Progress 12/01/16 to 08/31/20
Outputs
Target Audience: 1) Graduate and undergraduate students participating directly in research projects in the Amador-Noguez lab; 2) Undergraduates students taking the course Microbio526 'Physiology of microorganisms', which is taught by Dr. Amador-Noguez Changes/Problems:TheCovid-19 pandemic significantly impacted this project during its last year, specifically affecting the completion and submission of two manuscripts. We anticipate submission of these two manuscripts this year. What opportunities for training and professional development has the project provided? The research project supported graduate student (Tippapha Pisithkul, CMB), who graduated last year and current graduate student (Lauren Lucas, MDTP). This project also supported training of several undergraduates in the laboratory, as follows: Microbio 299 Independent study- Fall 2018-2019: 2 students (Anuchit Rupanya, Chavin Buasakdi) Spring 2018-2019: (Chavin Buasakdi) Microbio 681 and 682 - Senior Honors Thesis- Fall 2016-2017: 1 student (Tai Chaimarit) Spring 2016-2017: 1 student (Tai Chaimarit) How have the results been disseminated to communities of interest? The results for this project have been disseminated in scientific papers, national conferences, and meetings. In addition, we have two more publications, currently in preparation, that will be submitted during the summer and fall of 2021. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The overarching goal of this project was to characterize global metabolic remodeling during biofilm development using cutting-edge LC-MS-based metabolomic and proteomic approaches and, in combination with computational modeling, identifytranscriptional regulatory network responsible for metabolic remodeling during biofilm developmentusing cutting-edge LC-MS-based metabolomic and proteomic approaches, in combination with quantitative computational modeling. As detailed in previous annual reports, all goals in objectives 1 and 2 were successfully accomplished, with the onlyexception of item 3 in goal 2, which was only partially completed. Below is asummary of major accomplisments resulting from this proposal, which overall exceeded the original goals of this proposal by expanding into new related research areas. Systems-level analysis of metabolic remodeling during biofilm development of B. subtilis. We performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. We found a surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations, including increased TCA cycle activity, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron homeostasis, and a switch from acetate to acetoin fermentation, were previously unrecognized as biofilm-associated. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provided insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This work represents the most comprehensive and systematic characterization of metabolic alterations during biofilm development ever undertaken (Pisithkul et al, 2019): Pisithkul T, Schroeder JW, Trujillo A, Yeesin P, Chaiamarit Tai, Coon JJ, Wang JD, Amador-Noguez D. (2018) Metabolic remodeling during biofilm development of Bacillus subtilis. mBio, May 21;10(3). pii: e00623-19 Remodeling of lipid metabolism is required for B. subtilis biofilm development. We used LC-MS-based lipidomics to reveal that B. subtilis biofilm development is associated with complex dynamic changes in lipid membrane composition. We then used 13C-tracers to quantitative rates of synthesis of individual fatty acids and phospholipids during biofilm development. We then showed that knocking out certain fatty acid and lipid synthesis or degradation genes severely impairs biofilm development but show no adverse effects during planktonic growth. These results revealed that specific alterations to fatty acid and lipid metabolism, and the resulting changes in lipid membrane composition, are required for robust biofilm development in B. subtilis (Lucas et al, in preparation). Development of improved LC-MS-based methods to detect and quantitate B. subtilis alarmones. (p)ppGpp is a highly conserved bacterial alarmone which regulates many aspects of cellular physiology and metabolism. The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp are highly conserved bacterial alarmones that regulate many aspects of cellular physiology and metabolism. We developed an improved LC-MS-based method to detect a broad spectrum of alarmones and other small signaling molecules from bacterial cultures with high efficiency. We used this improved method to advance our understanding of the physiological role of alarmone synthetases (i.e. SasA) in B. subtilis (Fung et al, 2020) and to demonstrate the role of the nucleotide pGpp acts as a novel alarmone in B. subtilis (Yang et al, 2020). Fung DK, Yang J, Stevenson DM, Amador-Noguez D, Wang JD. (2020) Small Alarmone Synthetase SasA Expression Leads to Concomitant Accumulation of pGpp, ppApp, and AppppA in Bacillus subtilis. Front Microbiol. 2020 Sep 2;11:2083. Yang J, Anderson BW, Turdiev A, Turdiev H, Stevenson DM, Amador-Noguez D, Lee VT, Wang JD (2020) The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p) ppGpp. Nat Commun. 2020 Oct 23;11(1):5388
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
Lipid remodeling during biofilm development in Bacillus Subtili.
Authors: Tippapha Pisithkul and Daniel Amador-Noguez
Expected submission date is summer 2021.
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
Metabolic flux analysis of lipid synthesis during biofilm development.
Authors: Lauren Lucas and Daniel Amador-Noguez
Expected submission date is summer 2021.
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience: 1) Graduate and undergraduate students participating directly in research projects in the Amador-Noguez lab 2) Undergraduates students taking the course 'Physiology of microorganisms', which is taught by Dr. Amador-Noguez Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The research project of agraduate student (Tippapha Pisithkul, CMB)that graduated last year was partially supported by this grant. The research projects of a current graduate student (Lauren Lucas, MDTP) were partially supported by this grant. This project also supported training of several undergraduates in the laboratory, as follows: Microbio 299 Independent study Fall 2018-2019: 2 students (Anuchit Rupanya, Chavin Buasakdi) Spring 2018-2019: (Chavin Buasakdi) Microbio 681 and 682 - Senior Honors Thesis Fall 2016-2017: 1 student (Tai Chaimarit) Spring 2016-2017: 1 student (Tai Chaimarit) How have the results been disseminated to communities of interest?The results for this project have been disseminated in national conferences and meetings. In addition, we have two publications currently in preparation that will be submitted during the summer and fall of this year. What do you plan to do during the next reporting period to accomplish the goals?Ongoin and future work for 2020: The work generated by this project will serve as the foundationfor ongoing and future work in our laboratory that will further elucidate the role of metabolism in bacterial biofilm development. One of the areas that we are currently pursuing is membrane lipid remodeling during B. subtilis biofilm development. The specific project we are currently working on are: 1) Quantitate biosynthesis rates for fatty acids and lipids inBacillus subtilisduring biofilm development. 2)Determine physiological relevance of each lipid class forB. subtilisbiofilm development. We will continue to explore associations between modified lipid profiles and resulting phenotypes will give insight into what role each lipid plays in biofilm development. 3) Understand population heterogeneity in the context of fatty acid and lipid biosynthesis and degradation. We will continue our work on using confocal laser scanning microscopy to image the whole, intact biofilm to determine which populations of cells are undergoing each metabolism.
Impacts
What was accomplished under these goals?
Goal 1: Characterize global metabolic remodeling during biofilm development. 1.Measure alterations in metabolite concentrations during biofilm development. We carried out an integrated metabolomic-transcriptomic-proteomic analysis that revealed a widespread and dynamic remodeling of metabolism duringB. subtilisbiofilm development that affected central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Some of the major highlights include: upregulation of the TCA cyle during early biofilm development;alterations in de novo nucleotide biosynthesis;transient upregulation of extracellular matrix (ECM) synthesis;widespread alterations in iron acquisition and metabolism, including upregulation of bacillibactin biosynthesis and transport, a switch in electron-transfer proteins from ferredoxin to flavodoxin and upregulation of pulcherrimin synthesis;Transition from fatty acid synthesis to degradation during biofilm development; andwe also found that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. 2.Quantitate changes in metabolic flux and pathway utilization during biofilm development To further investigate changes in TCA cycle activity stated above, we performed dynamic isotope tracer experiments at 8, 16, 24, and 32 hours using13C-glycerol. In agreement with increased levels of TCA cycle intermediates and increased enzyme abundance, dynamic labeling experiments indicated that carbon flux into the TCA cycle, both via citrate synthase and anaplerotic reactions, increased considerably at 16 hours before decreasing somewhat at 24 and 32 hours. Increased carbon flux into the TCA cycle was matched by increased flux into lower glycolytic intermediates. Taken together, our data show that TCA cycle activity is rapidly upregulated during early biofilm development. Upregulation of the TCA cycle was concurrent with increased levels of nucleotides, deoxynucleotides, and their biosynthetic intermediates.The intracellular levels of nucleotides (NTPs), their biosynthetic precursors, and their deoxy counterparts (dNTPs) followed highly similar profiles during biofilm development. They all increased early in biofilm development, reached a peak at 16 hours, and declined gradually afterward. This coordinated pattern indicated upregulation of nucleotide biosynthesis during early biofilm growth. In agreement with this, dynamic13C-glycerol tracer experiments revealed an increased rate of13C-carbon incorporation into purines and pyrimidines at 16 hours. Goal 2: Identify and experimentally validate components of the transcriptional regulatory network responsible for metabolic remodeling during biofilm development The close agreement between metabolomic, transcriptomic, proteomic, and isotope tracer measurements indicated that metabolic remodeling during biofilm development was largely controlled at the transcriptional level.To identify the transcriptional regulators likely responsible for this metabolic remodeling, we performed a global analysis of transcription factor (TF) activity based on ourgene expression data by applying network component analysis in conjunction with recently published models of theB. subtilisglobal transcriptional network. Out of 227B. subtilisTFs, we identified 179 TFs with significant changes in activity (p < 0.01 with FDR = 1.27%) during biofilm development.This TF activity analysis identified transcriptional regulators likely responsible for the metabolic remodeling we have described during biofilm development. Our analysis also indicated differential utilization of sigma factors during biofilm development.Specifically, the inferred activity of four sigma factors,sE,sL,sB,andsD, changed drastically. The activity ofsEand sLincreased steadily over time,sBactivity decreased sharply between 12 to 20 hours, andsDactivity decreased gradually. Transcript levels of these four sigma factors followed closely their activity profiles and, with the exceptionsL, their protein abundance was also well correlated with their activity. The specific project we are currently working on are: 1) Quantitate biosynthesis rates for fatty acids and lipids inBacillus subtilisduring biofilm development. We are using isotope tracers (13C-glycerol, 13C-glutamate,15N-glutamate, and18O-phosphate) to quantitatein vivorates of fatty acid and lipid synthesis during biofilm development. Measurement of fatty acid synthesis rates is being carried out by tracking the dynamic incorporation of13C from glycerol or glutamate into saponified fatty acid using LC-MS/MS. This analysis will provide information on changes in fatty acid chain lengths, branching, and saturation. In addition, we are monitoring13C incorporation into fatty acid intermediates bound to acyl-carrier proteins (ACPs). Measurement of lipid synthesis rates is also been done by tracking incorporation of13C-carbons,15N-glutamate, or18O-phosphate into extracted phospholipids. To estimate fatty acid and lipid synthesis rates from dynamic isotope tracer data, we are using isotopically nonstationary metabolic flux analysis (INST-MFA). This analysis will distinguish whether changes in membrane lipid composition are due tode novosynthesis or recycling/modification of existing lipids and fatty acids. 2)Determine physiological relevance of each lipid class forB. subtilisbiofilm development.We are systematically deleting membrane lipid synthesis genes to determine which lipid classes matter most to robust biofilm development via assessment of biofilm appearance, cell count, dry weight, spore fraction, lipid profile, and cell morphology through all stages of biofilm growth. Associations between modified lipid profiles and resulting phenotypes will give insight into what role each lipid plays in biofilm development. 3) Understand population heterogeneity in the context of fatty acid and lipid biosynthesis and degradation. Cells throughout the biofilm may have varying expression of fatty acid synthesis and degradation pathways. We are creating transcriptional reporters for operons in both fatty acid biosynthesis and degradation pathways to visualize where these transcriptional changes are occurring in theB. subtilisbiofilm. For biosynthesis we are creating a reporter strain for the fapR-plsX-fabD-fabG operon and for degradation the fadN-fadA-fadE operon. Each will have a differently colored fluorescent protein that will allow for simultaneous imaging of both metabolic processes. We are using confocal laser scanning microscopy to image the whole, intact biofilm to determine which populations of cells are undergoing each metabolism.
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2020
Citation:
Lipid remodeling during biofilm development in Bacillus Subtili.
Authors: Tippapha Pisithkul and Daniel Amador-Noguez
Expected submission date is summer 2020
- Type:
Journal Articles
Status:
Other
Year Published:
2020
Citation:
Metabolic flux analysis of lipid synthesis during biofilm development.
Authors: Lauren Lucas and Daniel Amador-Noguez
Expected submission date is summer 2020
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Metabolic remodeling during biofilm development of Bacillus subtilis
Oral Presentation. 2019 Molecular Genetics of Bacteria & Phages Meeting. Madison, WI August 2019
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Metabolic remodeling during biofilm development of Bacillus subtilis
Oral presentation. ASM Microbe 2019. San Francisco, CA June 2019
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Changes in lipid membrane composition during biofilm development in B. subtilis
Oral Presentation. CSHL Metabolomics Seminar. Cold Spring Harbor Laboratory, NY June 2019
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience: 1) Graduate and undergraduate students participating directly in research projects in the Amador-Noguez lab 2) Undergraduates students taking the course 'Physiology of microorganisms', which is taught by Dr. Amador-Noguez Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A graduate student is being trained on this project. How have the results been disseminated to communities of interest? The results will be disseminated as publications in scientific journals. What do you plan to do during the next reporting period to accomplish the goals? We will continue to work on the goals.
Impacts
What was accomplished under these goals?
Goal 1: Characterize global metabolic remodeling during biofilm development. 1.Measure alterations in metabolite concentrations during biofilm development. We carried out an integrated metabolomic-transcriptomic-proteomic analysis that revealed a widespread and dynamic remodeling of metabolism during B. subtilis biofilm development that affected central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Some of the major highlights include: 1) Upregulation of the TCA cycle during early biofilm development 2) Alterations in de novo nucleotide biosynthesis 3) Transient upregulation of extracellular matrix (ECM) synthesis 4) Widespread alterations in iron acquisition and metabolism, including upregulation of bacillibactin biosynthesis and transport, a switch in electron-transfer proteins from ferredoxin to flavodoxin and upregulation of pulcherrimin synthesis 5) Transition from fatty acid synthesis to degradation during biofilm development 6) Finally, we also found that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. 2.Quantitate changes in metabolic flux and pathway utilization during biofilm development To further investigate changes in TCA cycle activity stated above, we performed dynamic isotope tracer experiments at 8, 16, 24, and 32 hours using 13C-glycerol. In agreement with increased levels of TCA cycle intermediates and increased enzyme abundance, dynamic labeling experiments indicated that carbon flux into the TCA cycle, both via citrate synthase and anaplerotic reactions, increased considerably at 16 hours before decreasing somewhat at 24 and 32 hours. Increased carbon flux into the TCA cycle was matched by increased flux into lower glycolytic intermediates. Taken together, our data show that TCA cycle activity is rapidly upregulated during early biofilm development. Upregulation of the TCA cycle was concurrent with increased levels of nucleotides, deoxynucleotides, and their biosynthetic intermediates. The intracellular levels of nucleotides (NTPs), their biosynthetic precursors, and their deoxy counterparts (dNTPs) followed highly similar profiles during biofilm development. They all increased early in biofilm development, reached a peak at 16 hours, and declined gradually afterward. This coordinated pattern indicated upregulation of nucleotide biosynthesis during early biofilm growth. In agreement with this, dynamic 13C-glycerol tracer experiments revealed an increased rate of 13C-carbon incorporation into purines and pyrimidines at 16 hours. Goal 2: Identify and experimentally validate components of the transcriptional regulatory network responsible for metabolic remodeling during biofilm development. The close agreement between metabolomic, transcriptomic, proteomic, and isotope tracer measurements indicated that metabolic remodeling during biofilm development was largely controlled at the transcriptional level. To identify the transcriptional regulators likely responsible for this metabolic remodeling, we performed a global analysis of transcription factor (TF) activity based on our gene expression data by applying network component analysis in conjunction with recently published models of the B. subtilis global transcriptional network. Out of 227 B. subtilis TFs, we identified 179 TFs with significant changes in activity (p < 0.01 with FDR = 1.27%) during biofilm development. This TF activity analysis identified transcriptional regulators likely responsible for the metabolic remodeling we have described during biofilm development. Upregulation of TCA cycle genes during early biofilm development appeared driven by the simultaneous decrease in activity of the carbon catabolite control proteins CcpA and CcpC, and the pleiotropic global regulator CodY, all three of which negatively regulate expression of genes in this pathway. Transcription of ECM genes is controlled by core components of the biofilm regulatory network. Their initial increase in transcription was likely driven by increased Spo0A activity and the resulting decrease in activity of the transcriptional repressors AbrB and SinR. Their subsequent decrease in transcript levels at later time points may be due to the recovery of SinR activity and decreased activity of transcriptional activators such as RemA, LutR, and DegU. The increased expression of bacillibactin synthesis genes (dhbACEBF operon) at the beginning of biofilm formation was likely driven by the simultaneous decrease in activity of the transcriptional repressors Fur and AbrB. The concomitant increase in iron-bound bacillibactin transporter genes (feuABC operon) may be similarly explained by decreased Fur activity together with increased Btr activity. Decreased Fur activity is also likely responsible for the upregulation of Flavodoxin genes (ykuNOP), whose expression profile is nearly identical to that of bacillibactin synthesis and transport genes. The switch-like transition from fatty acid and phospholipid biosynthesis to fatty acid beta-oxidation that occurred between 12 to 16 hours during biofilm development was likely driven by simultaneous but opposite changes in activity of the transcriptional repressors FapR and FadR. FapR, whose inferred activity increased sharply between 12-16 hours, represses expression of multiple operons encoding fatty acid biosynthetic genes. FadR, whose inferred activity decreased steadily over time, is a general repressor of fatty acid beta-oxidation pathways. Increased fatty acid degradation was also likely driven by decreased CcpA activity while decreased activity of the positive regulator ComA may have also contributed to decreased fatty acid synthes. Some transcription factors displayed a global role in regulating metabolic remodeling during biofilm development. Specifically, our results indicate that the global metabolic regulator CcpA, which governs transcription of over 250 genes, plays an important role at regulating the TCA cycle, fatty acid beta-oxidation, and acetoin production during biofilm development. Our analysis also indicated differential utilization of sigma factors during biofilm development. Specifically, the inferred activity of four sigma factors, sE, sL, sB, and sD, changed drastically. The activity of sE and sL increased steadily over time, sB activity decreased sharply between 12 to 20 hours, and sD activity decreased gradually. Transcript levels of these four sigma factors followed closely their activity profiles and, with the exception sL, their protein abundance was also well correlated with their activity.
Publications
|
Progress 12/01/16 to 09/30/17
Outputs
Target Audience:1) Undergraduate students participating directly in research projects in the Amador-Noguez lab 2) Undergraduates students taking the course 'Physilogy of microorganisms', which is taugh by Dr. Amador-Noguez. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project is now supporting the thesis work of a new graduate student in my laboratory. In addition, this project has also supported teaching and mentoring of undergraduate research students in the lab. How have the results been disseminated to communities of interest?The results will be disseminated as publications in scientific journals before the end of 2018. What do you plan to do during the next reporting period to accomplish the goals?We are making steady progress in this project. We will continue to follow the initial proposal to acomplish the goals of the next reporting period.
Impacts
What was accomplished under these goals?
IMPACT Despite its importance, we still know very little about the physiology and metabolism of bacterial biofilm as most research conducted to date has been focused on cultures of 'free-living' (planktonic) bacteria. Using the common soil bacterium Bacillus subtilis as a model system, the work in this proposal will provide the first systematic investigation of metabolism during bacterial biofilm formation and development, and as such, it has the potential to transform our understanding of bacterial metabolism and biofilm developmental processes. Our work will be performed by utilizing state-of-the-art tools (metabolomics) that can provide a holistic and quantitative view of all relevant aspects of metabolism related to biofilm formation and development. The metabolomics experiments proposed here represent a novel approach that has not previously been used to investigate biofilm formation in any bacteria. We therefore anticipate that this research plan will generate new fundamental insights into the process, control, and metabolism of bacterial biofilms. Given the ubiquity of bacterial biofilms in natural environments and agricultural settings, the knowledge generated by this project will be broadly applicable and have a significant impact on agriculture and food safety. ACOMPLISHMENTS Goal 1: Characterize global metabolic remodeling during biofilm development- 1. Measure alterations in metabolite concentrations during biofilm development. Intracellular metabolomic analysis of biofilm samples were performed using a set of LC-MS methods that enable measurement of over 200 known metabolites. Of the analyzed metabolites, 166 were reproducibly measured and 127 displayed significant alterations in their intracellular levels over the course of biofilm formation and development (p<0.05; ANOVA). Our analysis revealed remarkably dynamic changes during biofilm development in most of the measured metabolic pathways. These alterations encompassed both primary metabolic pathways such as glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, nucleotide and amino acid biosynthesis, as well as secondary metabolic pathways such as acetoin, pulcherrimin, and bacillibactin biosynthesis, among others. While the profiles of metabolites belonging to the same class or to the same pathway did not always display a coordinated behavior, numerous trends were evident. For example, in primary metabolism, the levels of most TCA cycle intermediates displayed a sharp peak during early biofilm formation (12 to 16 hours) followed by a steep decrease in subsequent development. Amino acids closely linked to primary metabolism (glutamate, glutamine, aspartate, and alanine) displayed a rapid decrease in early biofilm development; prephenate-derived aromatic amino acids (phenylalanine and tyrosine) increased steadily over time; and branched-chain amino acids (leucine, Isoleucine, and valine) levels peaked during mid-biofilm development (~20 hours). Nucleotide triphosphates (ATP, GTP, UTP, CTP), and nucleotide biosynthetic precursors, displayed maximum levels around 16-24 hours while the levels of nucleotide salvage intermediates (e.g. nucleosides and nucleobases) increased in late biofilm development. In secondary metabolism, N-acetylated amino acids (e.g. N-acetyl-glutamine and N-acetyl-glutamate) and nucleotide sugars (e.g. UDP-Glc, UDP-GlcNAc, and ADP-Glc) displayed a transient, sharp increase during early biofilm development. In addition to the intracellular metabolites, we also examined extracellular metabolites in the spent biofilm-inducing medium. We observed significant amount of TCA cycle intermediates, de novo UMP biosynthetic precursors (i.e. dihydroorotate and orotate), fermentation products, and small peptide derivatives. Interestingly, the levels of fermentation products and the small peptide derivatives accumulated over the course of biofilm growth. 2. Quantitate changes in metabolic flux and pathway utilization during biofilm development- We have implemented a methodology for using 13C-isotope tracers to quantitate metabolic fluxes during biofilm development. This year we will apply these methods to investigate changes in metabolic activity in a variety of growth conditions and mutant strains. Goal 2: Identify and experimentally validate components of the transcriptional regulatory network responsible for metabolic remodeling during biofilm development- 1. Determine correlations between metabolic remodeling and transcriptional changes in the biofilm regulatory network- In parallel to metabolomic measurements, we performed gene expression analysis (mRNAseq) during biofilm formation and development. Using Illumina Hiseq2000, we obtained over 10 million reads per library. The sequences were mapped to 4,348 unique genes in the genome: 4,246 on the chromosome and 102 genes on the macroplasmid. Out of the 4,348 mapped genes, 2,477 genes (~57% of the genome) had significant alterations in transcript levels. Mirroring our metabolomics data, RNA-seq data analysis showed highly structured and dynamic changes in primary and secondary metabolism genes as well as transcription regulators and genes involved in cell cycle and cellular component biogenesis during biofilm development. Genes involved in cell wall synthesis, bacteriocin biosynthesis, acetoin biosynthesis, and sporulation were enriched in those with continuous increased expression over time. Among those with decreasing transcriptional expression were flagellar biogenesis and organization and motility genes. Glycolytic and chemotactic genes had generally high expression levels during planktonic and early biofilm growth (8-16 hours of growth in the biofilm-inducing medium). The polyketide and polysaccharide (including exopolysaccharide (eps)) biosynthetic genes were upregulated specifically during 16 hours of growth. Lastly, genes involved in iron homeostasis and antibiotic biosynthesis were generally upregulated from 16-24 hours of growth. 2. Elucidate causal relationships between changes in metabolism and activation of specific components in the biofilm transcriptional regulatory network We hypothesized that metabolism associated with a relatively slow, developmental processes such as biofilm development, is transcriptionally regulated. We then sought to identify key factors underlying those transcriptional changes. Using our RNA-sequencing data in combination with established algorithms that integrate knowledge of the B. subtilis transcriptional network, we inferred activities of 227 B. subtilis transcription factors (TFs) over the course of biofilm growth. These results recapitulate know behaviors in the biofilm transcriptional regulatory network but also suggest a critical role for novel transcription factors not previously associated with biofilm development, such as metabolic regulators involved in carbon and nitrogen metabolism. We are currently analyzing these results to elucidate causal relationships between changes in metabolism and activation of these newly discovered components of the biofilm transcriptional regulatory network. 3. Identify common metabolic effects of biofilm-activating factors and signals- This year, we will be testing effects of biofilm-activating factors and signals commonly found in the soil or in root exudates.
Publications
|
|