Progress 09/15/05 to 09/14/08
Outputs
OUTPUTS: The probiotic bacterium Lactobacillus acidophilus NCFM employs a variety of mechanisms to survive passage and compete within the gastrointestinal tract. One important mechanism is likely cell-to-cell communication, mediated by autoinducer-2 (AI-2), to signal and adapt to changing environmental conditions. Genome analysis of L. acidophilus NCFM showed that the bacterium encodes the genetic elements for the synthetic pathway for AI-2 production. During this study it was shown that AI-2 is first produced during the early growth stage and accumulates throughout logarithmic growth phase through expression of the AI-2 synthase, LuxS. Using two approaches the LuxS gene, responsible for AI-2 synthesis, was inactivated: (i) via insertional inactivation using a plasmid integration vector; and (ii) via a luxX gene deletion using a double crossover recombination event to remove the gene from the L. acidophilus chromosome. Both the integration and deletion approaches created isogenic mutants in the LuxS gene that could be analyzed for production of AI-2 (primary project objective) and any changes in phenotype (primary project objective) when compared to the AI-2 producing parental culture. Phenotypic microarray (PM, 1152 assays) experiments were performed for NCFM and a luxS deletion mutant using twelve 96 well PM arrays (containing osmolytes, pH ranges and chemical substrates). Adherence experiments with the LuxS mutant and NCFM were also conducted in vitro with Caco-2 intestinal epithelial cells. Global transcriptional arrays compared gene expression at varying growth phases for the parent and LuxS mutant. The transcriptional and phenotypic results for the mutants generated were reported at the 2008 American Academy of Microbiology Annual Meeting and the 9th International Symposium on Lactic Acid Bacteria held in the Netherlands in September 2008. The data generated by this study are being disseminated by seminars, presentations and posters at various national and international meetings, and via publications that are in press for 2008/2009. PARTICIPANTS: Dr. Sarah OFlaherty, Post-doctoral research associate. Responsible for adherence assays with Caco-2 intestinal epithelial cells and the set up, conduct and interpretation of the phenotypic array platform on the wild-type vs LuxS mutants. Provided training for entry level graduate students in functional genomic techniques (gene integration/deletion), adherence assays, and phenotypic assays. Dr. Andrea Azcarate-Peril, Senior Scientist, Department of Food Science, NC State University. Responsible for establishing the microarray platforms used in this study, bioinformatic analysis of the data, and training of students and post-doctoral scientists on microarray technologies. Dr. B. Logan Buck, Ph.D. candidate, Department of Microbiology, and NIH-Biotechnology Fellow, NC State University. Ph.D. Thesis involved the detection of AI-2 production in Lactobacillus acidophilus, functional genomic analysis of the pathway for synthesis, and targeted the genetic knockout of luxS which is responsible for synthesis of the AI-2 signaling molecule. The luxS negative mutant was compared to the parent in the microarray analysis. Training provided by the project included functional genomics for gene inactivation and deletion, microarray hybridizations and bioinformatic analyses. Training and professional development during this period included participation at international meetings and presentation of the work conducted in this project. These meetings included - American Dairy Science Association Meeting (July 2008) where a major symposium presentation was made by Dr. Azcarate-Peril on the microarray platform used in this study. 9th International Symposium on Lactic Acid Bacteria (Sept 2008) where Dr. OFlaherty presented a poster on "Whole genome expression analysis of human intestinal epithelial cells in response to Lactobacillus acidophilus". Partnering organizations included Dairy Management Inc., NC Dairy Foundation and the Southeast Dairy Foods Research Center. TARGET AUDIENCES: Target audiences are the following: 1. Scientists and researchers working with lactic acid bacteria used as starter cultures and probiotics. 2. Industries that manufacture and distribute probiotic cultures in foods or as dietary supplements 3. 4. Commercial culture manufacturers that provide probiotic cultures to the food, dairy, and dietary supplement industries. Target audiences are: 1. Scientific audiences for gram positive commensal bacteria and their roles in the GI tract. 2. Members of the community of scientists working with lactic acid bacteria important to food fermentation, bioprocessing and health. 3. Scientists and industries working with probiotic bacteria to promote health and well being, and define the mechanisms underlying those activities. 4. Commercial culture manufacturers that provide probiotic cultures to the food, dairy, and dietary supplement industries. 5. Public/Consumers interested in mechanisms by which probiotic cultures impact general health and well being. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The bacterium Lactobacillus acidophilus NCFM employs a variety of mechanisms to survive passage and compete within the gastrointestinal tract. One important mechanism is likely cell-to-cell communication, mediated by autoinducer-2 (AI-2), to signal and adapt to changing environmental conditions. AI-2 is produced by L. acidophilus NCFM and during this study it was shown that AI-2 is first produced during the early growth stage and accumulates throughout logarithmic growth phase through expression of the AI-2 synthase, LuxS. Using two approaches the LuxS gene, responsible for AI-2 synthesis, was inactivated: (i) via insertional inactivation using a plasmid integration vector; and (ii) via a luxX gene deletion using a double crossover recombination event to remove the gene from the L. acidophilus chromosome. Both approaches eliminated the ability of L. acidophlus NCFM to produce AI-2. Phenotypic analysis of the LuxS negative mutants revealed a 58 % decrease in adherence to Caco-2 cells when the bacterial cells were used for adhesion assays directly from logarithmic phase cultures. Phenotypic microarray (PM) experiments for NCFM and a luxS deletion mutant were performed with twelve 96 well PM arrays (containing osmolytes, pH ranges and chemical substrates). Results indicated that only 2 of 1152 wells showed minimal differences in growth in the chemical substrates plumbagin and piperacillin, an oxidizing agent and a lactam antibiotic, respectively. A whole genome microarray analysis of NCFM and a LuxS- mutant strain was performed at early, middle, and late-logarithmic phases to investigate global gene expression influenced by AI-2 during growth. The highest number of genes were differentially expressed between the mutant and wild-type at early-log phase (OD600 0.2), and included genes related to growth, metabolism, and stress response. Few genes showed differential expression at mid-log phase (OD600 0.7), but included a transporter (LBA1796) and response regulator (LBA1798) for bacteriocin production. No differentially expressed genes were detected at late-log phase (OD600 1.2) under the conditions tested. Of the differentially expressed genes, most were positively influenced by LuxS. Examination of the stress response of NCFM revealed that upon exposure to heat and bile stress, the LuxS- mutant was more sensitive than the wild type when populations were harvested from early or middle-log phase. These results agreed with the overexpression of heat and bile-stress related genes in the wild type compared to the mutant strain during the early growth stages. AI-2 signaling during the early exponential phase appears to prepare the developing cell population for stress and interactive conditions naturally encountered during planktonic growth. This study provided the first report directly linking AI-2 production with adherence functions in a probiotic lactobacilli. Microarray analysis of the impact of a luxS mutation throughout the bacterial growth phase was also shown to impact the survivability of the bacterium in the later stages of stationary phase growth where low pH and nutrient depletion are encountered.
Publications
- Buck, B.L., A.Azcarate-Peril, and T.R. Klaenhammer. 2008. Role of autoinducer 2 on the adhesion ability of Lactobacillus acidophilus. J. Appl. Microbiology. In Press.
- Yong Jun Goh, M. Andrea Azcarate-Peril, Sarah OFlaherty, Evelyn Durmaz, Florence Valence, Daniel Molle, Sylvie Lortal, and Todd R. Klaenhammer. 2008. Development of a upp-based Counterselective Gene Replacement System for Study of the S-layer protein SlpX in Lactobacillus acidophilus NCFM. Appl. Environ. Microbiol. In Press.
- Goh, Y.J., E. Durmaz, F. Valence, D. Molle, S. Lortal, T.R. Klaenhammer. 2008. A markerless gene replacement system for Lactobacillus acidophilus NCFM with UPP-Based counterselection strategy. Proceedings of the 9th Symposium of Lactic Acid Bacteria. August 31-September 4th, 2008.
- OFlaherty, S. and T. R. Klaenhammer. 2008. Whole genome expression analysis of human intestinal epithelial cells in response to Lactobacillus acidophilus. 2008. Proceedings of the 9th Symposium of Lactic Acid Bacteria. August 31-September 4th, 2008.
- Klaenhammer, T.R., E. Altermann, E. Pfeiler, B.L. Buck, T. Duong, Y.J. Goh, R. Tallon, S. OFlaherty, R. Barrangou, and M.A. Azcarate-Peril. 2008. Functional genomics of probiotic lactobacilli. J. Clinical Gastroenterology. 42: S160-2.
- Duong, T. and T.R. Klaenhammer. 2008. Functional genomics of lactic acid bacteria. In, Therapeutic Microbiology: Probiotics and Other Strategies. J. Versalovic and M. Wilson, Eds. Pp 193-204. ASM Press, Washington, DC.
- Goh, Y.J. and T.R. Klaenhammer. 2009. Genomic features of lactobacillus species. Frontiers of Bioscience. In Press.
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Progress 09/15/06 to 09/14/07
Outputs
OUTPUTS: The bacterium Lactobacillus acidophilus NCFM employs a variety of mechanisms to survive passage and compete within the gastrointestinal tract. One important mechanism is likely cell-to-cell communication, mediated by autoinducer-2 (AI-2), to signal and adapt to changing environmental conditions. AI-2 is produced by L. acidophilus NCFM and accumulates throughout logarithmic growth phase through expression of the AI-2 synthase, LuxS. A whole genome microarray analysis of L. acidophilus NCFM and a LuxS- mutant strain was performed at early, middle, and late-logarithmic phase to investigate global gene expression influenced by AI-2 during growth. Hybridizations were carried out in a round-robin design and data were analyzed using a two-stage mixed model ANOVA. The highest number of genes was differentially expressed between the mutant and wild-type at early-log phase (OD600 0.2), and included genes related to growth, metabolism, and stress response. Few genes showed differential
expression at mid-log phase (OD600 0.7), but included a transporter (LBA1796) and response regulator (LBA1798) of a bacteriocin production and transport operon. No differentially expressed genes were detected at late-log phase (OD600 1.2) under the conditions tested. Of the differentially expressed genes, most were positively influenced by LuxS. Examination of the stress response of L. acidophilus NCFM revealed that upon exposure to heat and bile stress, the LuxS- mutant was more sensitive than the wild type when populations were harvested from early or middle-log phase. These results agreed with the overexpression of heat and bile-stress related genes in the wild type compared to the mutant strain during the early growth stages. AI-2 signaling during the early exponential phase appears to prepare the developing cell population for stress and interactive conditions naturally encountered during planktonic growth. This study provided the first microarray analysis of the impact of a luxS
mutation throughout the bacterial growth phase
PARTICIPANTS: Dr. Andrea Azcarate-Peril, Senior Scientist, Department of Food Science, NC State University. Responsible for establishing the microarray platforms used in this study, bioinformatic analysis of the data, and training of students and post-doctoral scientists on microarray technologies. Dr. B. Logan Buck, Ph.D. candidate, Department of Microbiology, and NIH-Biotechnology Fellow, NC State University. Ph.D. Thesis involved the detection of AI-2 production in Lactobacillus acidophilus, functional genomic analysis of the pathway for synthesis, and targeted the genetic knockout of luxS which is responsible for synthesis of the AI-2 signaling molecule. The luxS negative mutant was compared to the parent in the microarray analysis. Training provided by the project included functional genomics for gene inactivation and deletion, microarray experiments and bioinformatic analyses. Partnering organizations included Dairy Management Inc., and the Southeast Dairy Foods Research
Center.
TARGET AUDIENCES: Target audiences are the following: 1. Scientists and researchers working with lactic acid bacteria used as starter cultures and probiotics. 2. Industries that manufacture and distribute probiotic cultures in foods or as dietary supplements 3. Public/Consumers interested interested in mechanisms by which probiotic cultures impact general health and well being.
Impacts
Discovery of genes and pathways that are regulated by quorum sensing and signaling molecules is expected to uncover critical mechanisms through which probiotic cultures survive gastric passage, interact with the intestinal epithelium, and communicate with other members of the microbiota. From the results obtained in this period, it was determined that the AI-2 signaling molecule is produced in mid-log growth phase, and prepares the culture for tolerance to stresses that may be encountered in the gastrointestinal tract. Understanding these mechanisms and elucidating how probiotic bacteria impact health is expected to fuel the expanded use of these cultures in foods and dietary supplements. Specialized genomic facilities were utilized in this project. Notably the College of Agriculture and Life Sciences Genomic Research Laboratory (GRL), at North Carolina State University. Microarray slides were printed and scanned at the GRL, and analyzed with JMP Genomics software,
provided to NCSU by SAS.
Publications
- Azcarte-Peril, M.A., B. Bruno-Barcena, H.M. Hassan, and T.R. Klaenhammer. 2006. Transcriptional and functional analysis of oxalyl-coenzyme A (CoA) decarboxylase and formyl-CoA transferase genes from Lactobacillus acidophilus. Appl. Environ. Microbiol. 72:1891-1899.
- Klaenhammer, T.R., Pfeiler, E., and Duong, T. 2007. Genomics and Proteomics of Food-Borne Microorganisms. In Doyle, Beuchat, and Montville (eds.). Food Microbiology: Fundamentals and Frontiers, 3rd edition. ASM Press, Washington, DC. pp 935-952.
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Progress 09/15/05 to 09/15/06
Outputs
Microarray analysis of the global transcriptional response of Lactobacillus acidophilus NCFM was conducted with cells that were adherent to Caco-2, differentiated intestinal epithelial cells. Several genes potentially involved with adhesion to the intestinal epithelium were identified. Most notably, putative genes linked to the synthesis of an interspecies signaling molecule, autoinducer-2 (AI-2), were over expressed. Examination of the L. acidophilus NCFM genome revealed the complete pathway for AI-2 synthesis. AI-2 activity was detected in L. acidophilus NCFM during stationary growth phase using the Vibrio harveyi BB170 assay system. Using site-specific integration, an isogenic mutation was created in luxS and the resulting mutant of L. acidophilus NCFM did not produce AI-2. A 58 % decrease in adherence to Caco-2 cells was observed in the LuxS- mutant when the cells were used for adhesion directly from logarithmic phase cultures. A deletion of the luxS gene was also
created and found not to produce AI-2. This mutant will be used to analyze and compare the global transcriptional responses of lactobacilli producing, or not producing AI-2. The involvement of the AI-2 signaling molecule in adhesion represents one of the first phenotypes attributed to LuxS in lactobacilli. Other recent reports have implicated AI-2 in biofilm formation in lactobacilli.
Impacts
Discovery of genes and pathways that are regulated by quorum sensing and signaling molecules is expected to uncover critical mechanisms through which probiotic cultures survive gastric passage, interact with the intestinal epithelium, and communicate with other members of the microbiota. Understanding these mechanisms and elucidating how probiotic bacteria impact health is expected to fuel the expanded use of these cultures in foods and dietary supplements.
Publications
- Klaenhammer, T.R., R. Barrangou, B.L. Buck, M.A. Azcarate-Peril, E.Altermann. 2005. Genomic features of lactic acid bacteria effecting bioprocessing and health. FEMS Microbiology Reviews. 29:393-409.
- Azcarate Peril, M.A., O. McAuliffe, E. Altermann, S.Lick, W. M. Russell, and T.R. Klaenhammer. 2005. Microarray Analysis of a Two Component Regulatory System Involved in Acid Resistance and Proteolytic Activity in Lactobacillus acidophilus. Appl. Envir. Microbiol. 71: 5794 5804.
- Barrangou, R., M.A. Azcarate-Peril, T.Duong, S.B. Conners, R.M. Kelly, and T.R. Klaenhammer. 2006. Global analysis of carbohydrate utilization by Lactobacillus acidophilus using cDNA microarrays. Proc. Nat. Acad. Sci. USA 103: 3816-3821.
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