1 Novel antibacterial proteins from the microbial communities associated with the 1 sponge ...

20 Pages·2010·0.3 MB·English

AEM Accepts, published online ahead of print on 23 December 2010
Appl. Environ. Microbiol. doi:10.1128/AEM.02038-10
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Novel antibacterial proteins from the microbial communities associated with the
2 sponge Cymbastela concentrica and the green alga Ulva australis
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4 Pui Yi YUNG1,3, Catherine BURKE1,4, Matt LEWIS2, Staffan KJELLEBERG1,5, Torsten
5 THOMAS1* D
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7 1Centre for Marine Bio-Innovation and School of Biotechnology and Biomolecular d
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8 Sciences, University of New South Wales, Sydney, Australia f
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9 2J. Craig Venter Institute, Rockville, Maryland, USA
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10 3 NUS Environmental Research Institute and Department of Biological Sciences, National p
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11 University of Singapore, Singapore
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12 4The ithree Institute, University of Technology, Sydney, Australia s
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13 5Singapore Centre on Life Sciences Engineering, Nanyang Technological University, o
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14 Singapore o
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16 *To whom correspondence should be address:
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17 Torsten Thomas 2
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18 Tel: [phone]; Fax: [phone]; Email: [email protected] 9

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19 Address: Centre for Marine Bio-Innovation and School of Biotechnology and g
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20 Biomolecular Sciences, University of New South Wales, Australia s
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22 Section: Invertebrate microbiology
23 Keywords: antibacterial proteins, metagenomics, sponge bacteria, algal bacteria
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1 Short version of the title for use as a running head: Antibacterial proteins from sponge
2 and algal bacteria
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1 Abstract
2 The functional metagenomic screening of the microbial communities associated with a
3 temperate marine sponge and a green alga identified three novel hydrolytic enzymes with
4 antibacterial activities. The results suggest that uncultured alpha- and gamma-
5 proteobacteria contain new classes of proteins that may be a source of antibacterial D
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6 agents. n
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1 As a result of the rising number of multi-drug resistant bacteria, recent years have
2 witnessed an increased demand for novel antibiotic compounds. Indeed, examples of
3 multiple resistances have been reported for strains of Streptococcus pneumoniae and
4 Staphylococcus aureus across Asia, South America, Australia and Europe (10, 11, 25, 30,
5 45, 53). D
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7 In order to address the multidrug resistance in bacteria, sessile marine invertebrates have d
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8 been explored for the presence of antibiotics and have proven to be a rich source of such f
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9 novel compounds (13, 17, 21, 37, 41). For example, more than 200 new bioactive
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10 metabolites have been reported from sponges per year in the last decade (51).
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11 Unfortunately, compounds from marine sources are often only available in low quantities
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12 thus hampering further development into commercial products (18, 21, 23, 24). Due to s
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13 the fact that numerous natural products isolated from marine invertebrates show
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14 structural similarities to known metabolites of microbial origin (41, 44, 47), bioactive o
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15 screening has also focused on microorganisms associated with such host surfaces. For a
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16 example, the antibacterial peptide-polyketide andrimid was found in the extract of a
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17 sponge as well as in a Vibrio sp. isolated from this host (39). Several bacterial strains 2
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18 from the surface of the alga Ulva australis are also known to produce an array of 9

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19 compounds effective against bacteria, fungi, diatoms and other biofouling organisms (15, g
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20 16, 19, 42). These observations suggest that surface-associated microbial communities s
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21 carry a large potential for new antibiotics and bioactives.
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1 Isolation of bioactives from environmental bacteria, however, faces the limitation that
2 many strains are recalcitrant to culturing (1, 33, 48, 51), and this might be particularly
3 true for obligate or facultative symbionts. To access the uncultured majority of the
4 microbial world (43), functional metagenomic approaches have been developed that
5 allow for the expression of environmental DNA from uncultured organisms in surrogate D
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6 hosts (6, 22, 32, 35, 36, 46). Functional screening of metagenomic libraries has led to the n
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7 discovery of several novel bioactives and metabolic pathways (5, 36, 52), but the search d
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8 for new antibiotics has mainly focussed on soil-derived samples. f
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10 In this study, we explored functional metagenomic libraries from the microbial
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11 communities associated with two marine living surfaces, the temperate marine sponge
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12 Cymbastela concentrica and the green alga Ulva australis, for the presence of s
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13 antibacterial activities. We screened these libraries for the inhibition of a range of target
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14 strains, identified novel antibacterial genes, characterised their activities and determined o
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15 their phylogenetic origin (for further details on the Material and Methods see the a
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16 Supplementary Materials).
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17 2
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18 Functional screening of fosmid libraries identified three clones (two from C. concentrica 9

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19 (Cc) and one from U. australis (Ua)) that showed antibacterial activity against the marine g
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20 Bacillus strain Cc6. All clones lacked zones of inhibition in the absence of inducers (i.e. s
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21 arabinose or IPTG), indicating that genes from the fosmid insert were responsible to the
22 antibacterial activity. The clearance zones had radii of 0.5, 0.8, 0.3 cm for fosmid clones
23 CcAb1, CcAb2, UaAb1, respectively, while the positive control, CBAA11 (8), had a
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1 radius of 0.2 cm (Figure S1). The clone CcAb1 showed further activity against S. aureus
2 and Alteromonas sp. CCSH174 (inhibition zone of 0.5 and 0.6 cm, respectively) and
3 UaAb1 was active towards S. aureus and K. pneumoniae (both 0.2 cm inhibition).
4 CcAb2 did not exhibit antibacterial activity against additional target strains. These
5 results show that the microbial community associated with the two marine eukaryotes D
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6 contains genes, which encode for antibacterial activities against bacteria from both n
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7 environmental and clinical settings. d
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9 Through random transposon mutagenesis six, eight and three mutants were identified to
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10 have lost their antibacterial activities for CcAb1, CcAb2 and UaAb1, respectively.
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11 Details of the ORFs identified, namely abg1, abg2 and abg3, are shown in Table 1. All
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12 three genes had a clearly recognisable -10 and -35 box of bacterial promoters, suggesting s
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13 that they are under the control of their own promoters. In addition, Abg1 and Abg2 also
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14 contained predicted signal peptides (31 aa: o
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15 MSASTCLRREYFHCFRVLLIASVLLSGNILA and 26 aa: a
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16 MNILNKKLLSILLTVATLFL VTVASA, respectively), which indicate that they are
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17 secreted. Complementation of the abg1 and abg2 genes into E. coli containing either their 2
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18 respective transposon mutant fosmids or the empty pCC1FOS vector (the host fosmid of 9

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19 the libraries) restored their antibacterial properties, showing that the genes were solely g
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20 responsible for the activities (data not shown). Subcloning of abg3 from UaAb1 was not s
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21 successful, despite several attempts, and hence subsequent functional characterizations
22 were performed on the original fosmid and its transposon mutants.
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1 Annotations of abg1, abg2 and abg3 showed that they encode for novel enzymes, with
2 Abg1 having no significant homology to experimentally characterised proteins, while
3 Abg2 and Abg3 have moderate sequence homology to an esterase from Burkholderia
4 gladioli (accession no. Q9KX40) and a hydrolase from Acanthamoeba polyphaga,
5 respectively (Table 1). Abg1, 2 and 3 contain the conserved PFAM domains of GDSL- D
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6 like lipase, beta-lactamases and Abhydrolase_3, respectively (Table 1). Comparison of n
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7 the three proteins to the non-curated Swiss-Prot database showed homology to proteins d
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8 putatively annotated as lipolytic enzymes or beta-lactamases (Table S2). Together these f
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9 results indicate that the three proteins could have hydrolytic activities.
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11 To further define the postulated hydrolytic activities, we tested the degradation of the
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12 lipid analogue tributyrin. Abg1 and Abg 2 were capable of degrading tributryrin with s
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13 clearance zones of 0.7 and 0.4 cm, respectively. Fosmid clone UaAb1 also degraded
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14 tributyrin with clearance zone of 0.5 cm. The transposon mutant of UaAb1 with the o
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15 disrupted abg3 gene failed to degrade the substrate, indicating that Abg3 mediates a
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16 hydrolytic activity (Figure S2).
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18 Abg2 had similarity to beta-lactamase domains (Table 1) and proteins (Table S2), but 9

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19 when we exposed E. coli pBAD:Chlor-abg2 clone to five different beta-lactam antibiotics g
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20 no resistance was observed. This shows that Abg2 is unlikely to have true beta-lactamase s
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21 activity.
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1 The three proteins had less than 20 % pair-wise sequence identity to each other, yet
2 surprisingly, they all produced hydrolytic/lipolytic activities and conferred antibacterial
3 properties to E. coli. We therefore propose that these proteins represent three new classes
4 of antibacterial proteins.
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6 The fosmids containing the antibacterial genes were completely sequenced to gain insight n
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7 into their genomic context and phylogenetic origin. The antibacterial genes abg1, abg2 d
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8 and abg3 were positioned in ORF 17, 11 and 20 for CcAb1, CcAb2 and UaAb1, f
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9 respectively (further details in Table S3 and Figure S3). Phylogenetic prediction with
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10 Phylopythia algorithm (38) indicated that clone CcAb1 belongs to the class delta-
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11 proteobacteria, while taxonomic prediction with MEGAN (26) assigns more than 50 % of
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12 its ORFs to gamma-proteobacteria (Table 2). This clone had a high correlation index of s
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13 tetranucleotide composition (0.7) to a fosmid clone previously described to be derived
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14 from a novel gamma-proteobacterium in the bacterial community of the sponge C. o
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15 concentrica (54), Accession number GQ160460). It is therefore likely that the source of a
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16 the CcAb1 fosmid is a novel gamma-proteobacterium. Clone CcAb2 also had a high
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17 correlation index (0.68) to this gamma-proteobacterial sequence, while the Phylopythia 2
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18 algorithm and MEGAN analysis gave inconclusive results. We therefore postulate that 9

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19 fosmids CcAb1 and CcAb2 have been derived from the same organism. Both g
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20 Phylopythia and MEGAN analysis showed that clone UaAb1 is most likely derive from a s
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21 bacterium in the class alpha-proteobacteria, with the majority of ORFs taxonomically
22 assigned to the Sphingomonadales order (Table 2).
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1 We have here identified three novel hydrolytic enzymes from sponge- and algal-
2 associated microbial communities that are responsible for antibacterial activities. These
3 enzymes were identified to possibly originate from alpha- and gamma-proteobacteria,
4 highlighting the utility of screening functional metagenomic libraries for discovery of
5 novel antibacterial activities. Most of the antibacterial agents that have been identified by D
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6 metagenomic screening are small molecules, for example, palmitoylputrescine (7), n
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7 violacein (6), turbomycin A and B (20), indirubin and indigo (34). The results presented d
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8 in this study suggest the possibility of hydrolases as alternative sources of antibacterial f
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9 activity from host-associated microorganisms.
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11 Microbial hydrolytic enzymes (e.g. lipases and esterases) play a major role in
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12 biotechnological applications as detergents, in food processing, and stereospecific s
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13 organic synthesis, catalyzing both the hydrolysis and synthesis of long-chain acyl
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14 glycerols (2). Previous functional screening of microbial metagenomic libraries o
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15 associated with the sponge Alphysina aerophoba and Hytrios erecta also found novel a
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16 lipolytic enzymes (28, 40), however no antibiotic activity was reported. Lipases act on
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17 lipids to release fatty acids of different chain lengths, which are known to have a broad 2
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18 spectrum of antibacterial activity (12, 27). The mode of action is thought to be related to 9

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19 the detergent properties of these acids, which allow them to create pores, or at high g
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20 concentrations, to cause cell lysis through cell wall degradation (12). Free fatty acids s
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21 released through the action of lipases have been shown to protect human skin against
22 infection from opportunistic pathogens such as S. aureus (14) and to protect the
23 gastrointestinal tract against pathogens such as Helicobacter pylori, Enterococcus
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1 faecalis and Klebsiella pneumoniae (49, 50). Lipases have also been associated with
2 antibacterial activity in sandflies (4), suggesting a broad biological role for lipases in the
3 protection against bacterial infection. Further extensive biochemical characterisation is
4 necessary to define the substrate and product range of the hydrolytic enzymes identified
5 here. This will provide insight into the mode of action of these novel enzyme classes. D
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7 Free fatty acids with antimicrobial properties have also been identified from algae and d
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8 sponges (3, 9, 29, 31) and it is possible that the role of the hydrolases detected from the f
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9 sponge- and algal- associated microbial communities is the conversion of lipids excreted
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10 by the eukaryotic host, to free fatty acids with antibacterial properties. This in turn could
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11 prevent the colonisation or growth of certain bacteria and hence would have an impact on
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12 community composition of the host’s microbiota. s
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14 We thank Jason Koval for his technical support. This work was supported by the o
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15 Australian Research Council, the Betty and Moore Foundation and the Centre for Marine a
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16 Bio-Innovation.
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