Metagenomic analysis of the microbial community in fermented grape marc reveals that Lactobacillus fabifermentans is one of the dominant species: insights into its genome structure

Grape marc used for the production of distilled beverages undergoes prolonged storage which allows alcoholic fermentation to occur. Harsh conditions including low pH, limited oxygen and nutrients, temperature fluctuations, and high ethanol concentrations imposed by that environment create a strong selective pressure on microorganisms. A detailed characterization of the bacterial community during two time points of the fermentation process was performed using high-throughput sequencing of the V3–V6 16S rDNA hypervariable regions. The results revealed a marked reduction in the number of bacterial species after 30 days of incubation and made it possible to identify those species that are able to grow in that extreme environment. The genome sequence of Lactobacillus fabifermentans, one of the dominant species identified, was then analyzed using shotgun sequencing and comparative genomics. The results revealed that it is one of the largest genomes among the Lactobacillus sequenced and is characterized by a large number of genes involved in carbohydrate utilization and in the regulation of gene expression. The genome was shaped through a large number of gene duplication events, while lateral gene transfer contributed to a lesser extent with respect to other Lactobacillus species. According to genomic analysis, its carbohydrate utilization pattern and ability to form biofilm are the main genetic traits linked to the adaptation the species underwent permitting it to grow in fermenting grape marc.

[1]  P. Ribereau-gayon,et al.  Evolution of Acetic Acid Bacteria During Fermentation and Storage of Wine , 1984, Applied and environmental microbiology.

[2]  T. H. Lee,et al.  Practical Implications of Malolactic Fermentation: A Review , 1985, American Journal of Enology and Viticulture.

[3]  B. Chassy.  Prospects for improving economically significant Lactobacillus strains by ‘genetic technology’ , 1985 .

[4]  L. Nykänen.  Formation and Occurrence of Flavor Compounds in Wine and Distilled Alcoholic Beverages , 1986, American Journal of Enology and Viticulture.

[5]  L. Ingram,et al.  On the Evolution of Alcohol Tolerance in Microorganisms , 1987 .

[6]  José M. Cardoso Duarte,et al.  Perspectives in Biotechnology , 1987, NATO ASI Series.

[7]  Z. Weinberg,et al.  The effect of applying lactic bacteria at ensilage on the chemical and microbiological composition of vetch, wheat and alfalfa silages , 1988 .

[8]  D. Dubourdieu,et al.  Structure of an exocellular beta-D-glucan from Pediococcus sp., a wine lactic bacteria. , 1990, Carbohydrate research.

[9]  M. Collins,et al.  Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16S rRNA , 1991 .

[10]  G. Speranza,et al.  Absolute stereochemistry and enantiomeric excess of 2-butanol in distilled spirits of different origin , 1994 .

[11]  T. A. Roberts,et al.  A response surface study on the role of some environmental factors affecting the growth of Saccharomyces cerevisiae. , 1995, International journal of food microbiology.

[12]  B. Hahn-Hägerdal,et al.  l-lactic acid production from whole wheat flour hydrolysate using strains of Lactobacilli and Lactococci , 1997 .

[13]  M. Urdaci,et al.  DNA probe and PCR‐specific reaction for Lactobacillus plantarum , 1997, Journal of applied microbiology.

[14]  M. Borodovsky,et al.  GeneMark.hmm: new solutions for gene finding. , 1998, Nucleic acids research.

[15]  T. Hogg,et al.  Microbial and chemical changes during the spontaneous ensilage of grape pomace , 1999 .

[16]  S. Walker,et al.  Quantitative RT-PCR : Pitfalls and Potential , 1999 .

[17]  S. Lory,et al.  Complete genome sequence of Pseudomonas aeruginosa PAO 1 , an opportunistic pathogen , 2000 .

[18]  H. Kawasaki,et al.  Systematic study of the genus Acetobacter with descriptions of Acetobacter indonesiensis sp. nov., Acetobacter tropicalis sp. nov., Acetobacter orleanensis (Henneberg 1906) comb. nov., Acetobacter lovaniensis (Frateur 1950) comb. nov., and Acetobacter estunensis (Carr 1958) comb. nov. , 2000, The Journal of general and applied microbiology.

[19]  F. Malcata,et al.  Effect of time and temperature of fermentation on the microflora of grape pomace , 2000 .

[20]  F. Sesma,et al.  Citrate utilization by homo- and heterofermentative lactobacilli. , 2000, Microbiological research.

[21]  Kim Rutherford,et al.  Artemis: sequence visualization and annotation , 2000, Bioinform..

[22]  S. Lory,et al.  Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen , 2000, Nature.

[23]  H. Ochman,et al.  Lateral gene transfer and the nature of bacterial innovation , 2000, Nature.

[24]  S. Torriani,et al.  Differentiation of Lactobacillus plantarum,L. pentosus, and L. paraplantarum by recA Gene Sequence Analysis and Multiplex PCR Assay with recA Gene-Derived Primers , 2001, Applied and Environmental Microbiology.

[25]  E V Koonin,et al.  Lineage-specific gene expansions in bacterial and archaeal genomes. , 2001, Genome research.

[26]  P. Ribereau-gayon,et al.  Handbook of Enology , 2001 .

[27]  P. Hugenholtz.  Exploring prokaryotic diversity in the genomic era , 2002, Genome Biology.

[28]  J. Swings,et al.  Re-examination of the genus Acetobacter, with descriptions of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. , 2002, International journal of systematic and evolutionary microbiology.

[29]  M. Kleerebezem,et al.  Complete genome sequence of Lactobacillus plantarum WCFS1 , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[30]  A I Saeed,et al.  TM4: a free, open-source system for microarray data management and analysis. , 2003, BioTechniques.

[31]  R. Vogel,et al.  Exopolysaccharide and Kestose Production by Lactobacillus sanfranciscensis LTH2590 , 2003, Applied and Environmental Microbiology.

[32]  F. Rodríguez-Valera,et al.  Comparative genomics of gene-family size in closely related bacteria , 2004, Genome Biology.

[33]  L. Eberl,et al.  Influence of Polyphenols on Bacterial Biofilm Formation and Quorum-sensing , 2003, Zeitschrift fur Naturforschung. C, Journal of biosciences.

[34]  D. Cowan,et al.  Review and re-analysis of domain-specific 16S primers. , 2003, Journal of microbiological methods.

[35]  E. Nimwegen.  Scaling Laws in the Functional Content of Genomes , 2003, physics/0307001.

[36]  Dean Laslett,et al.  ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. , 2004, Nucleic acids research.

[37]  M. Moreno-Arribas,et al.  Wine volatile and amino acid composition after malolactic fermentation: effect of Oenococcus oeni and Lactobacillus plantarum starter cultures. , 2005, Journal of agricultural and food chemistry.

[38]  Hwa-Won Ryu,et al.  Lactic acid production from agricultural resources as cheap raw materials. , 2005, Bioresource technology.

[39]  Peter Schattner,et al.  The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs , 2005, Nucleic Acids Res..

[40]  T. Hansen.  Bergey's Manual of Systematic Bacteriology , 2005 .

[41]  Duane Szafron,et al.  BASys: a web server for automated bacterial genome annotation , 2005, Nucleic Acids Res..

[42]  Michiel Kleerebezem,et al.  Exploring Lactobacillus plantarum Genome Diversity by Using Microarrays , 2005, Journal of bacteriology.

[43]  Shukun Yu,et al.  A novel family of glucosyl 1,5-anhydro-d-fructose derivatives synthesised by transglucosylation with dextransucrase from Leuconostoc mesenteroides NRRL B-512F. , 2005, Carbohydrate research.

[44]  J. Oliver.  The viable but nonculturable state in bacteria. , 2005, Journal of microbiology.

[45]  Georgios S. Vernikos,et al.  Interpolated variable order motifs for identification of horizontally acquired DNA: revisiting the Salmonella pathogenicity islands , 2006, Bioinform..

[46]  Robert Barber,et al.  Prophage Finder: A Prophage Loci Prediction Tool for Prokaryotic Genome Sequences , 2006, Silico Biol..

[47]  P. Ribereau-gayon,et al.  The microbiology of wine and vinifications , 2006 .

[48]  Adam Godzik,et al.  Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences , 2006, Bioinform..

[49]  K. Komagata,et al.  Reclassification of Gluconacetobacter hansenii strains and proposals of Gluconacetobacter saccharivorans sp. nov. and Gluconacetobacter nataicola sp. nov. , 2006, International journal of systematic and evolutionary microbiology.

[50]  Pascal Ribéreau-Gayon,et al.  Handbook of Enology: The Microbiology of Wine and Vinifications , 2006 .

[51]  K. Shanmugam,et al.  Isolation and Characterization of Acid-Tolerant, Thermophilic Bacteria for Effective Fermentation of Biomass-Derived Sugars to Lactic Acid , 2006, Applied and Environmental Microbiology.

[52]  Sarah A. Teichmann,et al.  DBD: a transcription factor prediction database , 2005, Nucleic Acids Res..

[53]  M. Penttilä,et al.  Efficient Production of l-Lactic Acid from Xylose by Pichia stipitis , 2006, Applied and Environmental Microbiology.

[54]  G. Fleet,et al.  Lactic acid bacteria associated with wine grapes from several Australian vineyards , 2006, Journal of applied microbiology.

[55]  Y. Ohkouchi,et al.  Direct production of L+-lactic acid from starch and food wastes using Lactobacillus manihotivorans LMG18011. , 2006, Bioresource technology.

[56]  C. Webb,et al.  Xylanase and pectinase production by Aspergillus awamori on grape pomace in solid state fermentation , 2007 .

[57]  D. Gokhale,et al.  Utilization of Molasses Sugar for Lactic Acid Production by Lactobacillus delbrueckii subsp. delbrueckii Mutant Uc-3 in Batch Fermentation , 2007, Applied and Environmental Microbiology.

[58]  Rick L. Stevens,et al.  The RAST Server: Rapid Annotations using Subsystems Technology , 2008, BMC Genomics.

[59]  Ibtissem Grissa,et al.  CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats , 2007, Nucleic Acids Res..

[60]  D. Nielsen,et al.  The microbiology of Ghanaian cocoa fermentations analysed using culture-dependent and culture-independent methods. , 2007, International journal of food microbiology.

[61]  E. Birney,et al.  Velvet: algorithms for de novo short read assembly using de Bruijn graphs. , 2008, Genome research.

[62]  S. Furukawa,et al.  Mixed-Species Biofilm Formation by Lactic Acid Bacteria and Rice Wine Yeasts , 2008, Applied and Environmental Microbiology.

[63]  A. Sangrador-Vegas,et al.  Genome Sequence of Lactobacillus helveticus, an Organism Distinguished by Selective Gene Loss and Insertion Sequence Element Expansion , 2007, Journal of bacteriology.

[64]  Sarah A. Teichmann,et al.  DBD––taxonomically broad transcription factor predictions: new content and functionality , 2007, Nucleic Acids Res..

[65]  J. Alonso,et al.  SSF production of lactic acid from cellulosic biosludges. , 2008, Bioresource technology.

[66]  Andreas Wilke,et al.  phylogenetic and functional analysis of metagenomes , 2022 .

[67]  Ilse Cleenwerck,et al.  Polyphasic taxonomy of acetic acid bacteria: an overview of the currently applied methodology. , 2008, International journal of food microbiology.

[68]  Yuzo Yamada,et al.  Genera and species in acetic acid bacteria. , 2008, International journal of food microbiology.

[69]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[70]  M. Vergassola,et al.  The Listeria transcriptional landscape from saprophytism to virulence , 2009, Nature.

[71]  Steven J. M. Jones,et al.  Abyss: a Parallel Assembler for Short Read Sequence Data Material Supplemental Open Access , 2022 .

[72]  H. Uchiyama,et al.  Stress resistance of biofilm and planktonic Lactobacillus plantarum subsp. plantarum JCM 1149. , 2009, Food microbiology.

[73]  Tracy K. Teal,et al.  Systematic artifacts in metagenomes from complex microbial communities , 2009, The ISME Journal.

[74]  James R. Cole,et al.  The Ribosomal Database Project: improved alignments and new tools for rRNA analysis , 2008, Nucleic Acids Res..

[75]  M. Kjos,et al.  An overview of the mosaic bacteriocin pln loci from Lactobacillus plantarum , 2009, Peptides.

[76]  J. Steele,et al.  Genome Sequence and Comparative Genome Analysis of Lactobacillus casei: Insights into Their Niche-Associated Evolution , 2009, Genome biology and evolution.

[77]  P. Vandamme,et al.  Lactobacillus fabifermentans sp. nov. and Lactobacillus cacaonum sp. nov., isolated from Ghanaian cocoa fermentations. , 2009, International journal of systematic and evolutionary microbiology.

[78]  Robert G. Beiko,et al.  Identifying biologically relevant differences between metagenomic communities , 2010, Bioinform..

[79]  M. Kleerebezem,et al.  Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches. , 2010, Environmental microbiology.

[80]  S. Garcia,et al.  Survival of Lactobacillus casei (LC-1) adhered to prebiotic vegetal fibers , 2010 .

[81]  N. Perna,et al.  progressiveMauve: Multiple Genome Alignment with Gene Gain, Loss and Rearrangement , 2010, PloS one.

[82]  S. D. De Keersmaecker,et al.  Adaptation factors of the probiotic Lactobacillus rhamnosus GG. , 2010, Beneficial microbes.

[83]  Brad T. Sherman,et al.  Gene duplications in prokaryotes can be associated with environmental adaptation , 2010, BMC Genomics.

[84]  Christian Brion,et al.  FSY1, a horizontally transferred gene in the Saccharomyces cerevisiae EC1118 wine yeast strain, encodes a high-affinity fructose/H+ symporter. , 2010, Microbiology.

[85]  Ying Xu,et al.  cBar: a computer program to distinguish plasmid-derived from chromosome-derived sequence fragments in metagenomics data , 2010, Bioinform..

[86]  Cuiqing Ma,et al.  Efficient production of L-lactic acid from cassava powder by Lactobacillus rhamnosus. , 2010, Bioresource technology.

[87]  L. Holm,et al.  The Pfam protein families database , 2005, Nucleic Acids Res..

[88]  Patrick J. Biggs,et al.  SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data , 2010, BMC Bioinformatics.

[89]  F. Fontana,et al.  Effects of yeast inoculation on volatile compound production by grape marcs , 2011, Annals of Microbiology.

[90]  H. Hemmi,et al.  Alterations in D-amino acid concentrations and microbial community structures during the fermentation of red and white wines. , 2011, Journal of bioscience and bioengineering.

[91]  E. Mateo,et al.  Diversity of acetic acid bacteria present in healthy grapes from the Canary Islands. , 2011, International journal of food microbiology.

[92]  P. Vandamme,et al.  Spontaneous organic cocoa bean box fermentations in Brazil are characterized by a restricted species diversity of lactic acid bacteria and acetic acid bacteria. , 2011, Food microbiology.

[93]  A. Schieber,et al.  Structure–function relationships of the antibacterial activity of phenolic acids and their metabolism by lactic acid bacteria , 2011, Journal of applied microbiology.

[94]  M. Sogin,et al.  Water mass‐specificity of bacterial communities in the North Atlantic revealed by massively parallel sequencing , 2011, Molecular ecology.

[95]  Narmada Thanki,et al.  CDD: a Conserved Domain Database for the functional annotation of proteins , 2010, Nucleic Acids Res..

[96]  S. Campanaro,et al.  The RNA polymerase subunits E/F from the Antarctic archaeon Methanococcoides burtonii bind to specific species of mRNA. , 2011, Environmental microbiology.

[97]  M. Kleerebezem,et al.  Complete Resequencing and Reannotation of the Lactobacillus plantarum WCFS1 Genome , 2012, Journal of bacteriology.

[98]  B. Trost,et al.  Complete Genome Sequence of the Beer Spoilage Organism Pediococcus claussenii ATCC BAA-344T , 2012, Journal of bacteriology.

[99]  F. Fontana,et al.  Acidification of grape marc for alcoholic beverage production: effects on indigenous microflora and aroma profile after distillation. , 2012, International journal of food microbiology.

[100]  Nicholas A. Bokulich,et al.  Next-Generation Sequencing Reveals Significant Bacterial Diversity of Botrytized Wine , 2012, PloS one.

[101]  G. Garrity.  Bergey’s Manual® of Systematic Bacteriology , 2012, Springer New York.

[102]  Jane M. McCarthy,et al.  Comparative analysis of the Oenococcus oeni pan genome reveals genetic diversity in industrially-relevant pathways , 2012, BMC Genomics.

[103]  S. Campanaro,et al.  The transcriptional landscape of the deep-sea bacterium Photobacterium profundum in both a toxR mutant and its parental strain , 2012, BMC Genomics.

[104]  Sean R. Eddy,et al.  Rfam 11.0: 10 years of RNA families , 2012, Nucleic Acids Res..

[105]  Vanessa Pittet,et al.  Transcriptome Sequence and Plasmid Copy Number Analysis of the Brewery Isolate Pediococcus claussenii ATCC BAA-344T during Growth in Beer , 2013, PloS one.

[106]  K. Howell,et al.  Biodiversity, dynamics and ecology of bacterial community during grape marc storage for the production of grappa. , 2013, International journal of food microbiology.

[107]  Oscar P. Kuipers,et al.  BAGEL3: automated identification of genes encoding bacteriocins and (non-)bactericidal posttranslationally modified peptides , 2013, Nucleic Acids Res..

[108]  P. O’Toole,et al.  Catabolic flexibility of mammalian-associated lactobacilli , 2013, Microbial Cell Factories.

[109]  Kai Wang,et al.  Diversity of culturable root-associated/endophytic bacteria and their chitinolytic and aflatoxin inhibition activity of peanut plant in China , 2012, World Journal of Microbiology and Biotechnology.

[110]  Stan J. J. Brouns,et al.  Comparative Genomic and Functional Analysis of 100 Lactobacillus rhamnosus Strains and Their Comparison with Strain GG , 2013, PLoS genetics.

[111]  Favaro Lorenzo,et al.  Grape marcs as unexplored source of new yeasts for future biotechnological applications , 2013, World journal of microbiology & biotechnology.

[112]  R. Siezen,et al.  Lactobacillus paracasei Comparative Genomics: Towards Species Pan-Genome Definition and Exploitation of Diversity , 2013, PloS one.

[113]  Genome Sequence of Lactobacillus fabifermentans Strain T30PCM01, Isolated from Fermenting Grape Marc , 2014, Genome Announcements.

[114]  S. Campanaro,et al.  The impact of genomic variability on gene expression in environmental Saccharomyces cerevisiae strains. , 2014, Environmental microbiology.