Download Identification of an antibacterial protein by functional screening of a

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Identification of an antibacterial protein by functional screening of a human oral metagenomic
library
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Preeti Arivaradarajan1, Gunasekaran Paramasamy1, Sean P. Nair2, Elaine Allan2, Peter Mullany2#
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Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences,
Madurai Kamaraj University, Madurai, India.
Department of Microbial Diseases, UCL Eastman Dental Institute, London, United Kingdom.
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#Corresponding author
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Mailing address: Department of Microbial Diseases, UCL Eastman Dental Institute, 256 Gray’s Inn
Road, University College London, WC1X 8LD, United Kingdom.
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Phone: 44(0)203 456 1223. Fax: 44 (0)203 456 1127.
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E-mail: [email protected].
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ABSTRACT
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Screening of a bacterial artificial chromosome (BAC) library (19,200 clones) containing metagenomic
DNA from human plaque and saliva allowed the isolation of four antimicrobial producing clones.
Three of these clones were pigmented and encoded Glutamyl-tRNA reductase (GluTR) homologues,
an enzyme which catalyses the first step in the C5 pathway leading to tetrapyrole synthesis, and one
had antibacterial activity with no colony pigmentation. The latter clone contained a BAC with an
insert size of 15.6 kb. Initial attempts to localise the gene(s) responsible for antimicrobial activity by
sub-cloning into pUC based vectors failed, so a new expression plasmid for toxic gene expression
(pTGEX) was designed which enabled localisation of the antibacterial activity to a 4.7 kb HindIII
fragment. Transposon mutagenesis localised 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) and N. mucosa (accession number
WP_003745926). An E. coli clone expressing Abp1 exhibited antibacterial activity against Bacillus
subtilis BS78H, Staphylococcus epidermidis NCTC 11964 and Staphylococcus aureus NCTC 12493
(MRSA). However no antibacterial activity against Pseudomonas aeruginosa ATCC 9027, Neisseria
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subflava ATCC A1078, Escherichia coli K12 JM109, Fusobacterium nucleatum ATCC 25586, Prevotella
intermedia ATCC 25611, Veillonella parvula ATCC 10790 and Lactobacillus casei NCTC 6375 was
observed.
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INTRODUCTION
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After the industrialization of penicillin production a plethora of antibiotics came into the market
(Fernandes, 2006). However overuse of antibiotics acts as a selection pressure resulting in the
emergence of resistant bacteria. This is exacerbated by the fact that antibiotic resistance genes are
often borne on mobile genetic elements which may promote their rapid dissemination. Transfer of
resistance genes to common pathogens makes otherwise treatable diseases untreatable, for a
recent review (Juhas, 2015). Thus there is an urgent need for new antimicrobials.
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Microorganisms are known producers of antimicrobials (Tawiah et al., 2012) however access to all
antimicrobial producers is hindered by the fact that not all are cultivable in the laboratory.
Metagenomics allows access to the genetic potential of whole microbial communities in an
environmental sample (Mullany, 2014) and has allowed the isolation of novel antimicrobial products,
for example, investigation of the microbial communities associated with the marine sponge,
Cymbastela concentric, and the green alga, Ulva australis, led to the identification of three novel
hydrolytic enzymes with antibacterial activity (Yung et al., 2011). The pigments, indirubin and indigo,
were discovered to have antibacterial activity by screening a fosmid library of a forest soil
metagenome (Lim et al., 2005). The broad spectrum antibiotics, turbomycin A and B, from a soil
metagenomic library were reported by Gillespie et al. (2002). The antibiotics, violacein and
palmitoylputrerescine, were isolated from soil and bromeliad tank water metagenomic libraries,
respectively (Brady et al., 2001; Brady & Clardy, 2004). Palmitoylputrerescine was not found in a
previous screen for antibacterials amongst cultivable bacteria, indicating that metagenomic
screening allows access to natural products that cannot be found using other methods. Recently a
novel method for growing previously uncultivable soil bacteria has been developed which enabled
the isolation of a new antibiotic, teixobactin, further emphasising the potential of non-cultivable
bacteria as a rich source of useful products (Ling et al., 2015).
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In this study we investigated the human oral metagenome by functional screening for antimicrobial
production. A novel antibacterial protein was isolated from a BAC library of human plaque and saliva
metagenomic DNA. Also a vector for efficient cloning and stable maintenance of toxic genes was
designed and used.
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MATERIALS AND METHODS
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Bacterial cultures and vectors used.
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Escherichia coli strains and plasmids used in this study are listed in Table 1. The indicator bacteria
tested were: Bacillus subtilis BS78H (a chloramphenicol resistant derivative of CU2189),
Staphylococcus epidermidis NCTC 11964, Staphylococcus aureus NCTC 12493 (MRSA), Pseudomonas
aeruginosa ATCC 9027, Neisseria subflava ATCC A1078, Escherichia coli K12 JM109, Fusobacterium
nucleatum ATCC 25586, Prevotella intermedia ATCC 25611, Veillonella parvula ATCC 10790 and
Lactobacillus casei NCTC 6375.
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In order to mediate stable cloning of genes toxic to E. coli, the vector, pTGEX (plasmid for toxic gene
expression) was designed (Fig. 1). This was constructed by DNA2.0, USA to our specifications:
plasmid pJ421 containing a p15a (Chang & Cohen, 1978 )origin of replication was modified by
insertion of the pUC19 lacZ fragment containing a multiple cloning site allowing cloning flexibility
and blue white selection. DNA cloned into pTGEX is tightly controlled by an isopropyl-β-Dthiogalactopyranoside (IPTG)-inducible phage T5 promoter. The vector has transcription
terminators, synthesised by DNA 2.0 and placed upstream and downstream of the multiple cloning
sites.
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Functional screening.
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Clones from a previously described human oral metagenomic library (Warburton et al., 2009) were
grown on Luria Bertani (LB) agar containing chloramphenicol (12.5 µg/ml) and IPTG (0.5 mM) for 24
h at 37°C. Thereafter the plates were incubated at 22-25°C for 5 days. For the antibacterial assay, a
1% inoculum from a 16 h B. subtilis culture (in LB broth) was diluted to obtain an optical density at
600 nm (OD600) of 0.3. This suspension was diluted 1 in 100 in to 0.5% LB agar and overlaid on to the
LB agar plate containing the metagenomic clones. The plates were incubated for 24 h at 37°C
followed by 3-4 days incubation at 22-25°C and were scored for a zone of B. subtilis cell lysis.
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The antibacterial activity of clones showing activity against B. subtilis were also tested against S.
epidermidis, S. aureus, P. aeruginosa, N. subflava, E. coli and L. casei were grown aerobically at 37 °C
in brain-heart infusion (BHI) broth. Cultures of F. nucleatum, P. intermedia, and V. parvula were
grown in BHI broth in an anaerobic atmosphere (80% N2, 10% CO2, 10% H2).
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Subcloning and DNA sequencing.
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The pTGEX plasmid was used for subcloning in E. coli DH5α. Subclones from each experiment were
grown on LB agar containing kanamycin (30 µg/ml) and IPTG (1 mM) for 12 h at 37°C followed by 12
h incubation at 22-25°C and thereafter scored for antibacterial activity by functional screening as
described above. The insert DNA from the subclone showing antibacterial activity was sequenced
using pTGEX sequencing primers, pTGEXFP (5’-TTACGAGCTTCATGCACAG-3’) and pTGEXRP (5’AGGGTTATTGTCTCATGAGC-3’) which flank the cloning site.
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Bioinformatics analysis.
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DNA sequences were analysed using tools available at the National Centre for Biotechnology
Information (NCBI), ORF finder (http://www.ncbi.nlm.nih.gov/) and BLAST (Altschul et al., 1990). A
putative promoter was assigned using a promoter prediction program, BPROM
(http://linux1.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb). The
transcription terminator was recognized using the web server, ARNold (Naville et al., 2011). The
putative signal peptide and cleavage site was identified by the online tool, Prediction of Protein
Localization Sites (PSORT) (Gardy et al., 2005).
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Transposon mutagenesis.
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For transposon mutagenesis, the Entranceposon (an artificial Mu transposon) containing a
kanamycin resistance gene was used. Transposon mutant libraries were constructed following the
manufacturer’s instructions (Template Generation System II kit; TGS, F-702; Finnzyme, Finland). The
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transposition reaction mixture was transformed into the strains of E. coli EPI300-T1R carrying the
plasmids encoding antibacterials and transformants were selected on LB agar containing
chloramphenicol (12.5 µg/ml) and kanamycin (20 µg/ml). The transformants were grown for 12 h at
37°C, with shaking at 200 rpm in 96 well flat bottom plates with 110 µl LB broth containing
kanamycin. 5 µl of the 12 h culture was spotted on to LB agar supplemented with chloramphenicol
(12.5 µg/ml) and IPTG (0.5 mM) and the plates were incubated overnight at 37°C. After overnight
incubation, an overlay assay using B. subtilis was performed as described above and mutants not
showing a zone of inhibition were selected. To localise the transposon insertion site, plasmids
isolated from the mutants were digested with HindIII and analyzed by gel electrophoresis. Mutant
plasmids with single transposon insertions were sequenced using transposon specific primers (SeqW
and SeqE provided by the manufacturer) and the transposon insertion point identified.
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Nucleotide sequence submission.
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The nucleotide sequence of the insert from p112C was deposited in GenBank under the accession
number KF955286.
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RESULTS AND DISCUSSION
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Screening of a human plaque and saliva metagenomic library allowed the isolation of four clones
producing antibacterial products.
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19,200 human pooled plaque and saliva oral metagenomic clones were screened and three
brownish-red pigmented clones, BAC28G, BAC108J and BAC110F, containing BACs with insert sizes
of 17.7 kb, 5.4 kb and 21.3 kb, respectively, and one non-pigmented clone, HOAb112C, which
contained a BAC with an insert of 15.6 kb, gave zones of growth inhibition of B. subtilis.
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In vitro transposon mutagenesis of plasmids from the pigmented clones showed that the loss of
function mutants invariably had a transposon inserted in an ORF (of identical sequence in all three
pigmented clones) homologous to hemA which encodes glutamyl-tRNA reductase (GluTR), the first
enzyme in the tetrapyrrole C5 biosynthetic pathway ( Srivastava & Beale, 2005). The predicted
amino acid sequence from the metagenomic clones was 99% identical to GluTR of a number of
Neisseria species (e.g. N. mucosa WP_003748579), 96% identical to GluTR of V. parvula
(YP_003312383) and 91% identical to GluTR of Neisseria flavescens (WP_003679373). Pigmented
metagenomic clones with antibacterial activity have been found in gene libraries constructed from
rice paddy soil DNA and from metagenomic DNA from the marine sponge, Discodermia calyx (Kim et
al., 2009; He et al., 2012). Biochemical analysis of the pigments from the soil metagenome showed
that they were coproporphyrin and minor porphyin intermediates and the gene encoding GluTR
(hemA) was shown to be responsible for pigment production. As E. coli also contains a hemA gene it
is likely that the pigmentation is due to increased levels of GluTR as a result of either the increase in
copy number of hemA gene or increased expression in the metagenomic clone. Therefore it is likely
that the GluTR-encoding clones from the oral metagenome were also producing antibacterial
porphyrins.
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Sub-cloning of the antibacterial encoding genes required the construction of a new vector
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The non-pigmented clone, HOAb112C, contained a plasmid designated p112C with an insert of 15.6
kb. In order to localize the region responsible for antibacterial activity, subcloning of the three
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HindIII fragments (7 kb, 4.7 kb & 3.9 kb) was attempted. Despite multiple attempts, the gene
encoding antimicrobial activity could not be cloned into pUC based vectors. However, the three
HindIII fragments were successfully cloned in pTGEX (see materials and methods for vector details)
to generate pTGEX7, pTGEX4.7 and pTGEX3.9 and antibacterial activity was only observed in E. coli
containing pTGEX4.7 (Fig. 2).
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The utility of pTGEX was further demonstrated by sub-cloning the 5.4 kb insert from pBAC108J,
containing the gene involved in production of a pigmented antibacterial metabolite (see above) into
pTGEX to generate pTGEX5.4. The radius of the zone of inhibition in pTGEX5.4 was 0.4 cm while that
in the original BAC clone was 0.1 cm (data not shown). The larger zone of inhibition produced by
subcloning in pTGEX, may prove useful for the easier identification of clones producing antibacterial
products.
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DNA sequence analysis of a clone encoding a novel antibacterial protein.
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DNA sequence analysis of pTGEX4.7 showed that it contained six orfs (Fig. 3) with homology to genes
of Neisseria species: starting at the the 5’ end, the first partial orf showed 82% identity to the gene
fadD coding for long-chain-fatty -acid--CoA ligase (NCBI accession number CBN86964), the second
orf showed 86% identity to the gene trmU coding for tRNA (5-methylaminomethyl-2-thiouridylate)methyltransferase (accession number ADO32009), the third showed 83% identity to a gene coding
for a hypothetical protein (accession number ACF28991) which we subsequently designated
antibacterial protein1 (abp1). The fourth orf showed 73% identity to dgk coding for diacylglycerol
kinase (accession number CBN86961), the fifth orf displayed 84% identity to gshB coding for
glutathione synthetase (accession number ADZ01159) and the sixth partial orf showed 80% identity
to recN coding for DNA repair protein RecN (accession number CBN87706).
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To precisely identify the gene(s) responsible for antibacterial activity, p112C was subjected to in vitro
transposon mutagenesis. Two mutants, 112CTM3D10 and 112CTM1H7, which had lost antibacterial
activity and had a single transposon insertion were obtained. In 112CTM3D10, the transposon was
inserted in the 483 bp orf (abp1) whose translation product showed 94% identity to a hypothetical
protein (WP_003681960) of N. flavescens. Homology searches in the conserved domain database
showed that Abp1 belonged to a family of proteins (putative membrane or periplasmic proteins)
with unknown function. However, the mutagenesis results clearly showed that the gene encoded a
protein that elicited antibacterial activity. Interestingly, in 112CTM1H7, abp1 was intact and the
transposon insertion was upstream of the ORF within a predicted promoter resulting in loss of the
optimal spacing between the -10 and -35 boxes; supporting the idea that the predicted promoter is
driving the transcription of abp1. Downstream of the gene, a Rho-independent transcription
terminator was predicted (Fig. 4).
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At the N-terminus of Abp1 a 19 residue signal peptide was recognized by PSORT (Fig. 4) which is
predicted to direct the protein to the periplasmic space (certainty=0.93).
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HOAb112 has antimicrobial activity against a range of different bacteria
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HOAb112 had antibacterial activity against S. epidermidis NCTC 11964, S. aureus NCTC 12493 and B.
subtilis. However no antibacterial activity against P. aeruginosa ATCC 9027, N. subflava ATCC A1078,
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E. coli K12 JM109, F. nucleatum ATCC 25586, P. intermedia ATCC 25611, V. parvula ATCC 10790 or L.
casei NCTC 6375 was observed.
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The narrow antibacterial spectrum of Abp1 may be useful therapeutically.
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In summary, this work has shown that the human oral metagenome is a source of antimicrobial
agents. A potential problem in accessing these in functional metagenomic studies is their toxicity to
the host. To overcome this we have designed the vector pTGEX which allows the cloning of toxic
genes. In future work we plan to use this vector to construct gene libraries and screen for
antimicrobial production.
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ACKNOWLEDGEMENTS
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The financial support by Commonwealth Scholarship Commission in the United Kingdom through
Commonwealth Split-site Doctoral Scholarship is acknowledged. The authors are grateful to Dr.
Haitham Hussain from Department of Microbial Diseases, UCL Eastman Dental Institute, London for
providing Bacillus subtilis BS78H.
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TABLES
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TABLE 1 List of plasmids used in this study
Strains and Plasmid
Description
Source
Bacterial strains
E. coli DH5α
F− endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG
Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–
Invitrogen
E. coli BL21 (DE3)
F− ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene1
ind1 sam7 nin5])
Novagen
E. coli EPI300-T1R
F− mcrA Δ(mrr-hsdRMS-mcrBC) (StrR) Φ80dlacZΔM15 ΔlacX74
recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ− rpsL nupG trfA
tonAdhfr
Epicentre
pTGEX
KanR , T5 promoter, vector for cloning toxic genes lethal to the
host
This work
p112C
Plasmid from BAC clone HOAb112C
This work
pTGEX7
7 kb HindIII fragment from p112C cloned in pTGEX
This work
pTGEX4.7
4.7 kb HindIII fragment from p112C cloned in pTGEX
This work
pTGEX3.9
3.9 kb HindIII fragment from p112C cloned in pTGEX
This work
pET-21a(+)
AmpR , T7 promoter, heterologous protein overexpression vector
Novagen
pETabp1
abp1 with signal peptide cloned in pET-21a
This work
Plasmids
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FIGURES
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Fig. 1 pTGEX. The direction of the arrows shows the orientation of the genetic elements. The 4.3 kb
vector has a p15a origin of replication and a kanamycin resistance selection marker. The insert DNA
expression is driven by the T5 promoter. The lacO operator near the promoter along with the lacI
product ensures strict control over the T5 promoter. The multiple cloning site (MCS) located within
lacZ’ offers 14 restriction sites for ease of cloning.
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Fig. 2 Activity screening of p112C subclones in pTGEX vector expressed in E. coli DH5α. The vector
alone negative control and the 7 kb and 3.9 kb subclones did not show antagonistic activity against
the overlaid B. subtilis. However the 4.7 kb subclone clearly showed a zone of growth inhibition.
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Fig. 3 Gene organization of the 4.7 kb insert fragment of p112C. Four full length genes and two
terminal partial genes are located in the insert. The length of the genes and the protein product
coded by each of them are shown. All the six genes are oriented in the same direction.
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Fig. 4 Nucleotide sequence and the deduced amino acid sequence of abp1 with regulatory elements.
Putative promoter region -10 and -35 box is marked in blue and purple respectively. The Rhoindependent transcription terminator is marked at the end of the sequence with bases in green
representing the region forming the loop and those in red represent the region forming the stem of
the terminator. The free energy of the stem-loop region was -14.57 kcal/mol. The deduced amino
acid sequence is shown below the nucleotide sequence. A 19 residue signal peptide is shown in bold
at the N-terminus of the protein sequence.