RECTA: Regulon Identification Based on Comparative Genomics and Transcriptomics Analysis

Regulons, which serve as co-regulated gene groups contributing to the transcriptional regulation of microbial genomes, have the potential to aid in understanding of underlying regulatory mechanisms. In this study, we designed a novel computational pipeline, RECTA, for regulon prediction related to the gene regulatory network under certain conditions. To demonstrate the effectiveness of this tool, we implemented RECTA on Lactococcus lactis MG1363 data to elucidate acid-response regulons. Lactococcus lactis is one of the most important Gram-positive lactic acid-producing bacteria, widely used in food industry and has been proved to have advantages in oral delivery of drug and vaccine. The pipeline carries out differential gene expression, gene co-expression analysis, cis-regulatory motif finding, and comparative genomics to predict and validate regulons related to acid stress response. A total of 51 regulonswere identified, 14 of which have computational-verified significance. Among these 14 regulons, five of them were computationally predicted to be connected with acid stress response with (i) known transcriptional factors in MEME suite database successfully mapped in Lactococcus lactis MG1363; and (ii) differentially expressed genes between pH values of 6.5 (control) and 5.1 (treatment). Validated by 36 literature confirmed acid stress response related proteins and genes, 33 genes in Lactococcus lactis MG1363 were found having orthologous genes using BLAST, associated to six regulons. An acid response related regulatory network was constructed, involving two trans-membrane proteins, eight regulons (llrA, llrC, hllA, ccpA, NHP6A, rcfB, regulons #8 and #39), nine functional modules, and 33 genes with orthologous genes known to be associated to acid stress. Our RECTA pipeline provides an effective way to construct a reliable gene regulatory network through regulon elucidation. The predicted response pathways could serve as promising candidates for better acid tolerance engineering in Lactococcus lactis. RECTA has strong application power and can be effectively applied to other bacterial genomes where the elucidation of the transcriptional regulation network is needed.

[1]  N. Kaplan,et al.  D- and L-lactic acid dehydrogenases in Lactobacillus plantarum. , 1960, The Journal of biological chemistry.

[2]  J. Monod,et al.  [Operon: a group of genes with the expression coordinated by an operator]. , 1960, Comptes rendus hebdomadaires des seances de l'Academie des sciences.

[3]  D. Bauer.  Constructing Confidence Sets Using Rank Statistics , 1972 .

[4]  D. Kolodrubetz,et al.  Duplicated NHP6 genes of Saccharomyces cerevisiae encode proteins homologous to bovine high mobility group protein 1. , 1990, The Journal of biological chemistry.

[5]  R. Hutkins,et al.  pH Homeostasis in Lactic Acid Bacteria , 1993 .

[6]  A. Rincé,et al.  Cloning, expression, and nucleotide sequence of genes involved in production of lactococcin DR, a bacteriocin from lactococcus lactis subsp. lactis , 1994, Applied and environmental microbiology.

[7]  Y. Auffray,et al.  The Lactic Acid Stress Response of Lactococcus lactis subsp. lactis , 1996, Current Microbiology.

[8]  N. L. Glass,et al.  Transcriptional analysis of the , 1996 .

[9]  G. Jayaraman,et al.  Transcriptional analysis of the Streptococcus mutans hrcA, grpE and dnaK genes and regulation of expression in response to heat shock and environmental acidification , 1997, Molecular microbiology.

[10]  F. Tomita,et al.  Characterization of a mutant of Lactococcus lactis with reduced membrane-bound ATPase activity under acidic conditions. , 1998, Bioscience, biotechnology, and biochemistry.

[11]  G. Venemâ,et al.  A chloride‐inducible acid resistance mechanism in Lactococcus lactis and its regulation , 1998, Molecular microbiology.

[12]  G. Venemâ,et al.  Environmental stress responses in Lactococcus lactis , 1999 .

[13]  E. O'Sullivan,et al.  Relationship between Acid Tolerance, Cytoplasmic pH, and ATP and H+-ATPase Levels in Chemostat Cultures of Lactococcus lactis , 1999, Applied and Environmental Microbiology.

[14]  M. Givskov,et al.  Molecular characterization of the pH‐inducible and growth phase‐dependent promoter P170 of Lactococcus lactis , 1999, Molecular microbiology.

[15]  Pascal Hols,et al.  Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering , 1999, Nature Biotechnology.

[16]  H. Aso,et al.  Lactococcus lactis contains only one glutamate decarboxylase gene. , 1999, Microbiology.

[17]  S. Ehrlich,et al.  Six putative two-component regulatory systems isolated from Lactococcus lactis subsp. cremoris MG1363. , 2000, Microbiology.

[18]  P. R. Jensen,et al.  The Membrane-Bound H+-ATPase Complex Is Essential for Growth of Lactococcus lactis , 2000, Journal of bacteriology.

[19]  S. Ehrlich,et al.  Acid‐ and multistress‐resistant mutants of Lactococcus lactis : identification of intracellular stress signals , 2000, Molecular microbiology.

[20]  Elliot J. Lefkowitz,et al.  Genome of the Bacterium Streptococcus pneumoniae Strain R6 , 2001, Journal of bacteriology.

[21]  Mathieu Blanchette,et al.  Algorithms for phylogenetic footprinting , 2001, RECOMB.

[22]  G D Stormo,et al.  A comparative genomics approach to prediction of new members of regulons. , 2001, Genome research.

[23]  F. González-Candelas,et al.  Evolution of arginine deiminase (ADI) pathway genes. , 2002, Molecular phylogenetics and evolution.

[24]  F. Vogensen,et al.  Identification of proteins induced at low pH in Lactococcus lactis. , 2003, International journal of food microbiology.

[25]  C. Hill,et al.  Surviving the Acid Test: Responses of Gram-Positive Bacteria to Low pH , 2003, Microbiology and Molecular Biology Reviews.

[26]  Edgar Wingender,et al.  PRODORIC: prokaryotic database of gene regulation , 2003, Nucleic Acids Res..

[27]  T. Hindré,et al.  Regulation of lantibiotic lacticin 481 production at the transcriptional level by acid pH. , 2004, FEMS microbiology letters.

[28]  S. Ehrlich,et al.  Low-redundancy sequencing of the entire Lactococcus lactis IL1403 genome , 1999, Antonie van Leeuwenhoek.

[29]  Alain Dufour,et al.  Two acid‐inducible promoters from Lactococcus lactis require the cis‐acting ACiD‐box and the transcription regulator RcfB , 2005, Molecular microbiology.

[30]  S. Ehrlich,et al.  Transcriptional analysis of the cyclopropane fatty acid synthase gene of Lactococcus lactis MG1363 at low pH. , 2005, FEMS microbiology letters.

[31]  Lei Shen,et al.  Combining phylogenetic motif discovery and motif clustering to predict co-regulated genes , 2005, Bioinform..

[32]  E. Maguin,et al.  Genetic structure and transcriptional analysis of the arginine deiminase (ADI) cluster in Lactococcus lactis MG1363. , 2006, Canadian journal of microbiology.

[33]  William Stafford Noble,et al.  Quantifying similarity between motifs , 2007, Genome Biology.

[34]  F. Baneyx,et al.  Chaperone Hsp31 Contributes to Acid Resistance in Stationary-Phase Escherichia coli , 2006, Applied and Environmental Microbiology.

[35]  Ramasamy,et al.  Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral , 2006 .

[36]  A. T. Carter,et al.  Mucosal delivery of a pneumococcal vaccine using Lactococcus lactis affords protection against respiratory infection. , 2007, The Journal of infectious diseases.

[37]  A. Malki,et al.  Escherichia coli HdeB Is an Acid Stress Chaperone , 2006, Journal of bacteriology.

[38]  Inna Dubchak,et al.  RegTransBase—a database of regulatory sequences and interactions in a wide range of prokaryotic genomes , 2006, Nucleic Acids Res..

[39]  Alexander Goesmann,et al.  Complete Genome Sequence of the Prototype Lactic Acid Bacterium Lactococcus lactis subsp. cremoris MG1363 , 2007, Journal of bacteriology.

[40]  Aldert L. Zomer,et al.  Time-Resolved Determination of the CcpA Regulon of Lactococcus lactis subsp. cremoris MG1363 , 2006, Journal of bacteriology.

[41]  M. Nascimento,et al.  CcpA Regulates Central Metabolism and Virulence Gene Expression in Streptococcus mutans , 2008, Journal of bacteriology.

[42]  G. Grandi,et al.  CsrRS Regulates Group B Streptococcus Virulence Gene Expression in Response to Environmental pH: a New Perspective on Vaccine Development , 2009, Journal of bacteriology.

[43]  C. Hill,et al.  Molecular characterization of the arginine deiminase system in Listeria monocytogenes: regulation and role in acid tolerance. , 2009, Environmental microbiology.

[44]  I. Akyol,et al.  Regulation of the acid induciblercfB promoter inLactococcus lactis subsp.lactis , 2008, Annals of Microbiology.

[45]  Mikael Bodén,et al.  MEME Suite: tools for motif discovery and searching , 2009, Nucleic Acids Res..

[46]  E. Papoutsakis,et al.  Metabolite stress and tolerance in the production of biofuels and chemicals: Gene‐expression‐based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum , 2010, Biotechnology and bioengineering.

[47]  B. Poolman,et al.  Genome Sequences of Lactococcus lactis MG1363 (Revised) and NZ9000 and Comparative Physiological Studies , 2010, Journal of bacteriology.

[48]  Inna Dubchak,et al.  RegPredict: an integrated system for regulon inference in prokaryotes by comparative genomics approach , 2010, Nucleic Acids Res..

[49]  D. Stillman.  Nhp6: a small but powerful effector of chromatin structure in Saccharomyces cerevisiae. , 2010, Biochimica et biophysica acta.

[50]  Ying Xu,et al.  Integration of sequence-similarity and functional association information can overcome intrinsic problems in orthology mapping across bacterial genomes , 2011, Nucleic acids research.

[51]  A. Grover,et al.  Phylogenetic footprinting: a boost for microbial regulatory genomics , 2012, Protoplasma.

[52]  James C. W. Locke,et al.  Stochastic Pulse Regulation in Bacterial Stress Response , 2011, Science.

[53]  Ying Xu,et al.  A new framework for identifying cis-regulatory motifs in prokaryotes , 2010, Nucleic acids research.

[54]  Sebastian Bonhoeffer,et al.  Evolution of Stress Response in the Face of Unreliable Environmental Signals , 2012, PLoS Comput. Biol..

[55]  H. Kobayashi,et al.  Adenosine deamination increases the survival under acidic conditions in Escherichia coli , 2012, Journal of applied microbiology.

[56]  R. May,et al.  The CovS/CovR Acid Response Regulator Is Required for Intracellular Survival of Group B Streptococcus in Macrophages , 2012, Infection and Immunity.

[57]  D. Raoult,et al.  Obesity-associated gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii , 2011, International Journal of Obesity.

[58]  Ying Xu,et al.  An integrated toolkit for accurate prediction and analysis of cis-regulatory motifs at a genome scale , 2013, Bioinform..

[59]  Eberhard O. Voit,et al.  Metabolic and Transcriptional Analysis of Acid Stress in Lactococcus lactis, with a Focus on the Kinetics of Lactic Acid Pools , 2013, PloS one.

[60]  M. Elowitz,et al.  Functional Roles of Pulsing in Genetic Circuits , 2013, Science.

[61]  Catarina Costa,et al.  The YEASTRACT database: an upgraded information system for the analysis of gene and genomic transcription regulation in Saccharomyces cerevisiae , 2013, Nucleic Acids Res..

[62]  Xin Chen,et al.  DMINDA: an integrated web server for DNA motif identification and analyses , 2014, Nucleic Acids Res..

[63]  Guojun Li,et al.  Elucidation of Operon Structures across Closely Related Bacterial Genomes , 2014, PloS one.

[64]  Xin Chen,et al.  DOOR 2.0: presenting operons and their functions through dynamic and integrated views , 2013, Nucleic Acids Res..

[65]  Yan-jun Ma,et al.  Oral Administration of Recombinant Lactococcus lactis Expressing HSP65 and Tandemly Repeated P277 Reduces the Incidence of Type I Diabetes in Non-Obese Diabetic Mice , 2014, PloS one.

[66]  P. Lund,et al.  Coping with low pH: molecular strategies in neutralophilic bacteria. , 2014, FEMS microbiology reviews.

[67]  Xin Chen,et al.  Revisiting operons: an analysis of the landscape of transcriptional units in E. coli , 2015, BMC Bioinformatics.

[68]  C. Bauer,et al.  Analysis of the FnrL regulon in Rhodobacter capsulatus reveals limited regulon overlap with orthologues from Rhodobacter sphaeroides and Escherichia coli , 2015, BMC Genomics.

[69]  C. M. Oslowski.  Stress Responses , 2015, Methods in Molecular Biology.

[70]  Bingqiang Liu,et al.  Bacterial regulon modeling and prediction based on systematic cis regulatory motif analyses , 2016, Scientific Reports.

[71]  Z. Zeng,et al.  Recombinant Lactococcus lactis NZ9000 secretes a bioactive kisspeptin that inhibits proliferation and migration of human colon carcinoma HT-29 cells , 2016, Microbial Cell Factories.

[72]  David J. Arenillas,et al.  JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles , 2015, Nucleic Acids Res..

[73]  Bingqiang Liu,et al.  An integrative and applicable phylogenetic footprinting framework for cis-regulatory motifs identification in prokaryotic genomes , 2016, BMC Genomics.

[74]  J. Aerts,et al.  SCENIC: Single-cell regulatory network inference and clustering , 2017, Nature Methods.

[75]  Xin Chen,et al.  DMINDA 2.0: integrated and systematic views of regulatory DNA motif identification and analyses , 2017, Bioinform..

[76]  Yaoqi Zhou,et al.  Systems-level understanding of ethanol-induced stresses and adaptation in E. coli , 2017, Scientific Reports.

[77]  B. Spellerberg,et al.  Acid Stress Response Mechanisms of Group B Streptococci , 2017, Front. Cell. Infect. Microbiol..

[78]  Yang Li,et al.  An algorithmic perspective of de novo cis-regulatory motif finding based on ChIP-seq data , 2017, Briefings Bioinform..

[79]  Ying Xu,et al.  DOOR: a prokaryotic operon database for genome analyses and functional inference , 2019, Briefings Bioinform..

[80]  Lactococcus lactis , 2020, Definitions.