Identification of an antibacterial protein by functional screening of a human oral metagenomic library.
Screening of a bacterial artificial chromosome (BAC) library containing metagenomic DNA from human plaque and saliva allowed the isolation of four clones producing antimicrobial activity. Three of these were pigmented and encoded homologues of glutamyl-tRNA reductase (GluTR), an enzyme involved in the C5 pathway leading to tetrapyrole synthesis, and one clone had antibacterial activity with no pigmentation. The latter contained a BAC with an insert of 15.6 kb. Initial attempts to localize the gene(s) responsible for antimicrobial activity by subcloning into pUC-based vectors failed. A new plasmid for toxic gene expression (pTGEX) was designed enabling localization of the antibacterial activity to a 4.7-kb HindIII fragment. Transposon mutagenesis localized the gene to an open reading frame of 483 bp designated antibacterial protein1 (abp1). Abp1 was 94% identical to a hypothetical protein of Neisseria subflava (accession number WP_004519448.1). An Escherichia coli clone expressing Abp1 exhibited antibacterial activity against Bacillus subtilis BS78H, Staphylococcus epidermidis NCTC 11964 and B4268, and S. aureus NCTC 12493,ATCC 35696 and NCTC 11561. However, no antibacterial activity was observed against Pseudomonas aeruginosa ATCC 9027, N. subflava ATCC A1078, E. coli K12 JM109 and BL21(DE3) Fusobacterium nucleatum ATCC 25586 and NCTC 11326, Prevotella intermedia ATCC 25611, Veillonella parvula ATCC 10790 or Lactobacillus casei NCTC 6375.
Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India.
Preeti
P
Arivaradarajan
Philip J
PJ
Warburton
Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India.
Gunasekaran
G
Paramasamy
Department of Microbial Diseases, UCL Eastman Dental Institute, London WC1X 8LD, UK.
Sean P
SP
Nair
Department of Microbial Diseases, UCL Eastman Dental Institute, London WC1X 8LD, UK.
Elaine
E
Allan
Department of Microbial Diseases, UCL Eastman Dental Institute, London WC1X 8LD, UK p.mullany@ucl.ac.uk.
Peter
P
Mullany
D000445:Aldehyde Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects; D001419:Bacteria / Q000187:drug effects; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D003773:Dental Plaque / Q000382:microbiology*; D004926:Escherichia coli / Q000235:genetics; D048168:Escherichia coli K12 / Q000187:drug effects; D005822:Genetic Vectors; D015698:Genomic Library; D006801:Humans; D007780:Lacticaseibacillus casei / Q000187:drug effects; D054892:Metagenome; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009343:Neisseria / Q000737:chemistry*; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000187:drug effects; D012463:Saliva / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000187:drug effects; D013212:Staphylococcus epidermidis / Q000187:drug effects
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase
Functional metagenomics; antimicrobial agent; bacterial artificial chromosome