scholarly journals Engineering Bacillus subtilis as a Versatile and Stable Platform for Production of Nanobodies

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Mengdi Yang ◽  
Ge Zhu ◽  
George Korza ◽  
Xin Sun ◽  
Peter Setlow ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Extreme Environments ◽  
Protein Detection ◽  
Therapeutic Interventions ◽  
Whole Body ◽  
Competent Cell ◽  
Single Chain ◽  
Content Type ◽  
On Demand ◽  
Cell Extracts

ABSTRACT There is a growing need for a highly stable system to allow the production of biologics for diagnoses and therapeutic interventions on demand that could be used in extreme environments. Among the variety of biologics, nanobodies (Nbs) derived from single-chain variable antibody fragments from camelids have attracted great attention in recent years due to their small size and great stability with translational potentials in whole-body imaging and the development of new drugs. Intracellular expression using the bacterium Escherichia coli has been the predominant system to produce Nbs, and this requires lengthy steps for releasing intracellular proteins for purification as well as removal of endotoxins. Lyophilized, translationally competent cell extracts have also been explored as offering portability and long shelf life, but such extracts may be difficult to scale up and suffer from batch-to-batch variability. To address these problems, we present a new system to do the following: (i) engineer the spore-forming bacterium Bacillus subtilis to secrete Nbs that can target small molecules or protein antigens on mammalian cells, (ii) immobilize Nbs containing a cellulose-binding domain on a cellulose matrix for long-term storage and small-molecule capturing, (iii) directly use Nb-containing bacterial supernatant fluid to perform protein detection on cell surfaces, and (iv) convert engineered B. subtilis to spores that are resistant to most environmental extremes. In summary, our work may open a new paradigm for using B. subtilis as an extremely stable microbial factory to produce Nbs with applications in extreme environments on demand. IMPORTANCE It is highly desirable to produce biologics for diagnoses and therapeutic interventions on demand that could be used in a variety of settings. Among the many biologics, Nbs have attracted attention due to their small size, thermal stability, and broad utility in diagnoses, therapies, and fundamental research. Nbs originate from antibodies found in camelids, and >10 companies have invested in Nbs as potential drugs. Here, we present a system using cells of the bacterium Bacillus subtilis as a versatile platform for production of Nbs and then antigen detection via customized affinity columns. Importantly, B. subtilis carrying engineered genes for Nbs can form spores, which survive for years in a desiccated state. However, upon rehydration and exposure to nutrients, spores rapidly transition to growing cells which secrete encoded Nbs, thus allowing their manufacture and purification.

scholarly journals Single-Cell Microscopy Reveals That Levels of Cyclic di-GMP Vary amongBacillus subtilisSubpopulations

2019 ◽  
Vol 201 (16) ◽  
Author(s):  
Cordelia A. Weiss ◽  
Jakob A. Hoberg ◽  
Kuanqing Liu ◽  
Benjamin P. Tu ◽  
Wade C. Winkler
Keyword(s):  
Bacillus Subtilis ◽  
Single Cell ◽  
Relative Abundance ◽  
Biofilm Formation ◽  
Single Cells ◽  
Cell Types ◽  
Population Heterogeneity ◽  
Competent Cell ◽  
Gram Positive ◽  
Content Type

ABSTRACTThe synthesis of signaling molecules is one strategy bacteria employ to sense alterations in their environment and rapidly adjust to those changes. In Gram-negative bacteria, bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) regulates the transition from a unicellular motile state to a multicellular sessile state. However, c-di-GMP signaling has been less intensively studied in Gram-positive organisms. To that end, we constructed a fluorescentyfpreporter based on a c-di-GMP-responsive riboswitch to visualize the relative abundance of c-di-GMP for single cells of the Gram-positive model organismBacillus subtilis. Coupled with cell-type-specific fluorescent reporters, this riboswitch reporter revealed that c-di-GMP levels are markedly different amongB. subtiliscellular subpopulations. For example, cells that have made the decision to become matrix producers maintain higher intracellular c-di-GMP concentrations than motile cells. Similarly, we find that c-di-GMP levels differ between sporulating and competent cell types. These results suggest that biochemical measurements of c-di-GMP abundance are likely to be inaccurate for a bulk ensemble ofB. subtiliscells, as such measurements will average c-di-GMP levels across the population. Moreover, the significant variation in c-di-GMP levels between cell types hints that c-di-GMP might play an important role duringB. subtilisbiofilm formation. This study therefore emphasizes the importance of using single-cell approaches for analyzing metabolic trends within ensemble bacterial populations.IMPORTANCEMany bacteria have been shown to differentiate into genetically identical yet morphologically distinct cell types. Such population heterogeneity is especially prevalent among biofilms, where multicellular communities are primed for unexpected environmental conditions and can efficiently distribute metabolic responsibilities.Bacillus subtilisis a model system for studying population heterogeneity; however, a role for c-di-GMP in these processes has not been thoroughly investigated. Herein, we introduce a fluorescent reporter, based on a c-di-GMP-responsive riboswitch, to visualize the relative abundance of c-di-GMP for singleB. subtiliscells. Our analysis shows that c-di-GMP levels are conspicuously different amongB. subtiliscellular subtypes, suggesting a role for c-di-GMP during biofilm formation. These data highlight the utility of riboswitches as tools for imaging metabolic changes within individual bacterial cells. Analyses such as these offer new insight into c-di-GMP-regulated phenotypes, especially given that other biofilms also consist of multicellular communities.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

scholarly journals Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Robert S. Brzozowski ◽  
Brooke R. Tomlinson ◽  
Michael D. Sacco ◽  
Judy J. Chen ◽  
Anika N. Ali ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Cell Size ◽  
Unknown Function ◽  
Suppressor Mutation ◽  
Gram Positive ◽  
Content Type ◽  
Suppressor Mutations ◽  
Extragenic Suppressor ◽  
Cell Division Inhibition

ABSTRACT Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS. IMPORTANCE Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

scholarly journals Reduction of the Temperature Sensitivity of Halomonas hydrothermalis by Iron Starvation Combined with Microaerobic Conditions

2015 ◽  
Vol 81 (6) ◽  
pp. 2156-2162 ◽  
Author(s):  
Jesse P. Harrison ◽  
John E. Hallsworth ◽  
Charles S. Cockell
Keyword(s):  
Physicochemical Parameters ◽  
Marine Bacterium ◽  
Chemical Elements ◽  
Extreme Environments ◽  
Iron Availability ◽  
Iron Starvation ◽  
Specific Chemical ◽  
Content Type ◽  
Distribution And Ecology ◽  
Microaerobic Conditions

ABSTRACTThe limits to biological processes on Earth are determined by physicochemical parameters, such as extremes of temperature and low water availability. Research into microbial extremophiles has enhanced our understanding of the biophysical boundaries which define the biosphere. However, there remains a paucity of information on the degree to which rates of microbial multiplication within extreme environments are determined by the availability of specific chemical elements. Here, we show that iron availability and the composition of the gaseous phase (aerobic versus microaerobic) determine the susceptibility of a marine bacterium,Halomonas hydrothermalis, to suboptimal and elevated temperature and salinity by impacting rates of cell division (but not viability). In particular, iron starvation combined with microaerobic conditions (5% [vol/vol] O2, 10% [vol/vol] CO2, reduced pH) reduced sensitivity to temperature across the 13°C range tested. These data demonstrate that nutrient limitation interacts with physicochemical parameters to determine biological permissiveness for extreme environments. The interplay between resource availability and stress tolerance, therefore, may shape the distribution and ecology of microorganisms within Earth's biosphere.

scholarly journals Streptomycin-Induced Expression in Bacillus subtilis of YtnP, a Lactonase-Homologous Protein That Inhibits Development and Streptomycin Production in Streptomyces griseus

2011 ◽  
Vol 78 (2) ◽  
pp. 599-603 ◽  
Author(s):  
Johannes Schneider ◽  
Ana Yepes ◽  
Juan C. Garcia-Betancur ◽  
Isa Westedt ◽  
Benjamin Mielich ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signaling Pathway ◽  
Streptomyces Griseus ◽  
Aerial Mycelium ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Induced Expression ◽  
Content Type ◽  
Homologous Protein

ABSTRACTBacillus subtilisinduces expression of the geneytnPin the presence of the antimicrobial streptomycin, produced by the Gram-positive bacteriumStreptomyces griseus.ytnPencodes a lactonase-homologous protein that is able to inhibit the signaling pathway required for the streptomycin production and development of aerial mycelium inS. griseus.

scholarly journals Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

2016 ◽  
Vol 198 (21) ◽  
pp. 2925-2935 ◽  
Author(s):  
Heng Zhao ◽  
Yingjie Sun ◽  
Jason M. Peters ◽  
Carol A. Gross ◽  
Ethan C. Garner ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Repression ◽  
Cell Envelope ◽  
Turgor Pressure ◽  
Teichoic Acid ◽  
Lethal Gene ◽  
Genetic Redundancy ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Lipid Ii

ABSTRACTThe integrity of the bacterial cell envelope is essential to sustain life by countering the high turgor pressure of the cell and providing a barrier against chemical insults. InBacillus subtilis, synthesis of both peptidoglycan and wall teichoic acids requires a common C55lipid carrier, undecaprenyl-pyrophosphate (UPP), to ferry precursors across the cytoplasmic membrane. The synthesis and recycling of UPP requires a phosphatase to generate the monophosphate form Und-P, which is the substrate for peptidoglycan and wall teichoic acid synthases. Using an optimizedclusteredregularlyinterspacedshortpalindromicrepeat (CRISPR) system with catalytically inactive (“dead”)CRISPR-associated protein9(dCas9)-based transcriptional repression system (CRISPR interference [CRISPRi]), we demonstrate thatB. subtilisrequires either of two UPP phosphatases, UppP or BcrC, for viability. We show that a third predicted lipid phosphatase (YodM), with homology to diacylglycerol pyrophosphatases, can also support growth when overexpressed. Depletion of UPP phosphatase activity leads to morphological defects consistent with a failure of cell envelope synthesis and strongly activates the σM-dependent cell envelope stress response, includingbcrC, which encodes one of the two UPP phosphatases. These results highlight the utility of an optimized CRISPRi system for the investigation of synthetic lethal gene pairs, clarify the nature of theB. subtilisUPP-Pase enzymes, and provide further evidence linking the σMregulon to cell envelope homeostasis pathways.IMPORTANCEThe emergence of antibiotic resistance among bacterial pathogens is of critical concern and motivates efforts to develop new therapeutics and increase the utility of those already in use. The lipid II cycle is one of the most frequently targeted processes for antibiotics and has been intensively studied. Despite these efforts, some steps have remained poorly defined, partly due to genetic redundancy. CRISPRi provides a powerful tool to investigate the functions of essential genes and sets of genes. Here, we used an optimized CRISPRi system to demonstrate functional redundancy of two UPP phosphatases that are required for the conversion of the initially synthesized UPP lipid carrier to Und-P, the substrate for the synthesis of the initial lipid-linked precursors in peptidoglycan and wall teichoic acid synthesis.

Magnetovibrio blakemorei gen. nov., sp. nov., a magnetotactic bacterium ( Alphaproteobacteria : Rhodospirillaceae ) isolated from a salt marsh

2013 ◽  
Vol 63 (Pt_5) ◽  
pp. 1824-1833 ◽  
Author(s):  
Dennis A. Bazylinski ◽  
Timothy J. Williams ◽  
Christopher T. Lefèvre ◽  
Denis Trubitsyn ◽  
Jiasong Fang ◽  
...  
Keyword(s):  
Salt Marsh ◽  
Electron Acceptor ◽  
Anaerobic Growth ◽  
Rrna Gene ◽  
Magnetotactic Bacterium ◽  
Single Chain ◽  
Circular Chromosome ◽  
Terminal Electron Acceptor ◽  
Content Type ◽  
Link Type

A magnetotactic bacterium, designated strain MV-1T, was isolated from sulfide-rich sediments in a salt marsh near Boston, MA, USA. Cells of strain MV-1T were Gram-negative, and vibrioid to helicoid in morphology. Cells were motile by means of a single polar flagellum. The cells appeared to display a transitional state between axial and polar magnetotaxis: cells swam in both directions, but generally had longer excursions in one direction than the other. Cells possessed a single chain of magnetosomes containing truncated hexaoctahedral crystals of magnetite, positioned along the long axis of the cell. Strain MV-1T was a microaerophile that was also capable of anaerobic growth on some nitrogen oxides. Salinities greater than 10 % seawater were required for growth. Strain MV-1T exhibited chemolithoautotrophic growth on thiosulfate and sulfide with oxygen as the terminal electron acceptor (microaerobic growth) and on thiosulfate using nitrous oxide (N2O) as the terminal electron acceptor (anaerobic growth). Chemo-organoautotrophic and methylotrophic growth was supported by formate under microaerobic conditions. Autotrophic growth occurred via the Calvin–Benson–Bassham cycle. Chemo-organoheterotrophic growth was supported by various organic acids and amino acids, under microaerobic and anaerobic conditions. Optimal growth occurred at pH 7.0 and 26–28 °C. The genome of strain MV-1T consisted of a single, circular chromosome, about 3.7 Mb in size, with a G+C content of 52.9–53.5 mol%.Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MV-1T belongs to the family Rhodospirillaceae within the Alphaproteobacteria , but is not closely related to the genus Magnetospirillum . The name Magnetovibrio blakemorei gen. nov., sp. nov. is proposed for strain MV-1T. The type strain of Magnetovibrio blakemorei is MV-1T ( = ATCC BAA-1436T  = DSM 18854T).

Degradation of zearalenone in swine feed and feed ingredients by Bacillus subtilis ANSB01G

2014 ◽  
Vol 7 (2) ◽  
pp. 143-151 ◽  
Author(s):  
Y.P. Lei ◽  
L.H. Zhao ◽  
Q.G. Ma ◽  
J.Y. Zhang ◽  
T. Zhou ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Culture Supernatant ◽  
Antimicrobial Activities ◽  
Proteinase K ◽  
Rrna Gene ◽  
Distillers Grains With Solubles ◽  
Dried Distillers Grains ◽  
Cell Extracts ◽  
Gene Sequence Analysis ◽  
And Staphylococcus Aureus

Zearalenone (ZEA) and its derivatives are mycotoxins that can cause oestrogenic effects and impair the reproductive physiology of animals, especially in female swine. Strategies to reduce or eliminate ZEA contamination in foods and feeds are very much needed. Among 36 bacterial isolates obtained from a variety of animal intestinal chyme, mouldy foods and feeds, soils, etc., five isolates demonstrated the ability to reduce more than 50% of ZEA in a liquid medium; ANSB01G isolate taken from normal broiler intestinal chyme reduced ZEA the most, by 88.65%. Using physiological, biochemical, and 16S rRNA gene sequence analysis methods, the ANSB01G isolate was identified as Bacillus subtilis. Under simulated intestinal tract conditions, the ANSB01G B. subtilis isolate degraded 84.58, 66.34 and 83.04% of ZEA in naturally contaminated maize, dried distillers’ grains with solubles, and swine complete feed, respectively. The highest degradation of ZEA occurred when the mycotoxin was co-incubated with the whole bacterial culture, resulting in a reduction of 88.65%, followed by 75.60% using culture supernatant, 26.11% using cell extracts, and 15.06% using viable cells. Treatments consisting of both heating and addition of proteinase K significantly reduced the rate of ZEA degradation in the culture supernatant, indicating that the ZEA degradation might be enzymatic. B. subtilis ANSB01G displayed resistance to simulated gastrointestinal tract environments and antimicrobial activities against several common bacterial pathogens, including Escherichia coli, Salmonella typhimurium and Staphylococcus aureus. These properties of B. subtilis ANSB01G suggest the possibility of its potential to effectively degrade ZEA in feed and to develop functional feed products for livestock industries.

Zie ik het of zie ik het niet?

2021 ◽  
Vol 49 (1) ◽  
pp. 51-83
Author(s):  
Jorre Vannieuwenhuyze ◽  
Karen Donders ◽  
Ike Picone
Keyword(s):  
European Union ◽  
Government Regulation ◽  
Video On Demand ◽  
The European Union ◽  
Content Type ◽  
Consumption Behavior ◽  
Local Content ◽  
Positive Effects ◽  
On Demand ◽  
The Impact

Abstract Do I see or not? A study on the impact of placement on program consumption in an on-demand environment The European Union (2018) stipulates that Member States can implement rules to ensure the findability and visibility of local content in video- on- demand environments. Indeed, several countries are concerned that their own audiovisual programs or journalistic products will be consumed less in such environments. It is argued that, in such environments, media users completely decide themselves about their consumption agency, but such statements are also contested. In this research we analyze the impact of placement on the consumption of audiovisual programs in the video-on-demand environments of the Flemish broadcasters VRT and DPG. From experimental research we conclude that there is indeed a significant impact of placement on consumption behavior and that, in other words, manipulations by intermediary gatekeepers can have potentially negative and positive effects on the consumption of local content. Government regulation would therefore be a useful tool to safeguard the importance of proximity of content.

scholarly journals Complete Genome Sequence of Bacillus subtilis Strain WB800N, an Extracellular Protease-Deficient Derivative of Strain 168

2018 ◽  
Vol 7 (18) ◽  
Author(s):  
Haeyoung Jeong ◽  
Da-Eun Jeong ◽  
Seung-Hwan Park ◽  
Seong Joo Kim ◽  
Soo-Keun Choi
Keyword(s):  
Bacillus Subtilis ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Genetically Engineered ◽  
Subtilis Strain ◽  
Secretory Proteins ◽  
Extracellular Proteases ◽  
Content Type ◽  
Protease Deficient

Bacillus subtilis WB800N is a genetically engineered variant of B. subtilis 168, such that all extracellular proteases are disrupted, which enables WB800N to be widely used for the expression of secretory proteins. Here, we report the 4.2-Mb complete genome sequence of WB800N and present all of the disrupted gene structure.

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