A New Comparative-Genomics Approach for Defining Phenotype-Specific Indicators Reveals Specific Genetic Markers in Predatory Bacteria

Predatory bacteria seek and consume other live bacteria. Although belonging to taxonomically diverse groups, relatively few bacterial predator species are known. Consequently, it is difficult to assess the impact of predation within the bacterial realm. As no genetic signatures distinguishing them from non-predatory bacteria are known, genomic resources cannot be exploited to uncover novel predators. In order to identify genes specific to predatory bacteria, we developed a bioinformatic tool called DiffGene. This tool automatically identifies marker genes that are specific to phenotypic or taxonomic groups, by mapping the complete gene content of all available fully-sequenced genomes for the presence/absence of each gene in each genome. A putative ‘predator region’ of ~60 amino acids in the tryptophan 2,3-dioxygenase (TDO) protein was found to probably be a predator-specific marker. This region is found in all known obligate predator and a few facultative predator genomes, and is absent from most facultative predators and all non-predatory bacteria. We designed PCR primers that uniquely amplify a ~180bp-long sequence within the predators’ TDO gene, and validated them in monocultures as well as in metagenetic analysis of environmental wastewater samples. This marker, in addition to its usage in predator identification and phylogenetics, may finally permit reliable enumeration and cataloguing of predatory bacteria from environmental samples, as well as uncovering novel predators.

[1]  S. Green,et al.  Deconstructing the Polymerase Chain Reaction: Understanding and Correcting Bias Associated with Primer Degeneracies and Primer-Template Mismatches , 2015, PloS one.

[2]  Hirokazu Chiba,et al.  MBGD update 2015: microbial genome database for flexible ortholog analysis utilizing a diverse set of genomic data , 2014, Nucleic Acids Res..

[3]  A. Chatzinotas,et al.  Multiple micro-predators controlling bacterial communities in the environment. , 2014, Current opinion in biotechnology.

[4]  Koichiro Tamura,et al.  MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. , 2013, Molecular biology and evolution.

[5]  Arthur Brady,et al.  MetaRef: a pan-genomic database for comparative and community microbial genomics , 2013, Nucleic Acids Res..

[6]  Gail L. Rosen,et al.  POGO-DB—a database of pairwise-comparisons of genomes and conserved orthologous genes , 2013, Nucleic Acids Res..

[7]  U. Gophna,et al.  In and out: an analysis of epibiotic vs periplasmic bacterial predators , 2013, The ISME Journal.

[8]  Sarah L. Westcott,et al.  Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform , 2013, Applied and Environmental Microbiology.

[9]  E. Castro-Nallar,et al.  Pathogen typing in the genomics era: MLST and the future of molecular epidemiology. , 2013, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[10]  D. Kadouri,et al.  Predatory Bacteria: A Potential Ally against Multidrug-Resistant Gram-Negative Pathogens , 2013, PloS one.

[11]  Kurt E. Williamson,et al.  Estimates of viral abundance in soils are strongly influenced by extraction and enumeration methods , 2013, Biology and Fertility of Soils.

[12]  U. Gophna,et al.  By their genes ye shall know them: genomic signatures of predatory bacteria , 2012, The ISME Journal.

[13]  Hirokazu Chiba,et al.  MBGD update 2013: the microbial genome database for exploring the diversity of microbial world , 2012, Nucleic Acids Res..

[14]  Huan Chen,et al.  Predatory Bacteriovorax Communities Ordered by Various Prey Species , 2012, PloS one.

[15]  T. Gabaldón,et al.  Selection of Marker Genes Using Whole-Genome DNA Polymorphism Analysis , 2012, Evolutionary bioinformatics online.

[16]  P. Wang,et al.  Defining Function of Lipopolysaccharide O-antigen Ligase WaaL Using Chemoenzymatically Synthesized Substrates* , 2011, The Journal of Biological Chemistry.

[17]  T. Whittam,et al.  Obscured phylogeny and possible recombinational dormancy in Escherichia coli , 2011, BMC Evolutionary Biology.

[18]  F. Ekelund,et al.  The “soil microbial loop” is not always needed to explain protozoan stimulation of plants , 2009 .

[19]  David J Van Horn,et al.  Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities , 2009, Applied and Environmental Microbiology.

[20]  E. Jurkevitch,et al.  Predation between prokaryotes and the origin of eukaryotes , 2009, BioEssays : news and reviews in molecular, cellular and developmental biology.

[21]  F. Martin,et al.  The rhizosphere zoo: An overview of plant-associated communities of microorganisms, including phages, bacteria, archaea, and fungi, and of some of their structuring factors , 2009, Plant and Soil.

[22]  S. Jacquet,et al.  Seasonal and spatial variability of virio-, bacterio-, and picophytoplanktonic abundances in three peri-alpine lakes , 2009, Hydrobiologia.

[23]  Thijs J. G. Ettema,et al.  Signature Genes as a Phylogenomic Tool , 2008, Molecular biology and evolution.

[24]  J. Skolnick,et al.  The Mosaic Genome of Anaeromyxobacter dehalogenans Strain 2CP-C Suggests an Aerobic Common Ancestor to the Delta-Proteobacteria , 2008, PloS one.

[25]  P. Servais,et al.  Fate of heterotrophic bacteria in Lake Tanganyika (East Africa). , 2007, FEMS microbiology ecology.

[26]  Erko Stackebrandt,et al.  Niastella koreensis gen. nov., sp. nov. and Niastella yeongjuensis sp. nov., novel members of the phylum Bacteroidetes, isolated from soil cultivated with Korean ginseng. , 2006, International journal of systematic and evolutionary microbiology.

[27]  I. Uchiyama.  Hierarchical clustering algorithm for comprehensive orthologous-domain classification in multiple genomes , 2006, Nucleic acids research.

[28]  L. Aravind,et al.  The many faces of the helix-turn-helix domain: transcription regulation and beyond. , 2005, FEMS microbiology reviews.

[29]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[30]  W. Martin,et al.  Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes , 2004, Nature Reviews Genetics.

[31]  R. Truscott,et al.  Asp274 and His346 Are Essential for Heme Binding and Catalytic Function of Human Indoleamine 2,3-Dioxygenase* , 2003, Journal of Biological Chemistry.

[32]  C. Pedrós-Alió,et al.  Predatory prokaryotes: predation and primary consumption evolved in bacteria. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[33]  A. Cornish-Bowden.  Nomenclature for incompletely specified bases in nucleic acid sequences: recommendations 1984. , 1985, Nucleic acids research.

[34]  Peter H. A. Sneath,et al.  Numerical Taxonomy: The Principles and Practice of Numerical Classification , 1973 .

[35]  L. Sagan.  On the origin of mitosing cells , 1967, Journal of theoretical biology.

[36]  M. Maiden,et al.  Multi-locus sequence typing and the gene-by-gene approach to bacterial classification and analysis of population variation , 2014 .

[37]  A. Steinbüchel,et al.  Predatory Prokaryotes : Biology, Ecology and Evolution , 2007 .

[38]  E. Jurkevitch,et al.  Phylogenetic Diversity and Evolution of Predatory Prokaryotes , 2006 .

[39]  A. Sallal.  Lysis of cyanobacteria with Flexibacter spp isolated from domestic sewage. , 1994, Microbios.

[40]  L. Kedes,et al.  Nomenclature for incompletely specified bases in nucleic acid sequences. Recommendations 1984. Nomenclature Committee of the International Union of Biochemistry (NC-IUB). , 1986, Proceedings of the National Academy of Sciences of the United States of America.