scholarly journals Increased Fitness and Alteration of Metabolic Pathways during Bacillus subtilis Evolution in the Laboratory

2011 ◽  
Vol 77 (12) ◽  
pp. 4105-4118 ◽  
Author(s):  
Heather Maughan ◽  
Wayne L. Nicholson
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Pathways ◽  
High Performance ◽  
Colony Morphology ◽  
Flagellar Motility ◽  
Content Type ◽  
Batch Cultures ◽  
Fitness Advantage ◽  
Acetoin Production ◽  
Small Colony

ABSTRACTFive batch cultures ofBacillus subtiliswere subjected to evolution in the laboratory for 6,000 generations under conditions repressing sporulation in complex liquid medium containing glucose. Between generations 1,000 and 2,000, variants with a distinct small-colony morphology arose and swept through four of the five populations that had been previously noted for their loss of sporulation (H. Maughan et al., Genetics 177:937-948, 2007). To better understand the nature of adaptation in these variants, individual strains were isolated from one population before (WN715) and after (WN716) the sweep. In addition to colony morphology, strains WN715 and WN716 differed in their motility, aerotaxis, and cell morphology. Competition experiments showed that strain WN716 had evolved a distinct fitness advantage over the ancestral strain and strain WN715 during growth and the transition to the postexponential growth phase, which was more pronounced when WN715 was present in the coculture. Microarray analyses revealed candidate genes in which mutations may have produced some of the observed phenotypes. For example, loss of motility in WN716 was accompanied by decreased transcription of all flagellar, motility, and chemotaxis genes on the microarray. Transcription ofalsSandalsDwas also lower in strain WN716, and the predicted loss of acetoin production and enhanced acetate production was confirmed by high-performance liquid chromatography (HPLC) analysis. The results suggested that the derived colony morphology of strain WN716 was associated with increased fitness, the alteration of several metabolic pathways, and the loss of a typical postexponential-phase response.

scholarly journals Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis

2019 ◽  
Vol 48 (2) ◽  
pp. 996-1009 ◽  
Author(s):  
Yaokang Wu ◽  
Taichi Chen ◽  
Yanfeng Liu ◽  
Rongzhen Tian ◽  
Xueqin Lv ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Pathways ◽  
Metabolic Flux ◽  
Target Genes ◽  
Fine Tuning ◽  
Dual Control ◽  
Genetic Circuits ◽  
Dynamic Regulation ◽  
Batch Bioreactor ◽  
Acetoin Production

Abstract Dynamic regulation is an effective strategy for fine-tuning metabolic pathways in order to maximize target product synthesis. However, achieving dynamic and autonomous up- and down-regulation of the metabolic modules of interest simultaneously, still remains a great challenge. In this work, we created an autonomous dual-control (ADC) system, by combining CRISPRi-based NOT gates with novel biosensors of a key metabolite in the pathway of interest. By sensing the levels of the intermediate glucosamine-6-phosphate (GlcN6P) and self-adjusting the expression levels of the target genes accordingly with the GlcN6P biosensor and ADC system enabled feedback circuits, the metabolic flux towards the production of the high value nutraceutical N-acetylglucosamine (GlcNAc) could be balanced and optimized in Bacillus subtilis. As a result, the GlcNAc titer in a 15-l fed-batch bioreactor increased from 59.9 g/l to 97.1 g/l with acetoin production and 81.7 g/l to 131.6 g/l without acetoin production, indicating the robustness and stability of the synthetic circuits in a large bioreactor system. Remarkably, this self-regulatory methodology does not require any external level of control such as the use of inducer molecules or switching fermentation/environmental conditions. Moreover, the proposed programmable genetic circuits may be expanded to engineer other microbial cells and metabolic pathways.

scholarly journals Nonribosomal Peptide Synthase Gene Clusters for Lipopeptide Biosynthesis in Bacillus subtilis 916 and Their Phenotypic Functions

2014 ◽  
Vol 81 (1) ◽  
pp. 422-431 ◽  
Author(s):  
Chuping Luo ◽  
Xuehui Liu ◽  
Huafei Zhou ◽  
Xiaoyu Wang ◽  
Zhiyi Chen
Keyword(s):  
Bacillus Subtilis ◽  
Antifungal Activity ◽  
Hemolytic Activity ◽  
Biofilm Formation ◽  
Gene Clusters ◽  
Colony Morphology ◽  
Swarming Motility ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Phenotypic Features

ABSTRACTBacilluscyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features ofBacillusstrains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology.Bacillus subtilis916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome ofB. subtilis916 contains four nonribosomal peptide synthase (NRPS) gene clusters,srf,bmy,fen, andloc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studyingB. subtilis916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activityin vitro, the strain mutated insrfAAhad significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other thanfenresulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion,B. subtilis916 coproduces four families of LPs which contribute to the phenotypic features ofB. subtilis916 in an intricate way.

scholarly journals Rough diamonds in emerging markets: legacy, competitiveness, and sustained high performance

2019 ◽  
Vol 26 (3) ◽  
pp. 363-386
Author(s):  
Seung Ho Park ◽  
Gerardo R. Ungson
Keyword(s):  
Human Capital ◽  
Emerging Markets ◽  
High Performance ◽  
Core Competencies ◽  
Market Potential ◽  
Government Support ◽  
Screening Tests ◽  
Institutional Voids ◽  
Content Type ◽  
Over Time

Purpose The purpose of this paper is to uncover the underlying drivers of sustained high performing companies based on a field study of 127 companies in Brazilian, Russian, Indian and Chinese (BRIC) and Association of Southeast Asian Nations (ASEAN) emerging markets. Understanding these companies provides a complementary way of appraising the growth, development and transformation of emerging markets. The authors synthesize the findings in an overarching framework that covers six strategies for building and sustaining legacy that leads to the succession of intergenerational wealth over time: overcoming institutional voids, inclusive markets, deepening localization, nurturing government support, building core competencies and harnessing human capital. The authors relate these strategies to different levels of development using Prahalad and Hart’s BOP framework. Design/methodology/approach This study examines the underlying drivers of sustained high-performance companies based on field studies from an initial set of 105,260 BRIC companies and close to 500 companies in ASEAN. The methods employed four screening tests to arrive at a selection of the highest-performing firms: 70 firms in the BRIC nations and 58 firms from ASEAN. Following the selection, the authors constructed cases using primary interviews and secondary data, with the assistance of Ernst & Young and with academic colleagues in Manila. These studies were originally conducted in two separate time periods and reported accordingly. This paper synthesizes the findings of these two studies to arrive at an extended integrative framework. Findings From the cases, the authors examine six strategies for building and sustaining legacy that lead to high performance over time: overcoming institutional voids, creating inclusive markets, deepening localization, nurturing government support, building core competencies and harnessing human capital. To address the evolving state of institutional voids in these countries, the authors employ similar methods to hypothesize the placement of these strategies in the context of the world economic pyramid, initially formulated as the “bottom of the pyramid” framework. Originality/value This paper synthesizes and extends the authors’ previous works by proposing the concept of legacy to describe the emergence and succession of local exemplary firms in emerging markets. This study aims to complement extant measures of nation-growth based primarily on GDP. The paper also extends the literature on institutional voids in shifting the focus from the mix of voids to their evolving state. Altogether, the paper provides a complementary narrative on assessing the market potential of emerging markets by adopting several categories of performance.

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 In VitroActivities of Daptomycin-, Vancomycin-, and Teicoplanin-Loaded Polymethylmethacrylate against Methicillin-Susceptible, Methicillin-Resistant, and Vancomycin-Intermediate Strains of Staphylococcus aureus

2011 ◽  
Vol 55 (12) ◽  
pp. 5480-5484 ◽  
Author(s):  
Yuhan Chang ◽  
Wen-Chien Chen ◽  
Pang-Hsin Hsieh ◽  
Dave W. Chen ◽  
Mel S. Lee ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
High Performance ◽  
Low Dose ◽  
Antibacterial Activities ◽  
High Dose ◽  
Bone Cements ◽  
Antibacterial Effects ◽  
Methicillin Resistant ◽  
Content Type

ABSTRACTThe objective of this study was to evaluate the antibacterial effects of polymethylmethacrylate (PMMA) bone cements loaded with daptomycin, vancomycin, and teicoplanin against methicillin-susceptibleStaphylococcus aureus(MSSA), methicillin-resistantStaphylococcus aureus(MRSA), and vancomycin-intermediateStaphylococcus aureus(VISA) strains. Standardized cement specimens made from 40 g PMMA loaded with 1 g (low-dose), 4 g (middle-dose) or 8 g (high-dose) antibiotics were tested for elution characteristics and antibacterial activities. The patterns of release of antibiotics from the cement specimens were evaluated usingin vitrobroth elution assay with high-performance liquid chromatography. The activities of broth elution fluid against differentStaphylococcus aureusstrains (MSSA, MRSA, and VISA) were then determined. The antibacterial activities of all the tested antibiotics were maintained after being mixed with PMMA. The cements loaded with higher dosages of antibiotics showed longer elution periods. Regardless of the antibiotic loading dose, the teicoplanin-loaded cements showed better elution efficacy and provided longer inhibitory periods against MSSA, MRSA, and VISA than cements loaded with the same dose of vancomycin or daptomycin. Regarding the choice of antibiotics for cement loading in the treatment ofStaphylococcus aureusinfection, teicoplanin was superior in terms of antibacterial effects.

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 Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17

2014 ◽  
Vol 58 (10) ◽  
pp. 6185-6196 ◽  
Author(s):  
Marius Spohn ◽  
Norbert Kirchner ◽  
Andreas Kulik ◽  
Angelika Jochim ◽  
Felix Wolf ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gene Cluster ◽  
High Performance ◽  
Pathogenic Bacteria ◽  
Genome Mining ◽  
Gene Clusters ◽  
Von Willebrand Disease ◽  
Biosynthesis Gene Cluster ◽  
Content Type ◽  
Bioinformatic Tools

ABSTRACTThe emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products isAmycolatopsis. However,Amycolatopsis japonicumdoes not produce an antibiotic under standard laboratory conditions. In contrast to mostAmycolatopsisstrains,A. japonicumis genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, thebbrgene fromAmycolatopsis balhimycina(bbrAba), intoA. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing ofA. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed thein silicoprediction that the recombinantA. japonicum/pRM4-bbrAbasynthesizes ristomycin A.

scholarly journals Osmoadaptation Strategy of the Most Halophilic Fungus, Wallemia ichthyophaga, Growing Optimally at Salinities above 15% NaCl

2013 ◽  
Vol 80 (1) ◽  
pp. 247-256 ◽  
Author(s):  
Janja Zajc ◽  
Tina Kogej ◽  
Erwin A. Galinski ◽  
José Ramos ◽  
Nina Gunde-Cimerman
Keyword(s):  
Biomass Production ◽  
Osmotic Shock ◽  
Model Organism ◽  
Salinity Range ◽  
Growth Media ◽  
Optimal Salinity ◽  
Content Type ◽  
Batch Cultures ◽  
Specific Growth Rates ◽  
Optimum Salinity

ABSTRACTWallemia ichthyophagais a fungus from the ancient basidiomycetous genusWallemia(Wallemiales, Wallemiomycetes) that grows only at salinities between 10% (wt/vol) NaCl and saturated NaCl solution. This obligate halophily is unique among fungi. The main goal of this study was to determine the optimal salinity range for growth of the halophilicW. ichthyophagaand to unravel its osmoadaptation strategy. Our results showed that growth on solid growth media was extremely slow and resulted in small colonies. On the other hand, in the liquid batch cultures, the specific growth rates ofW. ichthyophagawere higher, and the biomass production increased with increasing salinities. The optimum salinity range for growth ofW. ichthyophagawas between 15 and 20% (wt/vol) NaCl. At 10% NaCl, the biomass production and the growth rate were by far the lowest among all tested salinities. Furthermore, the cell wall content in the dry biomass was extremely high at salinities above 10%. Our results also showed that glycerol was the major osmotically regulated solute, since its accumulation increased with salinity and was diminished by hypo-osmotic shock. Besides glycerol, smaller amounts of arabitol and trace amounts of mannitol were also detected. In addition,W. ichthyophagamaintained relatively small intracellular amounts of potassium and sodium at constant salinities, but during hyperosmotic shock, the amounts of both cations increased significantly. Given our results and the recent availability of the genome sequence,W. ichthyophagashould become well established as a novel model organism for studies of halophily in eukaryotes.

scholarly journals Specific Control of Pseudomonas aeruginosa Surface-Associated Behaviors by Two c-di-GMP Diguanylate Cyclases

mBio ◽  
2010 ◽  
Vol 1 (4) ◽  
Author(s):  
Judith H. Merritt ◽  
Dae-Gon Ha ◽  
Kimberly N. Cowles ◽  
Wenyun Lu ◽  
Diana K. Morales ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Extracellular Polysaccharide ◽  
Diguanylate Cyclase ◽  
Flagellar Motility ◽  
Critical Question ◽  
Cyclic Diguanylate ◽  
Critical Signal ◽  
Content Type ◽  
Wide Range

ABSTRACT The signaling nucleotide cyclic diguanylate (c-di-GMP) regulates the transition between motile and sessile growth in a wide range of bacteria. Understanding how microbes control c-di-GMP metabolism to activate specific pathways is complicated by the apparent multifold redundancy of enzymes that synthesize and degrade this dinucleotide, and several models have been proposed to explain how bacteria coordinate the actions of these many enzymes. Here we report the identification of a diguanylate cyclase (DGC), RoeA, of Pseudomonas aeruginosa that promotes the production of extracellular polysaccharide (EPS) and contributes to biofilm formation, that is, the transition from planktonic to surface-dwelling cells. Our studies reveal that RoeA and the previously described DGC SadC make distinct contributions to biofilm formation, controlling polysaccharide production and flagellar motility, respectively. Measurement of total cellular levels of c-di-GMP in ∆roeA and ∆sadC mutants in two different genetic backgrounds revealed no correlation between levels of c-di-GMP and the observed phenotypic output with regard to swarming motility and EPS production. Our data strongly argue against a model wherein changes in total levels of c-di-GMP can account for the specific surface-related phenotypes of P. aeruginosa. IMPORTANCE A critical question in the study of cyclic diguanylate (c-di-GMP) signaling is how the bacterial cell integrates contributions of multiple c-di-GMP-metabolizing enzymes to mediate its cognate functional outputs. One leading model suggests that the effects of c-di-GMP must, in part, be localized subcellularly. The data presented here show that the phenotypes controlled by two different diguanylate cyclase (DGC) enzymes have discrete outputs despite the same total level of c-di-GMP. These data support and extend the model in which localized c-di-GMP signaling likely contributes to coordination of the action of the multiple proteins involved in the synthesis, degradation, and/or binding of this critical signal.

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.

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