Antonie van Leeuwenhoek DOI 10.1007/s10482-015-0469-4
ORIGINAL PAPER
Antibacterial activity and mutagenesis of sponge-associated Pseudomonas fluorescens H41 Lumeng Ye . Juliana F. Santos-Gandelman . Cristiane C. P. Hardoim . Isabelle George . Pierre Cornelis . Marinella S. Laport
Received: 15 February 2015 / Accepted: 30 April 2015 © Springer International Publishing Switzerland 2015
Abstract Marine sponges (phylum Porifera) are well known to harbour a complex and diverse bacterial community. Some of these sponge-associated bacteria have been shown to be the real producers of secondary metabolites with a wide range of activities from antimicrobials to anticancer agents. Previously, we revealed that the strain Pseudomonas fluorescens H41 isolated from the sponge Haliclona sp. (collected at the coast of Rio de Janeiro, Brazil) showed a strong antimicrobial activity against clinical and marine bacteria. Thus, in this study the genes involved in the antimicrobial activity of P. fluorescens H41 were identified. To this
end, a library of mutants was generated via miniTnphoA3 transposon mutagenesis and the resulting clones were characterized for their antimicrobial activity. It was demonstrated that genes involved in the biosynthesis of the pyoverdine siderophore are related to the inhibitory activity of P. fluorescens H41. Therefore, this strain might play an important role in the biocontrol of the host sponge. Keywords Antagonism · Antimicrobial · Pseudomonas fluorescens · Pyoverdine · Porifera · Brazil
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10482-0150469-4) contains supplementary material, which is available to authorized users. L. Ye · P. Cornelis Department of Bioengineering Sciences, Research Group of Microbiology and VIB, Vrije Universiteit Brussel (VUB), Brussels, Belgium J. F. Santos-Gandelman · C. C. P. Hardoim · M. S. Laport (&) Instituto de Microbiologia Paulo de Go´es, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil e-mail: [email protected] I. George · M. S. Laport De´partement de Biologie des Organismes, Laboratoire de Biologie Marine, Universite´ Libre de Bruxelles (ULB), Brussels, Belgium
Pseudomonads are ubiquitous Gram-negative Gammaproteobacteria known for their extreme versatility and adaptability. They are widely distributed in the environment, including soil and water, and they also occur in association with various host organisms where they participate in a variety of interactions (Silby et al. 2011). As sessile and filter-feeding metazoans, marine sponges represent an ecologically important and highly diverse component of marine benthic communities throughout the world. In high microbial abundance (HMA) sponges, up to 38 % of the sponge wet weight is composed of bacterial cells. This bacterial abundance surpasses that of surrounding seawater by 2–4 orders of magnitude (Hentschel et al. 2006). Most of these
123
Antonie van Leeuwenhoek
bacteria are symbiotic and are potentially a major source of novel secondary metabolites and other valuable compounds (Piel 2004). These symbiotic bacteria may play a role in digestion, waste removal and help in the nutritional process, either by intracellular digestion or by translocation of metabolites produced by photosynthesis, nitrification and nitrogen fixation, among other processes. They may also stabilise the endoskeleton of their host and participate in its chemical defence (Skariyachan et al. 2014). The composition of the bacterial communities associated with sponges is different from that which can be found in other natural environments. The sponge mesohyl provides a unique microenvironment capable of harbouring a wide range of bacterial species with unprecedented metabolic properties, representing a great potential in the search for new compounds of biotechnological interest (Wang 2006; Taylor et al. 2007; Santos-Gandelman et al. 2014). Over the last few years, marine ecosystems have been exploited for the isolation of novel bioactive compounds. These secondary metabolites include a variety of antibiotics, biosurfactants, quorum-sensing inhibitors, among others, whose production is often governed by interactions among microbial species (Santos-Gandelman et al. 2014). In a previous work, our group characterized phenotypically and phylogenetically several bacterial strains isolated from distinct sponge species collected at the coast of Rio de Janeiro (Brazil) that showed antimicrobial activities (Santos et al. 2010). The most active strain was identified as Pseudomonas fluorescens H41, which was isolated from the sponge Haliclona sp. This strain exhibited a strong antimicrobial activity against both Gram-negative and Gram-positive bacteria. The aim of this study was to identify genes associated with the antimicrobial activity of strain P. fluorescens H41. Moreover, the potential role of this antimicrobial activity within the bacterial communities associated with sponge was also addressed.
Materials and methods Bacteria used in this study Pseudomonas fluorescens H41 (16S ribosomal RNA gene sequence GenBank accession EU862080) was
123
isolated from a Haliclona sp. sponge collected at the Cagarras Archipelago, Rio de Janeiro, Brazil (Santos et al. 2010). The strain was grown in Brain Heart Infusion (BHI), Luria-Bertani (LB) or Casamino acid (CAA, low iron) media at 25 °C for 18 h. In the transposon mutagenesis assays (see below) of strain H41 with miniTnphoA3 transposon, Escherichia coli SM10 (ʎ pir) (pUT:miniTnphoA3) was grown in LB medium at 37 °C for 18 h. Fifty-eight marine bacterial strains previously identified (Santos et al. 2010; Santos-Gandelman et al. 2013) were grown in BHI medium at 25 °C for 24 h and used as indicators in the antimicrobial activity assays. Among those, 54 were isolated from distinct sponges, two from surrounding seawater and two from sediment. Sponges, seawater and sediment samples were collected by scuba diving at depths and temperatures ranging from 4 to 20 m and 18 to 25 °C, respectively, in the Cagarras archipelago (CA) (23°01′S, 43°11′W) and Praia Vermelha (PV) beach (22°57′S, 43°09′W), Rio de Janeiro, Brazil (Supplementary Table 1). The following reference strains were obtained from bacterial culture collection and clinical strains and were also used as indicators in the assays for antimicrobial activity: Staphylococcus aureus ATCC 29213, Staphylococcus hominis ATCC 27844, Enterococcus faecium ATCC 19434, E. coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, S. aureus (CA-MRSA, community-associated methicillin-resistant S. aureus) and S. hominis (resistant to ampicillin and penicillin). These strains were grown in BHI medium at 37 °C for 18 h. Transposon mutagenesis assays Mutagenesis was done by biparental mating with the donor strain E. coli SM10 (ʎ pir) containing the suicide delivery system pUT (de Lorenzo et al. 1990) and the transposon miniTnphoA3, as described previously (Pattery et al. 1999; de Chial et al. 2003). Since the pUT:miniTnphoA3 plasmid is a suicide vector and is lost from P. fluorescens H41, the only way for the miniTnphoA3 to be retained is to transpose from the pUT:miniTnphoA3 plasmid into the chromosome of the P. fluorescens H41. Transconjugants were selected on CAA medium plates supplemented with the appropriate antibiotics (50 µg/ml gentamicin and 25 µg/ml chloramphenicol).
Antonie van Leeuwenhoek
Mutants were screened on agar-BHI for their lack of antimicrobial activity against S. aureus ATCC 29213 as described previously (Marinho et al. 2009). Localisation of TnphoA3 insertions Chromosomal DNA was obtained from the selected mutants using the Genome DNA KIT (MP Biomedicals, Illkirch Cedex—France) according to the manufacturer’s protocol. For each mutant, 1 mg of genomic DNA was cut using 1 unit of the restriction enzyme TaqI (Biolabs Inc., Ipswich, USA). The reaction was performed at 65 ° C for 1 h. After inactivation of the restriction enzyme at 80 °C for 20 min, 100 ng of the digested DNA was ligated overnight with 200 units of T4 DNA ligase at 16 °C in 200 ml of ligation buffer (50 mM Tris–HCl, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 25 mg/ml BSA, pH 7.5). These conditions favour auto-ligation and circularization of the fragments. Then DNA was precipitated by adding 20 ml of 3 M sodium acetate (pH 5.2) and 500 ml of ethanol 100 %. This was incubated at –20 °C for 1 h, and after centrifugation, the DNA was obtained as a pellet. Pelleted DNA was washed with 70 % ethanol and, after further centrifugation, dried at 37 °C. This dried DNA was used for inverse-PCR of the circularized fragments generated from the chromosomal DNA obtained from each mutant. The site of transposon insertion was determined using inverse PCR with primers PhoA5 (5′-GCGG CAGTCTGATCACCCGTTA-3′), Gm1 (5′-TGGAC CAGTTGCGTGAGCGCATA-3′), PhoA4 (5′-GCAC CGCCGGGTGCAGTAATTAT-3′) and Gm2 (5′-TG TCAACTGGGTTCGTGCCTTC-3′) as described previously (de Chial et al. 2003). The sequence of the DNA flanking region of the transposon was determined at the VIB Genetic Service Facility (Antwerp, Belgium) using the primer pair Gm2 and PhoA4. TaqI-digested genomic bacterial DNA isolated from antagonistic activity-negative P. fluorescens mutants was used as template. PCR was performed using the PCR kit ExTakara (Takara Clontech, SaintGermain-en-Laye, France) in a total volume of 50 µl containing: 1 9 ExTakara buffer, 2.5 mM of each deoxyribonucleotide triphosphate, 1 U of ExTakara DNA polymerase, 20 pmol of each primer and 100 ng DNA. The following PCR conditions were used: initial denaturation step at 94 °C for 50 s, followed by 10 cycles at 94 °C for 10 s, 55 °C for 30 s and 68 °C for 2 min, then 20 cycles at 94 °C for 10 s, 55 °C for
30 s and 68 °C for 2 min, with a final elongation step of 72 °C for 10 min. The amplicons were purified using QIAquick PCR purification protocol (Qiagen, Venlo, The Netherlands) following manufacturer’s instructions. After purification, the amplicons were subjected for sequencing by the dideoxy termination method with the same primers as those used for the PCR reaction. In order to map the position of the inserted transposon, the obtained sequences were used as query in a BLAST search of the P. fluorescens A506 genome (www.pseudomonas.com). For a few mutants, the sequences were not present in the chromosome of P. fluorescens A506. In that case, blastn searches of the National Center for Biotechnology Information (NCBI) database were performed. For every mutant, the sequences obtained with each of the primer pairs matched in the same gene or intergenic region of the sequenced chromosome. This confirmed the reliability of the method used for mapping the insertions. Antimicrobial activity assay The assay for antimicrobial substance production was performed as described previously (Marinho et al. 2009). Briefly, 107 cells of the strains were spotted onto BHI-agar. After growth of each strain at 25 °C for 24–48 h, 105 cells of the indicator strains mixed with 3 ml of BHI soft agar were poured over the plates. Plates were incubated at 25 °C (marine strains) or 37 °C (reference and clinical strains) for 18 h and the diameter of the inhibition zone around the spotted strain was measured. An indicator strain was considered sensitive to the activity of the P. fluorescens H41 strain or mutant clone when it exhibited a clear inhibition zone with a diameter ≥8 mm. When the inhibition zone was \8 mm or when bacterial colonies grew inside the inhibition zone, the indicator strain was considered resistant. Results were converted to graphs using the Cytoscape 3.1.0 program (http://www.cytoscape.org). Phylogenetic inferences Sequences obtained for the pvdL and pvdD genes were quality inspected and edited with the Sequence Scanner software V.1 (Applied Biosystems, Foster City, USA). Sequences for both genes were obtained at the NCBI database. Then, each sequence recovered
123
Antonie van Leeuwenhoek
from NCBI was aligned with the respective sequence from this study via blast two sequences (bl2seq). The closest relatives were used for phylogenetic analysis. These sequences were aligned using Clustal X in the MEGA6 software (Tamura et al. 2013). The appropriated evolutionary model was determined using MEGA6 (Tamura et al. 2013) and found to be the general-time reversible model (GTR) (Rodriguez et al. 1990) with a discrete gamma-distribution of among-site rate variation (Γ4) and a proportion of invariant sites (I) for pvdL gene and GTR + Γ4 for pvdD gene. Maximum likelihood analysis was conducted using MEGA6 (Tamura et al. 2013).
the wild type H41 strain. The majority of the mutants showed an inhibition zone of 20 mm in diameter in at least six of the seven indicator strains, while the spectrum of activity of the mutants with reduced antimicrobial activity varied among the tested indicators. More details on these results can be found in the supplementary material (Supplementary Table 2). On the other hand, the four mutants with no antimicrobial activity against S. aureus ATCC 29213, also lost their inhibitory activity against all tested strains, including antimicrobial resistant CA-MRSA and S. hominis strains. Further, they also lost their swarming motility and pyoverdine production (greenish colour) under conditions of iron limitation (CAA medium) (Fig. 2).
Results
Determination of the point of insertion of the transposon and identification of the mutated gene
Functional screening of the transposon mutagenesis library A miniTnphoA3 mutagenesis library was constructed and 960 mutants were screened for antimicrobial activity against S. aureus ATCC 29213. Four mutants (7B5, 8B2, 6G3 and 8H6) had no antimicrobial activity, nine and 15 mutants had reduced and 100 % antimicrobial activity against S. aureus, respectively (Fig. 1). These twenty-eight mutants were selected and their spectrum of antimicrobial activity was further analysed. They were tested against other bacteria of medical relevance, which were inhibited as well by Fig. 1 Antimicrobial activity of Pseudomonas fluorescens H41, wild type and mutants, against Staphylococcus aureus ATCC 29213. Four mutants (7B5, 8B2, 6G3 and 8H6) had no antimicrobial activity (arrows) and nine mutants had reduced inhibitory activity (\100 %) compared to the wild type (20 mm = 100 %)
123
The flanking regions of the transposon insertion in the genome of each of the four mutants harbouring no antimicrobial activity were sequenced. Among the genes inactivated, one gene could be identified. Its sequence presented high similarity to the pyoverdine synthetase gene pvdL from P. fluorescens A506 (GenBank accession YP_006325133.1). Genes inactivated in mutants with reduced antimicrobial activity coded for the non-ribosomal peptide synthase PvdD, a di-/tricarboxylate transporter, a peptide ABC transporter, a putative
Antonie van Leeuwenhoek
Fig. 2 Pyoverdine production and swarming motility under conditions of iron limitation (CAA medium): a positive control with the wild type strain H41 and b negative test for a representative mutant (ΔpvdL) P. fluorescens 6G3
nucleotidyltransferase, a putative adenylate cyclase, a diguanylate cyclase (GGDEF) domain protein, an alkaline phosphatase, a quinolinate synthetase complex, a HNH endonuclease domain protein and a hypothetical protein. Even though the pvdL and pvdD genes were detected in several species of Pseudomonas, none of the sequences in the NCBI database were retrieved from the marine environment (data not shown). The phylogenetic inference of pvdL is depicted in Fig. 3a. This clearly demonstrated that the sequence obtained from P. fluorescens H41 is distinct from all closest relatives that in this case were originated from plant-associated strains. Figure 3b shows the phylogenetic analysis of the pvdD and again the vast majority of the sequences were retrieved from plant-associated strains. The closest relatives to P. fluorescens H41 sequences are two sequences recovered from plant-associated strains. The mutants 6G3 (ΔpvdL) and 1H4 (ΔpvdD) showed null and reduced antimicrobial activity, respectively. Thus, given the results obtained, it can be concluded that pvdL and pvdD, which are genes involved in the biosynthesis of pyoverdine, are related to the antimicrobial activity of P. fluorescens H41.
for their antimicrobial activity against bacteria isolated from 11 sponge species, from surrounding seawater and from sediment collected along the coast of Rio de Janeiro city, Brazil. Among the 58 strains tested as indicators, the wild type strain inhibited 39 strains (67.2 %) and the ΔpvdL mutant did not inhibit any of these indicator strains (Fig. 4). More details regarding these results can be found in the supplementary material (Supplementary Table 2). Among the strains inhibited by P. fluorescens H41, two were isolated from the same sponge species at a distinct sampling site (PV) and 35 were isolated from ten distinct sponge species collected at both sampling places (CA and PV). Moreover, one of the inhibited strains was isolated from seawater and another one from marine sediment. However, the wild type strain showed no inhibitory activity against H40, a P. fluorescens strain isolated from the same sponge specimen of Haliclona sp. collected at the sample location (CA). Interestingly, the wild type of P. fluorescens H41 inhibited 85 % of the bacterial strains isolated at the same site (CA) and inhibited only 57 % of the bacterial strains isolated from sponges at PV.
Comparative analysis of antimicrobial activity of wild type and ΔpvdL
Discussion
To further characterise the ecological role of P. fluorescens H41 in the sponge microbiota, the wild type strain and 6G3 mutant (ΔpvdL) were also tested
Marine isolates of Pseudomonas are found in diverse ecosystems, including coastal regions, deep sea and more extreme environments. Marine pseudomonads have been detected in bacterioplankton, in sediments
123
Antonie van Leeuwenhoek
Fig. 3 Phylogenetic analyses of the pvdL and pvdD genes: the Maximum Likelihood trees (a) of pvdL (-ln likelihood: −1777.2200) and (b) of pvdD (-ln likelihood: −2057.0918)
are shown. The sequences retrieved in this study are highlighted in bold. Numbers at tree nodes are bootstrap values in Maximum Likelihood and values above 75 are shown
and associated with other organisms (Engel et al. 2002). The diversity of Pseudomonas strains isolated from a wide range of marine ecosystems suggests that these organisms may produce novel and diverse bioactive substances (Isnansetyo and Kamei 2009). However, very little is known about Pseudomonas strains isolated from marine sponges and their production of bioactive compounds (Santos et al.
2010; Marinho et al. 2009; Isnansetyo and Kamei 2009). So far, the most promising antimicrobial substances isolated from sponge-associated Pseudomonas spp. are 2-undecyl-4-quinolone (BultelPonce´ et al. 1999), cyclic dipeptides (also known as diketopiperazines, DKPs) (Jayatilake et al. 1996; Zheng et al. 2005; Santos et al. 2015) and pyrone I (Singh et al. 2003).
123
Antonie van Leeuwenhoek
Fig. 4 Antimicrobial activity of the wild Pseudomonas fluorescens H41 strain (H41) and mutant (6G3) against marine bacteria. The arrows indicate that the wild type strain showed antibacterial activity and the numbers represent the inhibition zone in mm. Marine strains without connecting arrows were not inhibited and the mutant strain showed no inhibitory activity. Strains isolated from the same marine sponge,
sediment or seawater are filled with the same colour and those belonging to the same bacterial genus have the same edge colour of the circle. Strains were isolated from specimens from the Cagarras Archipelago (CA) (on the left side) and from the Praia Vermelha (PV) beach (on the right side), Rio de Janeiro, Brazil
In this context, Santos et al. (2010) characterized Pseudomonas strains with antimicrobial activities isolated from distinct sponge species. The most active strains were P. fluorescens H40 and H41 and P. aeruginosa H51 retrieved from Haliclona sp. They exhibited activity against important pathogenic bacteria such as methicillin-resistant S. aureus (MRSA and CA-MRSA), vancomycin-resistant enterococci (VRE) and multidrug-resistant Gram-negative bacilli (E. coli, Klebsiella and Pseudomonas species). Currently, these drug-resistant strains are considered a public health problem, because their resistance is spreading rapidly (WHO 2014). In order to identify genes related to the antimicrobial activity of P. fluorescens H41, transposon mutagenesis assays followed by functional screening of the library were performed. This study showed that genes involved in the biosynthesis of the siderophore pyoverdine, notably pvdL, are related to the inhibitory
activity of the sponge-associated strain P. fluorescens H41 against pathogenic and marine bacteria. The results were supported by the loss of pyoverdine production in ΔpvdL and ΔpvdD mutants. The phylogenetic analyses of the strain H41 pvdL and pvdD genes revealed that the closest relatives to these sequences were from plant-associated strains. This is most likely related to the lack of studies addressing the marine habitat. Additionally, Spencer et al. (2003) reported that the pyoverdine segment was the most hypervariable region of the genome of the P. aeruginosa strains. Likewise, Smith et al. (2005) showed that the central part of the pyoverdine region in P. aeruginosa could not be aligned among pyoverdine types. The authors also noticed that the pvdD gene was one of the most divergent genes in the pyoverdine region. Divergent pvdD sequences might occur in other species of pseudomonads and thus explain the difficult in finding sequences that
123
Antonie van Leeuwenhoek
matched the ones generated in this study. Smith et al. (2005) also suggested a history of horizontal gene transfer, which might take place within other Pseudomonas species, including the marine species from which very little is known. There has been considerable debate in the literature about the contribution of siderophores to antagonism displayed by fluorescent pseudomonads against plant pathogens (Matthijs et al. 2007) but this is the first study demonstrating that pyoverdine is also produced by sponge-associated pseudomonads. PvdL, a non-ribosomal peptide synthetase from pseudomonads, is involved in the biosynthesis of the chromophore of the siderophore pyoverdine (Mossialos et al. 2002). Many Pseudomonas strains produce siderophores as a strategy to sequester iron in the marine environment, where the iron level is extremely low (Hider and Kong 2010). This is needed to maintain important enzymatic processes (where iron is a cofactor) and can be a prerequisite for pathogenicity for many Pseudomonas strains (Mossialos et al. 2002). More recently, siderophores have been assessed as targets for the development of new anti-bacterial molecules, due to the importance of iron in bacterial metabolism and to the paucity of effective antibiotics against multidrug-resistant bacteria (Frangipani et al. 2014). Relatively little information is available on the siderophore pyoverdine in marine bacteria compared to bacteria from terrestrial environments as was revealed in the phylogenetic analyses of the pvdL and pvdD genes. Similar results were recently observed in a Pseudomonas putida strain isolated from a stream in Brussels, Belgium (Ye et al. 2014). This strain was found to produce a compound with antimicrobial activity against the opportunistic pathogens S. aureus, P. aeruginosa, and the plant pathogen Pseudomonas syringae. Intriguingly, it was demonstrated that the majority of transposon mutants, which were unable to produce the compound, had insertions in genes involved in the biosynthesis of pyoverdine, despite the fact that pyoverdine itself showed no antimicrobial activity (Ye et al. 2014). One possible explanation, mentioned by the authors, was that the proteins involved in the production of the antibiotic compound share the same ‘‘siderosome’’ platform as the pyoverdine biosynthetic machinery recently described in P. aeruginosa (Schalk and Guillon 2013).
123
In this study, the significance of pyoverdine synthesis by the sponge-associated P. fluorescens H41 in relation to activity against bacteria of medical importance is demonstrated. These results open up interesting avenues in the search for novel compounds against multi-resistant bacteria. However, the contribution of pyoverdine to the ecological success of its producer(s) is less clear. It is known that microbial interactions in environmental settings form the basis of several important ecological processes. Among such interactions, competition between species plays an important role in the structuring of microbial communities (Joshi et al. 2009). Our results showed that the inhibitory activity of P. fluorescens H41 was high against bacteria isolated from different species of marine sponges whereas no activity was observed against the strain isolated from the same specimen. Furthermore, P. fluorescens H41 showed high inhibitory activities while others spongeassociated P. fluorescens strains did not have the ability to produce the antimicrobial substance. Many bacteria produce antibiotics to gain a competitive advantage over other microorganisms inhabiting the same ecological niche (Webster and Blackall 2009). The role of bacteria–bacteria crosstalk, whether beneficial or inhibitory, remains an interesting topic for future studies. A good example is the P. fluorescens strain A506, which is a commercially available biological control agent (BlightBan A506; Nufarm Americas Inc., Sugar Land, TX) used for the suppression of fire blight on pear and apple trees (Lindow et al. 1996). In the marine environment, competition for nutrients and space drives the evolution of colonisation and growth strategies of marine microorganisms. In that context, antimicrobials and other secondary metabolites produced by microorganisms may aim to inhibit the growth of others by affecting their survival and reproduction (Burgess et al. 1999). Thus, considering the results obtained in this study, which demonstrated that pvdL is directly related to expression of antimicrobial activity in P. fluorescens H41, we suggest that bacteria producing siderophores and antibiotics favour sponge fitness. In this case, we speculate that P. fluorescens H41 strain colonises the sponge and provides antagonistic molecules (such as pyoverdine) that benefit the host as a self-defence against invasive and pathogenic bacteria. In return, the bacteria gain a protected and nutrient-rich habitat.
Antonie van Leeuwenhoek
Only a few studies have addressed the importance of biochemical properties of symbiotic microbes in relation to sponge physiology. Richardson et al. (2012), after experimental manipulation with antibiotics, showed that associated bacteria favour sponge growth and influence the composition of the bacterial community. It was also reported that ferric iron (Fe3+) plays an important role in sponge morphogenesis. Hence, the availability of iron may be a key factor in determining sponge growth (Krasko et al. 2002). This suggests the production of compounds such as siderophores by the sponge-associated communities might play important roles for the host fitness. Given these data, further studies are needed to determine the potential role of siderophores as drivers of sponge bacterial communities and of sponge fitness. Finally, our results suggest a biocontrol role of the pyoverdine produced by P. fluorescens in its host sponge. Acknowledgments This work was supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ) to M.S. Laport. We are also grateful to Science without Borders Program/CNPq for the post doctorate scholarship to M.S. Laport. Conflict of interest
The authors declare no conflict of
interest.
References Bultel-Ponce´ V, Berge J, Debitus C, Nicolas J, Guyot M (1999) Metabolites from the sponge associated bacterium Pseudomonas species. Mar Biotechnol 1:384–390 Burgess JG, Jordan EM, Bregu M, Mearns-Spragg A, Boyd KG (1999) Microbial antagonism: a neglected avenue of natural products research. J Biotechnol 70:27–32 de Chial M, Ghysels B, Beatson SA, Geoffroy V, Meyer JM, Pattery T, Baysse C, Chablain P, Parsons YN, Winstanley C, Cordwell SJ, Cornelis P (2003) Identification of type II and type III pyoverdine receptors from Pseudomonas aeruginosa. Microbiology 149:821–831 de Lorenzo V, Herrero M, Jakubzik U, Timmis KN (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria. J Bacteriol 172:6568–6572 Engel S, Jensen PR, Fenical W (2002) Chemical ecology of marine microbial defence. J Chem Ecol 28:1971–1985 Frangipani E, Bonchi C, Minandri F, Imperi F, Visca P (2014) Pyochelin potentiates the inhibitory activity of gallium on Pseudomonas aeruginosa. Antimicrob Agents Chemother 58:5572–5575
Hentschel U, Usher KM, Taylor MW (2006) Marine sponges as microbial fermenters. FEMS Microbiol Ecol 55:167– 177 Hider RC, Kong XL (2010) Chemistry and biology of siderophores. Nat Prod Rep 27:637–657 Isnansetyo A, Kamei Y (2009) Bioactive substances produced by marine isolates of Pseudomonas. J Ind Microbiol Biotechnol 36:1239–1248 Jayatilake GS, Thornton MP, Leonard AC, Grimwade JE, Baker BJ (1996) Metabolites from an Antarctic sponge associated bacterium, Pseudomonas aeruginosa. J Nat Prod 59:293–296 Joshi H, Dave R, Venugopalan VP (2009) Competition triggers plasmid-mediated enhancement of substrate utilisation in Pseudomonas putida. PLoS ONE 4:e6065 Krasko A, Schro¨der HC, Batel R, Grebenjuk VA, Steffen R, Mu¨ller IM, Mu¨ller WE (2002) Iron induces proliferation and morphogenesis in primmorphs from the marine sponge Suberites domuncula. DNA Cell Biol 21:67–80 Lindow SE, McGourty G, Elkins R (1996) Interactions of antibiotics with Pseudomonas fluorescens strain A506 in the control of fire blight and frost injury to pear. Phytopathology 86:841–848 Marinho PR, Moreira AP, Pellegrino FL, Muricy G, Bastos MC, Santos KR, Giambiagi-deMarval M, Laport MS (2009) Marine Pseudomonas putida: a potential source of antimicrobial substances against antibiotic-resistant bacteria. Mem Inst Oswaldo Cruz 104:678–682 Matthijs S, Tehrani KA, Laus G, Jackson RW, Cooper RM, Cornelis P (2007) Thioquinolobactin, a Pseudomonas siderophore with antifungal and anti-Pythium activity. Environ Microbiol 9:425–434 Mossialos D, Ochsner U, Baysse C, Chablain P, Pirnay JP, Koedam N, Budzikiewicz H, Ferna´ndez DU, Scha¨fer M, Ravel J, Cornelis P (2002) Identification of new, conserved, non-ribosomal peptide synthetases from fluorescent pseudomonads involved in the biosynthesis of the siderophore pyoverdine. Mol Microbiol 45:1673–1685 Pattery T, Hernalsteens JP, de Greve H (1999) Identification and molecular characterization of a novel Salmonella enteritidis pathogenicity islet encoding an ABC transporter. Mol Microbiol 33:791–805 Piel J (2004) Metabolites from symbiotic bacteria. Nat Prod Rep 21:519–538 Richardson C, Hill M, Marks C, Runyen-Janecky L, Hill A (2012) Experimental manipulation of sponge/bacterial symbionts community composition with antibiotics: sponge cell aggregates as a unique tool to study animal/ microorganism symbiosis. FEMS Microbiol Ecol 81:407– 418 Rodriguez F, Oliver JL, Marin A, Medina JR (1990) The general stochastic model of nucleotide substitution. J Theor Biol 142:485–501 Santos OCS, Pontes PVML, Santos JFM, Muricy G, Giambiagi-deMarval M, Laport MS (2010) Isolation, characterization and phylogeny of sponge-associated bacteria with antimicrobial activities from Brazil. Res Microbiol 161:604–612 Santos OCS, Soares AR, Machado FLS, Romanos MTV, Muricy G, Giambiagi-deMarval M, Laport MS (2015) Investigation of biotechnological potential of sponge-
123
Antonie van Leeuwenhoek associated bacteria collected in Brazilian coast. Lett Appl Microbiol 60:140–147 Santos-Gandelman JF, Santos OCS, Pontes PVM, Andrade CL, Korenblum E, Muricy G, Giambiagi-deMarval M, Laport MS (2013) Community structure and characterization of sponge-associated bacteria from Brazilian coast. Mar Biotechnol 15:668–676 Santos-Gandelman JF, Giambiagi-deMarval M, Oelemann WMR, Laport MS (2014) Biotechnological potential of sponge-associated bacteria. Curr Pharm Biotechnol 15:143–155 Schalk IJ, Guillon L (2013) Pyoverdine biosynthesis and secretion in Pseudomonas aeruginosa: implications for metal homeostasis. Environ Microbiol 15:1661–1673 Silby MW, Winstanley C, Godfrey SAC, Levy SB, Jackson RW (2011) Pseudomonas genomes: diverse and adaptable. FEMS Microbiol Rev 35:652–680 Singh MP, Kong F, Janso JE, Arias DA, Suarez PA, Bernan VS, Petersen PJ, Weiss WJ, Carter G, Greenstein M (2003) Novel alpha-pyrones produced by a marine Pseudomonas sp. F92S91: taxonomy and biological activities. J Antibiot 56:1033–1044 Skariyachan S, Rao AG, Patil MR, Saikia B, Bharadwaj KN, Rao GS (2014) Antimicrobial potential of metabolites extracted from bacterial symbionts associated with marine sponges in coastal area of Gulf of Mannar Biosphere, India. Lett Appl Microbiol 58:231–241 Smith EE, Sims EH, Spencer DH, Rajinder K, Olson MV (2005) Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa. J Bacteriol 187:2138–2147
123
Spencer DH, Kas A, Smith EE, Raymond CK, Sims EH, Hastings M, Burns JL, Kaul R, Olson M (2003) Wholegenome sequence variation among multiple isolates of Pseudomonas aeruginosa. J Bacteriol 185:1316–1325 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729 Taylor MW, Radax R, Steger D, Wagner M (2007) Spongeassociated microorganisms: evolution, ecology, and biotechnological potential. Microbiol Mol Biol Rev 71:295–347 Wang G (2006) Diversity and biotechnological potential of the sponge-associated microbial consortia. J Ind Microbiol Biotechnol 33:545–551 Webster NS, Blackall LL (2009) What do we really know about sponge-microbial symbioses? ISME J 3:1–3 World Health Organization (2014) Antimicrobial resistance: global report on surveillance (2014) WHO Library Cataloguing-in-Publication Data 1-256. WHO Press, Geneva Ye L, Hildebrand F, Dingemans J, Ballet S, Laus G, Matthijs S, Berendsen R, Cornelis P (2014) Draft genome sequence analysis of a Pseudomonas putida W15Oct28 strain with antagonistic activity to Gram-positive and Pseudomonas sp. pathogens. PLoS ONE 9:e110038 Zheng L, Yan X, Xu J, Chen H, Lin W (2005) Hymeniacidon perleve associated bioactive bacterium Pseudomonas sp. NJ6-3-1. Prikl Biokhim Mikrobiol 41:35–39
ORIGINAL PAPER
Antibacterial activity and mutagenesis of sponge-associated Pseudomonas fluorescens H41 Lumeng Ye . Juliana F. Santos-Gandelman . Cristiane C. P. Hardoim . Isabelle George . Pierre Cornelis . Marinella S. Laport
Received: 15 February 2015 / Accepted: 30 April 2015 © Springer International Publishing Switzerland 2015
Abstract Marine sponges (phylum Porifera) are well known to harbour a complex and diverse bacterial community. Some of these sponge-associated bacteria have been shown to be the real producers of secondary metabolites with a wide range of activities from antimicrobials to anticancer agents. Previously, we revealed that the strain Pseudomonas fluorescens H41 isolated from the sponge Haliclona sp. (collected at the coast of Rio de Janeiro, Brazil) showed a strong antimicrobial activity against clinical and marine bacteria. Thus, in this study the genes involved in the antimicrobial activity of P. fluorescens H41 were identified. To this
end, a library of mutants was generated via miniTnphoA3 transposon mutagenesis and the resulting clones were characterized for their antimicrobial activity. It was demonstrated that genes involved in the biosynthesis of the pyoverdine siderophore are related to the inhibitory activity of P. fluorescens H41. Therefore, this strain might play an important role in the biocontrol of the host sponge. Keywords Antagonism · Antimicrobial · Pseudomonas fluorescens · Pyoverdine · Porifera · Brazil
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10482-0150469-4) contains supplementary material, which is available to authorized users. L. Ye · P. Cornelis Department of Bioengineering Sciences, Research Group of Microbiology and VIB, Vrije Universiteit Brussel (VUB), Brussels, Belgium J. F. Santos-Gandelman · C. C. P. Hardoim · M. S. Laport (&) Instituto de Microbiologia Paulo de Go´es, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil e-mail: [email protected] I. George · M. S. Laport De´partement de Biologie des Organismes, Laboratoire de Biologie Marine, Universite´ Libre de Bruxelles (ULB), Brussels, Belgium
Pseudomonads are ubiquitous Gram-negative Gammaproteobacteria known for their extreme versatility and adaptability. They are widely distributed in the environment, including soil and water, and they also occur in association with various host organisms where they participate in a variety of interactions (Silby et al. 2011). As sessile and filter-feeding metazoans, marine sponges represent an ecologically important and highly diverse component of marine benthic communities throughout the world. In high microbial abundance (HMA) sponges, up to 38 % of the sponge wet weight is composed of bacterial cells. This bacterial abundance surpasses that of surrounding seawater by 2–4 orders of magnitude (Hentschel et al. 2006). Most of these
123
Antonie van Leeuwenhoek
bacteria are symbiotic and are potentially a major source of novel secondary metabolites and other valuable compounds (Piel 2004). These symbiotic bacteria may play a role in digestion, waste removal and help in the nutritional process, either by intracellular digestion or by translocation of metabolites produced by photosynthesis, nitrification and nitrogen fixation, among other processes. They may also stabilise the endoskeleton of their host and participate in its chemical defence (Skariyachan et al. 2014). The composition of the bacterial communities associated with sponges is different from that which can be found in other natural environments. The sponge mesohyl provides a unique microenvironment capable of harbouring a wide range of bacterial species with unprecedented metabolic properties, representing a great potential in the search for new compounds of biotechnological interest (Wang 2006; Taylor et al. 2007; Santos-Gandelman et al. 2014). Over the last few years, marine ecosystems have been exploited for the isolation of novel bioactive compounds. These secondary metabolites include a variety of antibiotics, biosurfactants, quorum-sensing inhibitors, among others, whose production is often governed by interactions among microbial species (Santos-Gandelman et al. 2014). In a previous work, our group characterized phenotypically and phylogenetically several bacterial strains isolated from distinct sponge species collected at the coast of Rio de Janeiro (Brazil) that showed antimicrobial activities (Santos et al. 2010). The most active strain was identified as Pseudomonas fluorescens H41, which was isolated from the sponge Haliclona sp. This strain exhibited a strong antimicrobial activity against both Gram-negative and Gram-positive bacteria. The aim of this study was to identify genes associated with the antimicrobial activity of strain P. fluorescens H41. Moreover, the potential role of this antimicrobial activity within the bacterial communities associated with sponge was also addressed.
Materials and methods Bacteria used in this study Pseudomonas fluorescens H41 (16S ribosomal RNA gene sequence GenBank accession EU862080) was
123
isolated from a Haliclona sp. sponge collected at the Cagarras Archipelago, Rio de Janeiro, Brazil (Santos et al. 2010). The strain was grown in Brain Heart Infusion (BHI), Luria-Bertani (LB) or Casamino acid (CAA, low iron) media at 25 °C for 18 h. In the transposon mutagenesis assays (see below) of strain H41 with miniTnphoA3 transposon, Escherichia coli SM10 (ʎ pir) (pUT:miniTnphoA3) was grown in LB medium at 37 °C for 18 h. Fifty-eight marine bacterial strains previously identified (Santos et al. 2010; Santos-Gandelman et al. 2013) were grown in BHI medium at 25 °C for 24 h and used as indicators in the antimicrobial activity assays. Among those, 54 were isolated from distinct sponges, two from surrounding seawater and two from sediment. Sponges, seawater and sediment samples were collected by scuba diving at depths and temperatures ranging from 4 to 20 m and 18 to 25 °C, respectively, in the Cagarras archipelago (CA) (23°01′S, 43°11′W) and Praia Vermelha (PV) beach (22°57′S, 43°09′W), Rio de Janeiro, Brazil (Supplementary Table 1). The following reference strains were obtained from bacterial culture collection and clinical strains and were also used as indicators in the assays for antimicrobial activity: Staphylococcus aureus ATCC 29213, Staphylococcus hominis ATCC 27844, Enterococcus faecium ATCC 19434, E. coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, S. aureus (CA-MRSA, community-associated methicillin-resistant S. aureus) and S. hominis (resistant to ampicillin and penicillin). These strains were grown in BHI medium at 37 °C for 18 h. Transposon mutagenesis assays Mutagenesis was done by biparental mating with the donor strain E. coli SM10 (ʎ pir) containing the suicide delivery system pUT (de Lorenzo et al. 1990) and the transposon miniTnphoA3, as described previously (Pattery et al. 1999; de Chial et al. 2003). Since the pUT:miniTnphoA3 plasmid is a suicide vector and is lost from P. fluorescens H41, the only way for the miniTnphoA3 to be retained is to transpose from the pUT:miniTnphoA3 plasmid into the chromosome of the P. fluorescens H41. Transconjugants were selected on CAA medium plates supplemented with the appropriate antibiotics (50 µg/ml gentamicin and 25 µg/ml chloramphenicol).
Antonie van Leeuwenhoek
Mutants were screened on agar-BHI for their lack of antimicrobial activity against S. aureus ATCC 29213 as described previously (Marinho et al. 2009). Localisation of TnphoA3 insertions Chromosomal DNA was obtained from the selected mutants using the Genome DNA KIT (MP Biomedicals, Illkirch Cedex—France) according to the manufacturer’s protocol. For each mutant, 1 mg of genomic DNA was cut using 1 unit of the restriction enzyme TaqI (Biolabs Inc., Ipswich, USA). The reaction was performed at 65 ° C for 1 h. After inactivation of the restriction enzyme at 80 °C for 20 min, 100 ng of the digested DNA was ligated overnight with 200 units of T4 DNA ligase at 16 °C in 200 ml of ligation buffer (50 mM Tris–HCl, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 25 mg/ml BSA, pH 7.5). These conditions favour auto-ligation and circularization of the fragments. Then DNA was precipitated by adding 20 ml of 3 M sodium acetate (pH 5.2) and 500 ml of ethanol 100 %. This was incubated at –20 °C for 1 h, and after centrifugation, the DNA was obtained as a pellet. Pelleted DNA was washed with 70 % ethanol and, after further centrifugation, dried at 37 °C. This dried DNA was used for inverse-PCR of the circularized fragments generated from the chromosomal DNA obtained from each mutant. The site of transposon insertion was determined using inverse PCR with primers PhoA5 (5′-GCGG CAGTCTGATCACCCGTTA-3′), Gm1 (5′-TGGAC CAGTTGCGTGAGCGCATA-3′), PhoA4 (5′-GCAC CGCCGGGTGCAGTAATTAT-3′) and Gm2 (5′-TG TCAACTGGGTTCGTGCCTTC-3′) as described previously (de Chial et al. 2003). The sequence of the DNA flanking region of the transposon was determined at the VIB Genetic Service Facility (Antwerp, Belgium) using the primer pair Gm2 and PhoA4. TaqI-digested genomic bacterial DNA isolated from antagonistic activity-negative P. fluorescens mutants was used as template. PCR was performed using the PCR kit ExTakara (Takara Clontech, SaintGermain-en-Laye, France) in a total volume of 50 µl containing: 1 9 ExTakara buffer, 2.5 mM of each deoxyribonucleotide triphosphate, 1 U of ExTakara DNA polymerase, 20 pmol of each primer and 100 ng DNA. The following PCR conditions were used: initial denaturation step at 94 °C for 50 s, followed by 10 cycles at 94 °C for 10 s, 55 °C for 30 s and 68 °C for 2 min, then 20 cycles at 94 °C for 10 s, 55 °C for
30 s and 68 °C for 2 min, with a final elongation step of 72 °C for 10 min. The amplicons were purified using QIAquick PCR purification protocol (Qiagen, Venlo, The Netherlands) following manufacturer’s instructions. After purification, the amplicons were subjected for sequencing by the dideoxy termination method with the same primers as those used for the PCR reaction. In order to map the position of the inserted transposon, the obtained sequences were used as query in a BLAST search of the P. fluorescens A506 genome (www.pseudomonas.com). For a few mutants, the sequences were not present in the chromosome of P. fluorescens A506. In that case, blastn searches of the National Center for Biotechnology Information (NCBI) database were performed. For every mutant, the sequences obtained with each of the primer pairs matched in the same gene or intergenic region of the sequenced chromosome. This confirmed the reliability of the method used for mapping the insertions. Antimicrobial activity assay The assay for antimicrobial substance production was performed as described previously (Marinho et al. 2009). Briefly, 107 cells of the strains were spotted onto BHI-agar. After growth of each strain at 25 °C for 24–48 h, 105 cells of the indicator strains mixed with 3 ml of BHI soft agar were poured over the plates. Plates were incubated at 25 °C (marine strains) or 37 °C (reference and clinical strains) for 18 h and the diameter of the inhibition zone around the spotted strain was measured. An indicator strain was considered sensitive to the activity of the P. fluorescens H41 strain or mutant clone when it exhibited a clear inhibition zone with a diameter ≥8 mm. When the inhibition zone was \8 mm or when bacterial colonies grew inside the inhibition zone, the indicator strain was considered resistant. Results were converted to graphs using the Cytoscape 3.1.0 program (http://www.cytoscape.org). Phylogenetic inferences Sequences obtained for the pvdL and pvdD genes were quality inspected and edited with the Sequence Scanner software V.1 (Applied Biosystems, Foster City, USA). Sequences for both genes were obtained at the NCBI database. Then, each sequence recovered
123
Antonie van Leeuwenhoek
from NCBI was aligned with the respective sequence from this study via blast two sequences (bl2seq). The closest relatives were used for phylogenetic analysis. These sequences were aligned using Clustal X in the MEGA6 software (Tamura et al. 2013). The appropriated evolutionary model was determined using MEGA6 (Tamura et al. 2013) and found to be the general-time reversible model (GTR) (Rodriguez et al. 1990) with a discrete gamma-distribution of among-site rate variation (Γ4) and a proportion of invariant sites (I) for pvdL gene and GTR + Γ4 for pvdD gene. Maximum likelihood analysis was conducted using MEGA6 (Tamura et al. 2013).
the wild type H41 strain. The majority of the mutants showed an inhibition zone of 20 mm in diameter in at least six of the seven indicator strains, while the spectrum of activity of the mutants with reduced antimicrobial activity varied among the tested indicators. More details on these results can be found in the supplementary material (Supplementary Table 2). On the other hand, the four mutants with no antimicrobial activity against S. aureus ATCC 29213, also lost their inhibitory activity against all tested strains, including antimicrobial resistant CA-MRSA and S. hominis strains. Further, they also lost their swarming motility and pyoverdine production (greenish colour) under conditions of iron limitation (CAA medium) (Fig. 2).
Results
Determination of the point of insertion of the transposon and identification of the mutated gene
Functional screening of the transposon mutagenesis library A miniTnphoA3 mutagenesis library was constructed and 960 mutants were screened for antimicrobial activity against S. aureus ATCC 29213. Four mutants (7B5, 8B2, 6G3 and 8H6) had no antimicrobial activity, nine and 15 mutants had reduced and 100 % antimicrobial activity against S. aureus, respectively (Fig. 1). These twenty-eight mutants were selected and their spectrum of antimicrobial activity was further analysed. They were tested against other bacteria of medical relevance, which were inhibited as well by Fig. 1 Antimicrobial activity of Pseudomonas fluorescens H41, wild type and mutants, against Staphylococcus aureus ATCC 29213. Four mutants (7B5, 8B2, 6G3 and 8H6) had no antimicrobial activity (arrows) and nine mutants had reduced inhibitory activity (\100 %) compared to the wild type (20 mm = 100 %)
123
The flanking regions of the transposon insertion in the genome of each of the four mutants harbouring no antimicrobial activity were sequenced. Among the genes inactivated, one gene could be identified. Its sequence presented high similarity to the pyoverdine synthetase gene pvdL from P. fluorescens A506 (GenBank accession YP_006325133.1). Genes inactivated in mutants with reduced antimicrobial activity coded for the non-ribosomal peptide synthase PvdD, a di-/tricarboxylate transporter, a peptide ABC transporter, a putative
Antonie van Leeuwenhoek
Fig. 2 Pyoverdine production and swarming motility under conditions of iron limitation (CAA medium): a positive control with the wild type strain H41 and b negative test for a representative mutant (ΔpvdL) P. fluorescens 6G3
nucleotidyltransferase, a putative adenylate cyclase, a diguanylate cyclase (GGDEF) domain protein, an alkaline phosphatase, a quinolinate synthetase complex, a HNH endonuclease domain protein and a hypothetical protein. Even though the pvdL and pvdD genes were detected in several species of Pseudomonas, none of the sequences in the NCBI database were retrieved from the marine environment (data not shown). The phylogenetic inference of pvdL is depicted in Fig. 3a. This clearly demonstrated that the sequence obtained from P. fluorescens H41 is distinct from all closest relatives that in this case were originated from plant-associated strains. Figure 3b shows the phylogenetic analysis of the pvdD and again the vast majority of the sequences were retrieved from plant-associated strains. The closest relatives to P. fluorescens H41 sequences are two sequences recovered from plant-associated strains. The mutants 6G3 (ΔpvdL) and 1H4 (ΔpvdD) showed null and reduced antimicrobial activity, respectively. Thus, given the results obtained, it can be concluded that pvdL and pvdD, which are genes involved in the biosynthesis of pyoverdine, are related to the antimicrobial activity of P. fluorescens H41.
for their antimicrobial activity against bacteria isolated from 11 sponge species, from surrounding seawater and from sediment collected along the coast of Rio de Janeiro city, Brazil. Among the 58 strains tested as indicators, the wild type strain inhibited 39 strains (67.2 %) and the ΔpvdL mutant did not inhibit any of these indicator strains (Fig. 4). More details regarding these results can be found in the supplementary material (Supplementary Table 2). Among the strains inhibited by P. fluorescens H41, two were isolated from the same sponge species at a distinct sampling site (PV) and 35 were isolated from ten distinct sponge species collected at both sampling places (CA and PV). Moreover, one of the inhibited strains was isolated from seawater and another one from marine sediment. However, the wild type strain showed no inhibitory activity against H40, a P. fluorescens strain isolated from the same sponge specimen of Haliclona sp. collected at the sample location (CA). Interestingly, the wild type of P. fluorescens H41 inhibited 85 % of the bacterial strains isolated at the same site (CA) and inhibited only 57 % of the bacterial strains isolated from sponges at PV.
Comparative analysis of antimicrobial activity of wild type and ΔpvdL
Discussion
To further characterise the ecological role of P. fluorescens H41 in the sponge microbiota, the wild type strain and 6G3 mutant (ΔpvdL) were also tested
Marine isolates of Pseudomonas are found in diverse ecosystems, including coastal regions, deep sea and more extreme environments. Marine pseudomonads have been detected in bacterioplankton, in sediments
123
Antonie van Leeuwenhoek
Fig. 3 Phylogenetic analyses of the pvdL and pvdD genes: the Maximum Likelihood trees (a) of pvdL (-ln likelihood: −1777.2200) and (b) of pvdD (-ln likelihood: −2057.0918)
are shown. The sequences retrieved in this study are highlighted in bold. Numbers at tree nodes are bootstrap values in Maximum Likelihood and values above 75 are shown
and associated with other organisms (Engel et al. 2002). The diversity of Pseudomonas strains isolated from a wide range of marine ecosystems suggests that these organisms may produce novel and diverse bioactive substances (Isnansetyo and Kamei 2009). However, very little is known about Pseudomonas strains isolated from marine sponges and their production of bioactive compounds (Santos et al.
2010; Marinho et al. 2009; Isnansetyo and Kamei 2009). So far, the most promising antimicrobial substances isolated from sponge-associated Pseudomonas spp. are 2-undecyl-4-quinolone (BultelPonce´ et al. 1999), cyclic dipeptides (also known as diketopiperazines, DKPs) (Jayatilake et al. 1996; Zheng et al. 2005; Santos et al. 2015) and pyrone I (Singh et al. 2003).
123
Antonie van Leeuwenhoek
Fig. 4 Antimicrobial activity of the wild Pseudomonas fluorescens H41 strain (H41) and mutant (6G3) against marine bacteria. The arrows indicate that the wild type strain showed antibacterial activity and the numbers represent the inhibition zone in mm. Marine strains without connecting arrows were not inhibited and the mutant strain showed no inhibitory activity. Strains isolated from the same marine sponge,
sediment or seawater are filled with the same colour and those belonging to the same bacterial genus have the same edge colour of the circle. Strains were isolated from specimens from the Cagarras Archipelago (CA) (on the left side) and from the Praia Vermelha (PV) beach (on the right side), Rio de Janeiro, Brazil
In this context, Santos et al. (2010) characterized Pseudomonas strains with antimicrobial activities isolated from distinct sponge species. The most active strains were P. fluorescens H40 and H41 and P. aeruginosa H51 retrieved from Haliclona sp. They exhibited activity against important pathogenic bacteria such as methicillin-resistant S. aureus (MRSA and CA-MRSA), vancomycin-resistant enterococci (VRE) and multidrug-resistant Gram-negative bacilli (E. coli, Klebsiella and Pseudomonas species). Currently, these drug-resistant strains are considered a public health problem, because their resistance is spreading rapidly (WHO 2014). In order to identify genes related to the antimicrobial activity of P. fluorescens H41, transposon mutagenesis assays followed by functional screening of the library were performed. This study showed that genes involved in the biosynthesis of the siderophore pyoverdine, notably pvdL, are related to the inhibitory
activity of the sponge-associated strain P. fluorescens H41 against pathogenic and marine bacteria. The results were supported by the loss of pyoverdine production in ΔpvdL and ΔpvdD mutants. The phylogenetic analyses of the strain H41 pvdL and pvdD genes revealed that the closest relatives to these sequences were from plant-associated strains. This is most likely related to the lack of studies addressing the marine habitat. Additionally, Spencer et al. (2003) reported that the pyoverdine segment was the most hypervariable region of the genome of the P. aeruginosa strains. Likewise, Smith et al. (2005) showed that the central part of the pyoverdine region in P. aeruginosa could not be aligned among pyoverdine types. The authors also noticed that the pvdD gene was one of the most divergent genes in the pyoverdine region. Divergent pvdD sequences might occur in other species of pseudomonads and thus explain the difficult in finding sequences that
123
Antonie van Leeuwenhoek
matched the ones generated in this study. Smith et al. (2005) also suggested a history of horizontal gene transfer, which might take place within other Pseudomonas species, including the marine species from which very little is known. There has been considerable debate in the literature about the contribution of siderophores to antagonism displayed by fluorescent pseudomonads against plant pathogens (Matthijs et al. 2007) but this is the first study demonstrating that pyoverdine is also produced by sponge-associated pseudomonads. PvdL, a non-ribosomal peptide synthetase from pseudomonads, is involved in the biosynthesis of the chromophore of the siderophore pyoverdine (Mossialos et al. 2002). Many Pseudomonas strains produce siderophores as a strategy to sequester iron in the marine environment, where the iron level is extremely low (Hider and Kong 2010). This is needed to maintain important enzymatic processes (where iron is a cofactor) and can be a prerequisite for pathogenicity for many Pseudomonas strains (Mossialos et al. 2002). More recently, siderophores have been assessed as targets for the development of new anti-bacterial molecules, due to the importance of iron in bacterial metabolism and to the paucity of effective antibiotics against multidrug-resistant bacteria (Frangipani et al. 2014). Relatively little information is available on the siderophore pyoverdine in marine bacteria compared to bacteria from terrestrial environments as was revealed in the phylogenetic analyses of the pvdL and pvdD genes. Similar results were recently observed in a Pseudomonas putida strain isolated from a stream in Brussels, Belgium (Ye et al. 2014). This strain was found to produce a compound with antimicrobial activity against the opportunistic pathogens S. aureus, P. aeruginosa, and the plant pathogen Pseudomonas syringae. Intriguingly, it was demonstrated that the majority of transposon mutants, which were unable to produce the compound, had insertions in genes involved in the biosynthesis of pyoverdine, despite the fact that pyoverdine itself showed no antimicrobial activity (Ye et al. 2014). One possible explanation, mentioned by the authors, was that the proteins involved in the production of the antibiotic compound share the same ‘‘siderosome’’ platform as the pyoverdine biosynthetic machinery recently described in P. aeruginosa (Schalk and Guillon 2013).
123
In this study, the significance of pyoverdine synthesis by the sponge-associated P. fluorescens H41 in relation to activity against bacteria of medical importance is demonstrated. These results open up interesting avenues in the search for novel compounds against multi-resistant bacteria. However, the contribution of pyoverdine to the ecological success of its producer(s) is less clear. It is known that microbial interactions in environmental settings form the basis of several important ecological processes. Among such interactions, competition between species plays an important role in the structuring of microbial communities (Joshi et al. 2009). Our results showed that the inhibitory activity of P. fluorescens H41 was high against bacteria isolated from different species of marine sponges whereas no activity was observed against the strain isolated from the same specimen. Furthermore, P. fluorescens H41 showed high inhibitory activities while others spongeassociated P. fluorescens strains did not have the ability to produce the antimicrobial substance. Many bacteria produce antibiotics to gain a competitive advantage over other microorganisms inhabiting the same ecological niche (Webster and Blackall 2009). The role of bacteria–bacteria crosstalk, whether beneficial or inhibitory, remains an interesting topic for future studies. A good example is the P. fluorescens strain A506, which is a commercially available biological control agent (BlightBan A506; Nufarm Americas Inc., Sugar Land, TX) used for the suppression of fire blight on pear and apple trees (Lindow et al. 1996). In the marine environment, competition for nutrients and space drives the evolution of colonisation and growth strategies of marine microorganisms. In that context, antimicrobials and other secondary metabolites produced by microorganisms may aim to inhibit the growth of others by affecting their survival and reproduction (Burgess et al. 1999). Thus, considering the results obtained in this study, which demonstrated that pvdL is directly related to expression of antimicrobial activity in P. fluorescens H41, we suggest that bacteria producing siderophores and antibiotics favour sponge fitness. In this case, we speculate that P. fluorescens H41 strain colonises the sponge and provides antagonistic molecules (such as pyoverdine) that benefit the host as a self-defence against invasive and pathogenic bacteria. In return, the bacteria gain a protected and nutrient-rich habitat.
Antonie van Leeuwenhoek
Only a few studies have addressed the importance of biochemical properties of symbiotic microbes in relation to sponge physiology. Richardson et al. (2012), after experimental manipulation with antibiotics, showed that associated bacteria favour sponge growth and influence the composition of the bacterial community. It was also reported that ferric iron (Fe3+) plays an important role in sponge morphogenesis. Hence, the availability of iron may be a key factor in determining sponge growth (Krasko et al. 2002). This suggests the production of compounds such as siderophores by the sponge-associated communities might play important roles for the host fitness. Given these data, further studies are needed to determine the potential role of siderophores as drivers of sponge bacterial communities and of sponge fitness. Finally, our results suggest a biocontrol role of the pyoverdine produced by P. fluorescens in its host sponge. Acknowledgments This work was supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ) to M.S. Laport. We are also grateful to Science without Borders Program/CNPq for the post doctorate scholarship to M.S. Laport. Conflict of interest
The authors declare no conflict of
interest.
References Bultel-Ponce´ V, Berge J, Debitus C, Nicolas J, Guyot M (1999) Metabolites from the sponge associated bacterium Pseudomonas species. Mar Biotechnol 1:384–390 Burgess JG, Jordan EM, Bregu M, Mearns-Spragg A, Boyd KG (1999) Microbial antagonism: a neglected avenue of natural products research. J Biotechnol 70:27–32 de Chial M, Ghysels B, Beatson SA, Geoffroy V, Meyer JM, Pattery T, Baysse C, Chablain P, Parsons YN, Winstanley C, Cordwell SJ, Cornelis P (2003) Identification of type II and type III pyoverdine receptors from Pseudomonas aeruginosa. Microbiology 149:821–831 de Lorenzo V, Herrero M, Jakubzik U, Timmis KN (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria. J Bacteriol 172:6568–6572 Engel S, Jensen PR, Fenical W (2002) Chemical ecology of marine microbial defence. J Chem Ecol 28:1971–1985 Frangipani E, Bonchi C, Minandri F, Imperi F, Visca P (2014) Pyochelin potentiates the inhibitory activity of gallium on Pseudomonas aeruginosa. Antimicrob Agents Chemother 58:5572–5575
Hentschel U, Usher KM, Taylor MW (2006) Marine sponges as microbial fermenters. FEMS Microbiol Ecol 55:167– 177 Hider RC, Kong XL (2010) Chemistry and biology of siderophores. Nat Prod Rep 27:637–657 Isnansetyo A, Kamei Y (2009) Bioactive substances produced by marine isolates of Pseudomonas. J Ind Microbiol Biotechnol 36:1239–1248 Jayatilake GS, Thornton MP, Leonard AC, Grimwade JE, Baker BJ (1996) Metabolites from an Antarctic sponge associated bacterium, Pseudomonas aeruginosa. J Nat Prod 59:293–296 Joshi H, Dave R, Venugopalan VP (2009) Competition triggers plasmid-mediated enhancement of substrate utilisation in Pseudomonas putida. PLoS ONE 4:e6065 Krasko A, Schro¨der HC, Batel R, Grebenjuk VA, Steffen R, Mu¨ller IM, Mu¨ller WE (2002) Iron induces proliferation and morphogenesis in primmorphs from the marine sponge Suberites domuncula. DNA Cell Biol 21:67–80 Lindow SE, McGourty G, Elkins R (1996) Interactions of antibiotics with Pseudomonas fluorescens strain A506 in the control of fire blight and frost injury to pear. Phytopathology 86:841–848 Marinho PR, Moreira AP, Pellegrino FL, Muricy G, Bastos MC, Santos KR, Giambiagi-deMarval M, Laport MS (2009) Marine Pseudomonas putida: a potential source of antimicrobial substances against antibiotic-resistant bacteria. Mem Inst Oswaldo Cruz 104:678–682 Matthijs S, Tehrani KA, Laus G, Jackson RW, Cooper RM, Cornelis P (2007) Thioquinolobactin, a Pseudomonas siderophore with antifungal and anti-Pythium activity. Environ Microbiol 9:425–434 Mossialos D, Ochsner U, Baysse C, Chablain P, Pirnay JP, Koedam N, Budzikiewicz H, Ferna´ndez DU, Scha¨fer M, Ravel J, Cornelis P (2002) Identification of new, conserved, non-ribosomal peptide synthetases from fluorescent pseudomonads involved in the biosynthesis of the siderophore pyoverdine. Mol Microbiol 45:1673–1685 Pattery T, Hernalsteens JP, de Greve H (1999) Identification and molecular characterization of a novel Salmonella enteritidis pathogenicity islet encoding an ABC transporter. Mol Microbiol 33:791–805 Piel J (2004) Metabolites from symbiotic bacteria. Nat Prod Rep 21:519–538 Richardson C, Hill M, Marks C, Runyen-Janecky L, Hill A (2012) Experimental manipulation of sponge/bacterial symbionts community composition with antibiotics: sponge cell aggregates as a unique tool to study animal/ microorganism symbiosis. FEMS Microbiol Ecol 81:407– 418 Rodriguez F, Oliver JL, Marin A, Medina JR (1990) The general stochastic model of nucleotide substitution. J Theor Biol 142:485–501 Santos OCS, Pontes PVML, Santos JFM, Muricy G, Giambiagi-deMarval M, Laport MS (2010) Isolation, characterization and phylogeny of sponge-associated bacteria with antimicrobial activities from Brazil. Res Microbiol 161:604–612 Santos OCS, Soares AR, Machado FLS, Romanos MTV, Muricy G, Giambiagi-deMarval M, Laport MS (2015) Investigation of biotechnological potential of sponge-
123
Antonie van Leeuwenhoek associated bacteria collected in Brazilian coast. Lett Appl Microbiol 60:140–147 Santos-Gandelman JF, Santos OCS, Pontes PVM, Andrade CL, Korenblum E, Muricy G, Giambiagi-deMarval M, Laport MS (2013) Community structure and characterization of sponge-associated bacteria from Brazilian coast. Mar Biotechnol 15:668–676 Santos-Gandelman JF, Giambiagi-deMarval M, Oelemann WMR, Laport MS (2014) Biotechnological potential of sponge-associated bacteria. Curr Pharm Biotechnol 15:143–155 Schalk IJ, Guillon L (2013) Pyoverdine biosynthesis and secretion in Pseudomonas aeruginosa: implications for metal homeostasis. Environ Microbiol 15:1661–1673 Silby MW, Winstanley C, Godfrey SAC, Levy SB, Jackson RW (2011) Pseudomonas genomes: diverse and adaptable. FEMS Microbiol Rev 35:652–680 Singh MP, Kong F, Janso JE, Arias DA, Suarez PA, Bernan VS, Petersen PJ, Weiss WJ, Carter G, Greenstein M (2003) Novel alpha-pyrones produced by a marine Pseudomonas sp. F92S91: taxonomy and biological activities. J Antibiot 56:1033–1044 Skariyachan S, Rao AG, Patil MR, Saikia B, Bharadwaj KN, Rao GS (2014) Antimicrobial potential of metabolites extracted from bacterial symbionts associated with marine sponges in coastal area of Gulf of Mannar Biosphere, India. Lett Appl Microbiol 58:231–241 Smith EE, Sims EH, Spencer DH, Rajinder K, Olson MV (2005) Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa. J Bacteriol 187:2138–2147
123
Spencer DH, Kas A, Smith EE, Raymond CK, Sims EH, Hastings M, Burns JL, Kaul R, Olson M (2003) Wholegenome sequence variation among multiple isolates of Pseudomonas aeruginosa. J Bacteriol 185:1316–1325 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729 Taylor MW, Radax R, Steger D, Wagner M (2007) Spongeassociated microorganisms: evolution, ecology, and biotechnological potential. Microbiol Mol Biol Rev 71:295–347 Wang G (2006) Diversity and biotechnological potential of the sponge-associated microbial consortia. J Ind Microbiol Biotechnol 33:545–551 Webster NS, Blackall LL (2009) What do we really know about sponge-microbial symbioses? ISME J 3:1–3 World Health Organization (2014) Antimicrobial resistance: global report on surveillance (2014) WHO Library Cataloguing-in-Publication Data 1-256. WHO Press, Geneva Ye L, Hildebrand F, Dingemans J, Ballet S, Laus G, Matthijs S, Berendsen R, Cornelis P (2014) Draft genome sequence analysis of a Pseudomonas putida W15Oct28 strain with antagonistic activity to Gram-positive and Pseudomonas sp. pathogens. PLoS ONE 9:e110038 Zheng L, Yan X, Xu J, Chen H, Lin W (2005) Hymeniacidon perleve associated bioactive bacterium Pseudomonas sp. NJ6-3-1. Prikl Biokhim Mikrobiol 41:35–39
