Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins

Marciniak, Bogumiła C.; Trip, Hein; Veek, Patricia J van-der; Kuipers, Oscar P.

doi:10.1186/1475-2859-11-66

Cited by 22 publications

(23 citation statements)

References 75 publications

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“…However, this does not appear to causing significant levels of cell wall stress, as RT-qPCR measurements revealed similar levels of liaR expression in all strains regardless of Cel8A-LysM expression or the presence of protease inhibitors. This finding is generally consistent with microarray studies reported by Marciniak et al , which showed that only some types of secreted proteins cause the liaFRS operon to be upregulated (Marciniak, Trip et al 2012). …”

Section: Discussionsupporting

confidence: 92%

“…To investigate this issue, we used RT-qPCR to monitor the effect of protease inhibition and protein expression on membrane and cell envelope stress responses. Induction of the membrane stress response was determined by measuring cssR mRNA levels, which is part of the CssRS two-component response regulator that upregulates HtrA and HtrB protease levels during periods of secretory stress in B. subtilis (Hyyrylainen, Bolhuis et al 2001, Westers, Westers et al 2006, Marciniak, Trip et al 2012). The effect of protein expression on cssR mRNA levels was determined by comparing the parent and Cel8A-LysM expressing strains (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Stabilizing displayed proteins on vegetative Bacillus subtilis cells

Huang

Gosschalk

Kim

et al. 2018

Appl Microbiol Biotechnol

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Microbes engineered to display heterologous proteins could be useful biotechnological tools for protein engineering, lignocellulose degradation, biocatalysis, bioremediation and biosensing. Bacillus subtilis is a promising host to display proteins, as this model Gram-positive bacterium is genetically tractable and already used industrially to produce enzymes. To gain insight into the factors that affect displayed protein stability and copy-number, we systematically compared the ability of different protease-deficient B. subtilis strains (WB800, BRB07, BRB08 and BRB14) to display a Cel8A-LysM reporter protein in which the Clostridium thermocellum Cel8A endoglucanase is fused to LysM cell wall binding modules. Whole-cell cellulase measurements and fractionation experiments demonstrate that genetically eliminating extracytoplasmic bacterial proteases improves Cel8A-LysM displays levels. However, upon entering stationary phase, for all protease-deficient strains, the amount of displayed reporter dramatically decreases, presumably as a result of cellular autolysis. This problem can be partially overcome by adding chemical protease inhibitors, which significantly increase protein display levels. We conclude that strain BRB08 is well-suited for stably displaying our reporter protein, as genetic removal of its extracellular and cell wall-associated proteases leads to the highest levels of surface accumulated Cel8A-LysM without causing secretion stress or impairing growth. A two-step procedure is presented that enables the construction of enzyme coated vegetative B. subtilis cells that retain stable cell-associated enzyme activity for nearly 3 days. The results of this work could aid the development of whole cell display systems that have useful biotechnological applications.

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Section: Discussionsupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Stabilizing displayed proteins on vegetative Bacillus subtilis cells

Huang

Gosschalk

Kim

et al. 2018

Appl Microbiol Biotechnol

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“…All of these inducers are well-defined and readily available at low prices. Moreover, a recent study demonstrated that the Lia-system can also be induced by the overexpression of certain heterologous and secreted proteins, especially the universal shock protein USP45 from Lactococcus lactis and the TEM-1 β-lactamase from E. coli [39]. (iv) Lastly, an antibiotic-inducible LIKE-expression strain can easily be converted into a strong constitutive expression platform by the simple deletion of liaF , encoding the LiaRS-specific inhibitor protein [21-23].…”

Section: Discussionmentioning

confidence: 99%

The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

et al. 2012

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BackgroundBacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (PliaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min.ResultsBased on these traits of PliaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the “LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the PliaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the PliaI promoter.ConclusionsThe LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of PliaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available, and affordable inducers and (v) the convenient conversion of LIKE-derived inducible expression strains into strong constitutive protein production factories.

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“…An overlooked option is the possibility of an interaction between the expressed recombinant proteins and the host cellular machinery (e.g., RNA degradation and inhibition of cell growth caused by the expression of human RNase L containing "ankyrin repeat" motifs in E. coli) 28 .The issue of lower rARU yield could also be related to the unique sequence and the structural motifs present in the Clostridial DNA. It was recently shown that certain recombinant proteins (e.g., proteins of a different nature like soluble (xylanase and GFP) or inclusion body (Interferon β)) based on cellular localization (e.g., secreted proteins (NprE, XynA, Usp45, TEM-1 β-lactamase), membrane proteins (LmrA and XylP), or lipoproteins (MntA and YcdH)) caused different host cellular responses when overexpressed 29,30 . It is possible by using next generation sequencing tools to quantify the gene expression profile at different growth and production phases in order to identify changes related to substrate consumption, central carbon metabolism, and energy metabolism based on the sequence and structural features.…”

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confidence: 99%

Effect of restricted dissolved oxygen on expression of Clostridium difficile toxin A subunit from E. coli

Sharma

Phue

Khatipov

et al. 2020

Sci Rep

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The repeating unit of the C. difficile Toxin A (rARU, also known as CROPS [combined repetitive oligopeptides]) C-terminal region, was shown to elicit protective immunity against C. difficile and is under consideration as a possible vaccine against this pathogen. However, expression of recombinant rARU in E. coli using the standard vaccine production process was very low. Transcriptome and proteome analyses showed that at restricted dissolved oxygen (DO) the numbers of differentially expressed genes (DEGs) was 2.5-times lower than those expressed at unrestricted oxygen. Additionally, a 7.4-times smaller number of ribosome formation genes (needed for translation) were down-regulated as compared with unrestricted DO. Higher rARU expression at restricted DO was associated with up-regulation of 24 heat shock chaperones involved in protein folding and with the up-regulation of the global regulator RNA chaperone hfq. Cellular stress response leading to down-regulation of transcription, translation, and energy generating pathways at unrestricted DO were associated with lower rARU expression. Investigation of the C. difficile DNA sequence revealed the presence of cell wall binding profiles, which based on structural similarity prediction by BLASTp, can possibly interact with cellular proteins of E. coli such as the transcriptional repressor ulaR, and the ankyrins repeat proteins. At restricted DO, rARU mRNA was 5-fold higher and the protein expression 27-fold higher compared with unrestricted DO. The report shows a strategy for improved production of C. difficile vaccine candidate in E. coli by using restricted DO growth. This strategy could improve the expression of recombinant proteins from anaerobic origin or those with cell wall binding profiles.Clostridium difficile (C. difficile) is an anaerobic bacterial pathogen responsible for diarrhea and pseudomembranous colitis 1,2 . The bacteria produces two high molecular weight toxins, toxin A (308 kDa) and toxin B (269 kDa) 3,4 ; both contain a repeating region called rARU (CROPs) (104 kDa) in subunit A and rBRU (70 kDa) in subunit B 5,6 . Both Toxin A and Toxin B are responsible for clinical symptoms and are candidates for vaccine development 7,8 . Pfizer's genetically detoxified vaccine against C difficile is a toxoid mix of Toxin A and Toxin B expressed recombinantly is about to enter clinical trial phase III, and Valneva has a vaccine candidate of recombinant fusion protein of truncated portions of Toxin A and Toxin B, which completed a clinical trial phase II, with promising results 9-12 .In this report, we focused on improving the expression of the repeating unit region (rARU) of the C. difficile toxin A. The importance of rARU was shown in animal models where serum neutralizing antibodies to toxin A conferred immunity to this pathogen and antiserum against nontoxic recombinant peptide, containing the www.nature.com/scientificreports www.nature.com/scientificreports/ repeating units region (rARU), neutralized the enterotoxic and cytotoxic activity of the toxin 13,14 ...

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Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins

Cited by 22 publications

References 75 publications

Stabilizing displayed proteins on vegetative Bacillus subtilis cells

Stabilizing displayed proteins on vegetative Bacillus subtilis cells

The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

Effect of restricted dissolved oxygen on expression of Clostridium difficile toxin A subunit from E. coli

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