Rational Design of the N-Terminal Coding Sequence for Regulating Enzyme Expression in Bacillus subtilis

ABSTRACT The tet(L) gene of Bacillus subtilis confers low-level tetracycline (Tc) resistance. Previous work examining the >20-fold-inducible expression of tet(L) by Tc demonstrated a 12-fold translational induction. Here we show that the other component of tet(L) induction is at the level of mRNA stabilization. Addition of a subinhibitory concentration of Tc results in a two- to threefold increase in tet(L) mRNA stability. Using a plasmid-borne derivative of tet(L) with a large in-frame deletion of the coding sequence, the mechanism of Tc-induced stability was explored by measuring the decay of tet(L) mRNAs carrying specific mutations in the leader region. The results of these experiments, as well as experiments with a B. subtilis strain that is resistant to Tc due to a mutation in the ribosomal S10 protein, suggest different mechanisms for the effects of Tc on translation and on mRNA stability. The key role of the 5" end in determining mRNA stability was confirmed in these experiments. Surprisingly, the stability of several other B. subtilis mRNAs was also induced by Tc, which indicates that addition of Tc may result in a general stabilization of mRNA.

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Genetic optimisation of bacteria-induced calcite precipitation in Bacillus subtilis

Microbial Cell Factories ◽

10.1186/s12934-021-01704-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Timothy D. Hoffmann ◽

Kevin Paine ◽

Susanne Gebhard

Keyword(s):

Bacillus Subtilis ◽

Cell Surface ◽

Rational Design ◽

Molecular Mechanisms ◽

De Novo ◽

Contributing Factors ◽

Calcite Precipitation ◽

Nucleation Sites ◽

Nickel Uptake ◽

Extracellular Substances

Abstract Background Microbially induced calcite precipitation (MICP) is an ancient property of bacteria, which has recently gained considerable attention for biotechnological applications. It occurs as a by-product of bacterial metabolism and involves a combination of chemical changes in the extracellular environment, e.g. pH increase, and presence of nucleation sites on the cell surface or extracellular substances produced by the bacteria. However, the molecular mechanisms underpinning MICP and the interplay between the contributing factors remain poorly understood, thus placing barriers to the full biotechnological and synthetic biology exploitation of bacterial biomineralisation. Results In this study, we adopted a bottom-up approach of systematically engineering Bacillus subtilis, which has no detectable intrinsic MICP activity, for biomineralisation. We showed that heterologous production of urease can induce MICP by local increases in extracellular pH, and this can be enhanced by co-expression of urease accessory genes for urea and nickel uptake, depending on environmental conditions. MICP can be strongly enhanced by biofilm-promoting conditions, which appeared to be mainly driven by production of exopolysaccharide, while the protein component of the biofilm matrix was dispensable. Attempts to modulate the cell surface charge of B. subtilis had surprisingly minor effects, and our results suggest this organism may intrinsically have a very negative cell surface, potentially predisposing it for MICP activity. Conclusions Our findings give insights into the molecular mechanisms driving MICP in an application-relevant chassis organism and the genetic elements that can be used to engineer de novo or enhanced biomineralisation. This study also highlights mutual influences between the genetic drivers and the chemical composition of the surrounding environment in determining the speed, spatial distribution and resulting mineral crystals of MICP. Taken together, these data pave the way for future rational design of synthetic precipitator strains optimised for specific applications.

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Genetic Optimisation of Bacteria-Induced Calcite Precipitation in Bacillus subtilis

10.1101/2021.08.17.456648 ◽

2021 ◽

Author(s):

Timothy D Hoffmann ◽

Kevin Paine ◽

Susanne Gebhard

Keyword(s):

Bacillus Subtilis ◽

Cell Surface ◽

Rational Design ◽

Molecular Mechanisms ◽

De Novo ◽

Contributing Factors ◽

Calcite Precipitation ◽

Nucleation Sites ◽

Nickel Uptake ◽

Extracellular Substances

Background Microbially induced calcite precipitation (MICP) is an ancient property of bacteria, which has recently gained considerable attention for biotechnological applications. It occurs as a by-product of bacterial metabolism and involves a combination of chemical changes in the extracellular environment, e.g. pH increase, and presence of nucleation sites on the cell surface or extracellular substances produced by the bacteria. However, the molecular mechanisms underpinning MICP and the interplay between the contributing factors remain poorly understood, thus placing barriers to the full biotechnological and synthetic biology exploitation of bacterial biomineralisation. Results In this study, we adopted a bottom-up approach of systematically engineering Bacillus subtilis, which has no detectable intrinsic MICP activity, for biomineralisation. We showed that heterologous production of urease can induce MICP by local increases in extracellular pH, and this can be enhanced by co-expression of urease accessory genes for urea and nickel uptake, depending on environmental conditions. MICP can be strongly enhanced by biofilm-promoting conditions, which appeared to be mainly driven by production of exopolysaccharide, while the protein component of the biofilm matrix was dispensable. Attempts to modulate the cell surface charge of B. subtilis had surprisingly minor effects, and our results suggest this organism may intrinsically have a very negative cell surface, potentially predisposing it for MICP activity. Conclusions Our findings give insights into the molecular mechanisms driving MICP in an application-relevant chassis organism and the genetic elements that can be used to engineer de novo or enhanced biomineralisation. This study also highlights mutual influences between the genetic drivers and the chemical composition of the surrounding environment in determining the speed, spatial distribution and resulting mineral crystals of MICP. Taken together, these data pave the way for future rational design of synthetic precipitator strains optimised for specific applications.

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Influences of Various Peptide Linkers on the Thermotoga maritima MSB8 Nitrilase Displayed on the Spore Surface of Bacillus subtilis

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000454813 ◽

2017 ◽

Vol 27 (1) ◽

pp. 64-71 ◽

Cited By ~ 7

Author(s):

Huayou Chen ◽

Zhi Chen ◽

Bangguo Wu ◽

Jawad Ullah ◽

Tianxi Zhang ◽

...

Keyword(s):

Bacillus Subtilis ◽

Fusion Proteins ◽

Rational Design ◽

Thermotoga Maritima ◽

Sodium Dodecyl ◽

Ph Stability ◽

Activity Measurement ◽

Spore Surface ◽

Outer Coat ◽

The Stability

In the present study, fusion genes composed of Thermotoga maritima MSB8 nitrilase and Bacillus subtilis 168 outer coat protein CotG were constructed with various peptide linkers and displayed on B. subtilis DB 403 spores. The successful display of CotG-nit fusion proteins on the spore surface of B. subtilis was verified by Western blot analysis and activity measurement. It was demonstrated that the fusion with linker GGGGSEAAAKGGGGS presented the highest thermal and pH stability, which is 2.67- and 1.9-fold of the fusion without linker. In addition, fusion with flexible linker (GGGGS)3 demonstrated better thermal and pH stability than fusions with linkers GGGGS and (GGGGS)2. Fusion with rigid linker (EAAAK) demonstrated better thermal stability than fusions with linkers (EAAAK)2 and (EAAAK)3. Fusions with linker (EAAAK)2 demonstrated better pH stability than fusions with linkers (EAAAK) and (EAAAK)3. In the presence of 1 m<smlcap>M</smlcap> dithiothreitol, 1% (v/v) sodium dodecyl sulfate, and 20% (v/v) ethanol, the optimal linkers of the fusions were MGSSSN, GGGGSEAAAKGGGGS, and (GGGGS)3, respectively. In summary, our results showed that optimizing the peptide linkers with different type, length, and amino acid composition of the fusion proteins would be an efficient way to maintain the stability of fusion proteins and thus improve the nitrilase display efficiency, which could provide an effective method for rational design peptide linkers of displayed nitrilase on B. subtilis.

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Advances and prospects of Bacillus subtilis cellular factories: From rational design to industrial applications

Metabolic Engineering ◽

10.1016/j.ymben.2018.05.006 ◽

2018 ◽

Vol 50 ◽

pp. 109-121 ◽

Cited By ~ 56

Author(s):

Yang Gu ◽

Xianhao Xu ◽

Yaokang Wu ◽

Tengfei Niu ◽

Yanfeng Liu ◽

...

Keyword(s):

Bacillus Subtilis ◽

Rational Design ◽

Industrial Applications

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Interaction of intramembrane metalloprotease SpoIVFB with substrate Pro-σK

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1711467114 ◽

2017 ◽

Vol 114 (50) ◽

pp. E10677-E10686 ◽

Cited By ~ 6

Author(s):

Sabyasachi Halder ◽

Daniel Parrell ◽

Douglas Whitten ◽

Michael Feig ◽

Lee Kroos

Keyword(s):

Mass Spectrometry ◽

Bacillus Subtilis ◽

Rational Design ◽

Structural Model ◽

Cross Linking ◽

Membrane Domain ◽

Intramembrane Proteases ◽

Cbs Domain ◽

Partial Homology ◽

Chemical Cross Linking

Intramembrane proteases (IPs) cleave membrane-associated substrates in nearly all organisms and regulate diverse processes. A better understanding of how these enzymes interact with their substrates is necessary for rational design of IP modulators. We show that interaction ofBacillus subtilisIP SpoIVFB with its substrate Pro-σKdepends on particular residues in the interdomain linker of SpoIVFB. The linker plus either the N-terminal membrane domain or the C-terminal cystathione-β-synthase (CBS) domain of SpoIVFB was sufficient for the interaction but not for cleavage of Pro-σK. Chemical cross-linking and mass spectrometry of purified, inactive SpoIVFB–Pro-σKcomplex indicated residues of the two proteins in proximity. A structural model of the complex was built via partial homology and by using constraints based on cross-linking data. In the model, the Proregion of Pro-σKloops into the membrane domain of SpoIVFB, and the rest of Pro-σKinteracts extensively with the linker and the CBS domain of SpoIVFB. The extensive interaction is proposed to allow coordination between ATP binding by the CBS domain and Pro-σKcleavage by the membrane domain.

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Engineering a More Thermostable Blue Light Photo Receptor Bacillus subtilis YtvA LOV Domain by a Computer Aided Rational Design Method

PLoS Computational Biology ◽

10.1371/journal.pcbi.1003129 ◽

2013 ◽

Vol 9 (7) ◽

pp. e1003129 ◽

Cited By ~ 20

Author(s):

Xiangfei Song ◽

Yefei Wang ◽

Zhiyu Shu ◽

Jingbo Hong ◽

Tong Li ◽

...

Keyword(s):

Bacillus Subtilis ◽

Blue Light ◽

Rational Design ◽

Design Method ◽

Lov Domain ◽

Computer Aided

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Na+/H+ antiport activity conferred by Bacillus subtilis tetA(L), a 5' truncation product of tetA(L), and related plasmid genes upon Escherichia coli.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.4.852 ◽

1996 ◽

Vol 40 (4) ◽

pp. 852-857 ◽

Cited By ~ 10

Author(s):

J Cheng ◽

K Baldwin ◽

A A Guffanti ◽

T A Krulwich

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Recombinant Plasmid ◽

Tetracycline Resistance ◽

Coding Sequence ◽

E Coli ◽

22Na Uptake ◽

Plasmid Genes ◽

Related Plasmid ◽

Is10 Element

An Escherichia coli transformant expressing the Bacillus subtilis tetA(L) gene from a weak promoter was challenged by growth on medium with low, increasing tetracycline concentrations. Changes in the substrate preference ratios of the TetA(L)-mediated resistances and antiports were examined in view of recent findings suggesting that TetA(L) catalyzes efflux of Na+ in exchange for protons in addition to having the ability to catalyze metal-tetracycline/H+ antiport. After growth of the transformant on 1 microgram or more of tetracycline per ml for 12 to 15 h, the tetA(L) gene in the plasmid was found to be disrupted by an IS10 element 50 bp from the 5' end of the coding sequence. This disrupted recombinant plasmid, pKB1, conferred greater tetracycline resistance and higher levels of membrane metal-tetracycline/proton antiport than the original plasmid, pJTA1, but conferred lower NA+ resistance and Na+/H+ antiport levels than the original plasmid. The results indicate that the 5' end of the gene is necessary for optimal Na+/H+ antiport but that some such activity as well as robust tetracycline/H+ antiport persists in its absence. Two plasmid genes, tet(K) and qacA, were compared with tetA(L) vis-à-vis their abilities to enhance the Na+/H+ antiporter activity of everted vesicles from E. coli transformants. tet(K), which is more closely related to tetA(L), catalyzed 22Na+ uptake by energized vesicles, whereas the less closely related qacA gene did not.

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