistence by serving as a cofactor in antimicrobial-detoxifying enzymes. To test this, we performed a series of phenotypic, biochemical, and analytical chemistry studies in relation to the model soil bacteriumMycobacterium smegmatis. Mutant strains unable to synthesize or reduce F420were found to be more susceptible to a wide range of antibiotic and xenobiotic compounds. Compounds from three classes of antimicrobial compounds traditionally resisted by mycobacteria inhibited the growth of F420mutant strains at subnanomolar concentrations, namely, furanocoumarins (e.g., methoxsalen), arylmethanes (e.g., malachite green), and quinone analogues (e.g., menadione). We demonstrated that promiscuous F420H2-dependent reductases directly reduce these compounds by a mechanism consistent with hydride transfer. Moreover,M. smegmatisstrains unable to make F420H2lost the capacity to reduce and detoxify representatives of the furanocoumarin and arylmethane compound classes in whole-cell assays. In contrast, mutant strains were only slightly more susceptible to clinical antimycobacterials, and this appeared to be due to indirect effects of F420loss of function (e.g., redox imbalance) rather than loss of a detoxification system. Together, these data show that F420enhances antimicrobial resistance in mycobacteria and suggest that one function of the F420H2-dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify.IMPORTANCEThis study reveals that a unique microbial cofactor, F420, is critical for antimicrobial resistance in the environmental actinobacteriumMycobacterium smegmatis. We show that a superfamily of redox enzymes, the F420H2-dependent reductases, can reduce diverse antimicrobialsin vitroandin vivo.M. smegmatisstrains unable to make or reduce F420become sensitive to inhibition by these antimicrobial compounds. This suggests that mycobacteria have harnessed the unique properties of F420to reduce structurally diverse antimicrobials as part of the antibiotic arms race. The F420H2-dependent reductases that facilitate this process represent a new class of antimicrobial-detoxifying enzymes with potential applications in bioremediation and biocatalysis.

Complexity of scientific evidence in environmental forensic investigations

2015 ◽  
Vol 1 (4) ◽  
pp. 223-232 ◽  
Author(s):  
Val Spikmans
Keyword(s):  
Quality Control ◽  
Design Methodology ◽  
Scientific Evidence ◽  
Forensic Sciences ◽  
Limited Sample ◽  
Content Type ◽  
Large Size ◽  
Wide Range ◽  
Chain Of Custody ◽  
Forensic Investigations

Purpose – Environmental forensic investigations rely on the collection, analysis and interpretation of evidence from an environmental scene to assist in identifying the party responsible for the introduction of exogenous material. These investigations also try to elucidate if the environment and/or human health have been affected. The paper aims to discuss these issues. Design/methodology/approach – Environmental forensic investigations are considered a sub-category of the forensic sciences. The potential scientific evidence is subjected to the same rigour as for other forensic science disciplines, including quality control, accreditation, chain of custody and evidence integrity. The manner in which evidence is analysed and interpreted is also similar. Even though strong similarities can be drawn between environmental forensic investigations and the general forensic sciences, some important differences need to be understood. Findings – Environmental forensic investigations can be more complex than they first appear and identifying, analysing and interpreting scientific evidence is not always straightforward. It is crucial in the comprehension of the complexities of the environmental forensic discipline to understand the intricacies of the investigations, including the limited sample numbers, complex matrices, wide range of exogenous materials encountered, often large size of the scene, changes to the scene and, above all, the potential for degradation or transformation of evidence. In addition, scientific evidence is frequently used to gather intelligence rather than to provide knowledge that can be brought forward to determine guilt or innocence of an accused party. Originality/value – This paper explores the complexities of the discipline and discusses the difficulties that are encountered during environmental investigations.

scholarly journals Redirected Stress Responses in a Genome-Minimized ‘midi Bacillus ’ Strain with Enhanced Capacity for Protein Secretion

mSystems ◽  
2021 ◽  
Author(s):  
Rocío Aguilar Suárez ◽  
Minia Antelo-Varela ◽  
Sandra Maaß ◽  
Jolanda Neef ◽  
Dörte Becher ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bacillus Subtilis ◽  
Protein Secretion ◽  
Stress Responses ◽  
Human Pathogen ◽  
Content Type ◽  
Bacillus Strain ◽  
A Genome ◽  
Original Genome ◽  
Industrial Strains

Our present study showcases a genome-minimized nonpathogenic bacterium, the so-called midi Bacillus , as a chassis for the development of future industrial strains that serve in the production of high-value difficult-to-produce proteins. In particular, we explain how midi Bacillus , which lacks about one-third of the original genome, effectively secretes a protein of the major human pathogen Staphylococcus aureus that cannot be produced by the parental Bacillus subtilis strain.

scholarly journals Anaerobic α-Amylase Production and Secretion with Fumarate as the Final Electron Acceptor in Saccharomyces cerevisiae

2013 ◽  
Vol 79 (9) ◽  
pp. 2962-2967 ◽  
Author(s):  
Zihe Liu ◽  
Tobias Österlund ◽  
Jin Hou ◽  
Dina Petranovic ◽  
Jens Nielsen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Folding ◽  
Protein Secretion ◽  
Electron Acceptor ◽  
Anaerobic Growth ◽  
Anaerobic Conditions ◽  
Aerobic Conditions ◽  
Content Type ◽  
Final Electron ◽  
Final Electron Acceptor

ABSTRACTIn this study, we focus on production of heterologous α-amylase in the yeastSaccharomyces cerevisiaeunder anaerobic conditions. We compare the metabolic fluxes and transcriptional regulation under aerobic and anaerobic conditions, with the objective of identifying the final electron acceptor for protein folding under anaerobic conditions. We find that yeast produces more amylase under anaerobic conditions than under aerobic conditions, and we propose a model for electron transfer under anaerobic conditions. According to our model, during protein folding the electrons from the endoplasmic reticulum are transferred to fumarate as the final electron acceptor. This model is supported by findings that the addition of fumarate under anaerobic (but not aerobic) conditions improves cell growth, specifically in the α-amylase-producing strain, in which it is not used as a carbon source. Our results provide a model for the molecular mechanism of anaerobic protein secretion using fumarate as the final electron acceptor, which may allow for further engineering of yeast for improved protein secretion under anaerobic growth conditions.

scholarly journals Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis

2012 ◽  
Vol 79 (1) ◽  
pp. 347-356 ◽  
Author(s):  
Daphne T. W. Ng ◽  
Casim A. Sarkar
Keyword(s):  
Amino Acid ◽  
Lactococcus Lactis ◽  
Protein Secretion ◽  
Signal Peptide ◽  
Peptide Sequence ◽  
Signal Peptides ◽  
Biotechnological Production ◽  
Content Type ◽  
Amino Acid Mutagenesis ◽  
Native Signal Peptide

ABSTRACTLactococcus lactisis an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion fromL. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels ofStaphylococcus aureusnuclease and further evaluated them for secretion ofBacillus subtilisα-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineeredL. lactissignal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.

scholarly journals Escherichia coli SRP, Its Protein Subunit Ffh, and the Ffh M Domain Are Able To Selectively Limit Membrane Protein Expression When Overexpressed

mBio ◽  
2010 ◽  
Vol 1 (2) ◽  
Author(s):  
Ido Yosef ◽  
Elena S. Bochkareva ◽  
Eitan Bibi
Keyword(s):  
Escherichia Coli ◽  
Quality Control ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Translation ◽  
Mrna Levels ◽  
Content Type ◽  
E Coli ◽  
Quality Control Process

ABSTRACT The Escherichia coli signal recognition particle (SRP) system plays an important role in membrane protein biogenesis. Previous studies have suggested indirectly that in addition to its role during the targeting of ribosomes translating membrane proteins to translocons, the SRP might also have a quality control role in preventing premature synthesis of membrane proteins in the cytoplasm. This proposal was studied here using cells simultaneously overexpressing various membrane proteins and either SRP, the SRP protein Ffh, its 4.5S RNA, or the Ffh M domain. The results show that SRP, Ffh, and the M domain are all able to selectively inhibit the expression of membrane proteins. We observed no apparent changes in the steady-state mRNA levels or membrane protein stability, suggesting that inhibition may occur at the level of translation, possibly through the interaction between Ffh and ribosome-hydrophobic nascent chain complexes. Since E. coli SRP does not have a eukaryote-like translation arrest domain, we discuss other possible mechanisms by which this SRP might regulate membrane protein translation when overexpressed. IMPORTANCE The eukaryotic SRP slows down translation of SRP substrates by cytoplasmic ribosomes. This activity is important for preventing premature synthesis of secretory and membrane proteins in the cytoplasm. It is likely that an analogous quality control step would be required in all living cells. However, on the basis of its composition and domain structure and limited in vitro studies, it is believed that the E. coli SRP is unable to regulate ribosomes translating membrane proteins. Nevertheless, several in vivo studies have suggested otherwise. To address this issue further in vivo, we utilized unbalanced conditions under which E. coli simultaneously overexpresses SRP and each of several membrane or cytosolic proteins. Surprisingly, our results clearly show that the E. coli SRP is capable of regulating membrane protein synthesis and demonstrate that the M domain of Ffh mediates this activity. These results thus open the way for mechanistic characterization of this quality control process in bacteria.

scholarly journals Magnesium Suppresses Defects in the Formation of 70S Ribosomes as Well as in Sporulation Caused by Lack of Several Individual Ribosomal Proteins

2018 ◽  
Vol 200 (18) ◽  
Author(s):  
Genki Akanuma ◽  
Kotaro Yamazaki ◽  
Yuma Yagishi ◽  
Yuka Iizuka ◽  
Morio Ishizuka ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cell Proliferation ◽  
Bacillus Subtilis ◽  
Ribosomal Proteins ◽  
Living Cells ◽  
Content Type ◽  
Translational Activity ◽  
Mutant Strains ◽  
70S Ribosome ◽  
Ribosome Formation

ABSTRACTIndividually, the ribosomal proteins L1, L23, L36, and S6 are not essential for cell proliferation ofBacillus subtilis, but the absence of any one of these ribosomal proteins causes a defect in the formation of the 70S ribosomes and a reduced growth rate. In mutant strains individually lacking these ribosomal proteins, the cellular Mg2+content was significantly reduced. The deletion of YhdP, an exporter of Mg2+, and overexpression of MgtE, the main importer of Mg2+, increased the cellular Mg2+content and restored the formation of 70S ribosomes in these mutants. The increase in the cellular Mg2+content improved the growth rate and the cellular translational activity of the ΔrplA(L1) and the ΔrplW(L23) mutants but did not restore those of the ΔrpmJ(L36) and the ΔrpsF(S6) mutants. The lack of L1 caused a decrease in the production of Spo0A, the master regulator of sporulation, resulting in a decreased sporulation frequency. However, deletion ofyhdPand overexpression ofmgtEincreased the production of Spo0A and partially restored the sporulation frequency in the ΔrplA(L1) mutant. These results indicate that Mg2+can partly complement the function of several ribosomal proteins, probably by stabilizing the conformation of the ribosome.IMPORTANCEWe previously reported that an increase in cellular Mg2+content can suppress defects in 70S ribosome formation and growth rate caused by the absence of ribosomal protein L34. In the present study, we demonstrated that, even in mutants lacking individual ribosomal proteins other than L34 (L1, L23, L36, and S6), an increase in the cellular Mg2+content could restore 70S ribosome formation. Moreover, the defect in sporulation caused by the absence of L1 was also suppressed by an increase in the cellular Mg2+content. These findings indicate that at least part of the function of these ribosomal proteins can be complemented by Mg2+, which is essential for all living cells.

scholarly journals Combined Effect of Improved Cell Yield and Increased Specific Productivity Enhances Recombinant Enzyme Production in Genome-Reduced Bacillus subtilis Strain MGB874

2011 ◽  
Vol 77 (23) ◽  
pp. 8370-8381 ◽  
Author(s):  
Kenji Manabe ◽  
Yasushi Kageyama ◽  
Takuya Morimoto ◽  
Tadahiro Ozawa ◽  
Kazuhisa Sawada ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Enzyme Production ◽  
Cell Yield ◽  
Specific Productivity ◽  
Recombinant Enzymes ◽  
Content Type ◽  
Reduced Genome ◽  
Enhanced Production ◽  
Enzyme Productivity ◽  
Wide Range

ABSTRACTGenome reduction strategies to create genetically improved cellular biosynthesis machineries for proteins and other products have been pursued by use of a wide range of bacteria. We reported previously that the novelBacillus subtilisstrain MGB874, which was derived from strain 168 and has a total genomic deletion of 874 kb (20.7%), exhibits enhanced production of recombinant enzymes. However, it was not clear how the genomic reduction resulted in elevated enzyme production. Here we report that deletion of therocDEF-rocRregion, which is involved in arginine degradation, contributes to enhanced enzyme production in strain MGB874. Deletion of therocDEF-rocRregion caused drastic changes in glutamate metabolism, leading to improved cell yields with maintenance of enzyme productivity. Notably, the specific enzyme productivity was higher in the reduced-genome strain, with or without therocDEF-rocRregion, than in wild-type strain 168. The high specific productivity in strain MGB874 is likely attributable to the higher expression levels of the target gene resulting from an increased promoter activity and plasmid copy number. Thus, the combined effects of the improved cell yield by deletion of therocDEF-rocRregion and the increased specific productivity by deletion of another gene(s) or the genomic reduction itself enhanced the production of recombinant enzymes in MGB874. Our findings represent a good starting point for the further improvement ofB. subtilisreduced-genome strains as cell factories for the production of heterologous enzymes.

scholarly journals Cultivation in Space Flight Produces Minimal Alterations in the Susceptibility of Bacillus subtilis Cells to 72 Different Antibiotics and Growth-Inhibiting Compounds

2017 ◽  
Vol 83 (21) ◽  
Author(s):  
Michael D. Morrison ◽  
Patricia Fajardo-Cavazos ◽  
Wayne L. Nicholson
Keyword(s):  
Bacillus Subtilis ◽  
Space Flight ◽  
Space Station ◽  
Cesium Chloride ◽  
Phenotype Microarray ◽  
Content Type ◽  
Inhibitory Compounds ◽  
Long Duration ◽  
Human Space Flight ◽  
Wide Range

ABSTRACTPast results have suggested that bacterial antibiotic susceptibility is altered during space flight. To test this notion,Bacillus subtiliscells were cultivated in matched hardware, medium, and environmental conditions either in space flight microgravity on the International Space Station, termed flight (FL) samples, or at Earth-normal gravity, termed ground control (GC) samples. The susceptibility of FL and GC samples was compared to 72 antibiotics and growth-inhibitory compounds using the Omnilog phenotype microarray (PM) system. Only 9 compounds were identified by PM screening as exhibiting significant differences (P< 0.05, Student'sttest) in FL versus GC samples: 6-mercaptopurine, cesium chloride, enoxacin, lomefloxacin, manganese(II) chloride, nalidixic acid, penimepicycline, rolitetracycline, and trifluoperazine. Testing of the same compounds by standard broth dilution assay did not reveal statistically significant differences in the 50% inhibitory concentrations (IC50s) between FL and GC samples. The results indicate that the susceptibility ofB. subtiliscells to a wide range of antibiotics and growth inhibitors is not dramatically altered by space flight.IMPORTANCEThis study addresses a major concern of mission planners for human space flight, that bacteria accompanying astronauts on long-duration missions might develop a higher level of resistance to antibiotics due to exposure to the space flight environment. The results of this study do not support that notion.

scholarly journals Genomic and Chemical Diversity of Bacillus subtilis Secondary Metabolites against Plant Pathogenic Fungi

mSystems ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Heiko T. Kiesewalter ◽  
Carlos N. Lozano-Andrade ◽  
Mario Wibowo ◽  
Mikael L. Strube ◽  
Gergely Maróti ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Secondary Metabolites ◽  
Pathogenic Fungus ◽  
Gene Clusters ◽  
Chemical Diversity ◽  
Biosynthetic Gene ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Wide Range

ABSTRACT Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant growth-promoting and biocontrol abilities. These secondary metabolites include nonribosomal peptides with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates, and therefore, a comparative overview of secondary metabolites from various environmental B. subtilis strains is missing. In this study, we isolated 23 B. subtilis strains from 11 sampling sites, compared the fungal inhibition profiles of wild types and their nonribosomal peptide mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that nonribosomal peptide production varied among B. subtilis strains coisolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium spp., a combination of plipastatin and surfactin is required to hinder growth of Botrytis cinerea. Detailed genomic analysis revealed that altered nonribosomal peptide production profiles in specific isolates are due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical nonribosomal peptide detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis. IMPORTANCE Secondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which nonribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate the prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack the production of certain secondary metabolites.

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