- Research article
- Open Access
Genome analysis of Pseudoalteromonas flavipulchra JG1 reveals various survival advantages in marine environment
© Yu et al.; licensee BioMed Central Ltd. 2013
- Received: 1 July 2013
- Accepted: 14 October 2013
- Published: 16 October 2013
Competition between bacteria for habitat and resources is very common in the natural environment and is considered to be a selective force for survival. Many strains of the genus Pseudoalteromonas were confirmed to produce bioactive compounds that provide those advantages over their competitors. In our previous study, P. flavipulchra JG1 was found to synthesize a Pseudoalteromonas flavipulchra antibacterial Protein (PfaP) with L-amino acid oxidase activity and five small chemical compounds, which were the main competitive agents of the strain. In addition, the genome of this bacterium has been previously sequenced as Whole Genome Shotgun project (PMID: 22740664). In this study, more extensive genomic analysis was performed to identify specific genes or gene clusters which related to its competitive feature, and further experiments were carried out to confirm the physiological roles of these genes when competing with other microorganisms in marine environment.
The antibacterial protein PfaP may also participate in the biosynthesis of 6-bromoindolyl-3-acetic acid, indicating a synergistic effect between the antibacterial macromolecule and small molecules. Chitinases and quorum quenching enzymes present in P. flavipulchra, which coincide with great chitinase and acyl homoserine lactones degrading activities of strain JG1, suggest other potential mechanisms contribute to antibacterial/antifungal activities. Moreover, movability and rapid response mechanisms to phosphorus starvation and other stresses, such as antibiotic, oxidative and heavy metal stress, enable JG1 to adapt to deleterious, fluctuating and oligotrophic marine environments.
The genome of P. flavipulchra JG1 exhibits significant genetic advantages against other microorganisms, encoding antimicrobial agents as well as abilities to adapt to various adverse environments. Genes involved in synthesis of various antimicrobial substances enriches the antagonistic mechanisms of P. flavipulchra JG1 and affords several admissible biocontrol procedures in aquaculture. Furthermore, JG1 also evolves a range of mechanisms adapting the adverse marine environment or multidrug rearing conditions. The analysis of the genome of P. flavipulchra JG1 provides a better understanding of its competitive properties and also an extensive application prospect.
- Pseudoalteromonas flavipulchra
- Genome analysis
- Antibacterial metabolites
- Quorum quenching
- Survival advantages
The genus Pseudoalteromonas was established in 1995 and was found to be ubiquitous in the marine environment. Numerous Pseudoalteromonas strains were isolated from the polar region, inshore waters or surfaces of marine organism, and were shown to synthesize a range of bioactive molecules[2–4]. The production of molecules that are active against a variety of target organisms appears to be an important characteristic for this genus and may greatly benefit Pseudoalteromonas cells in their competition for nutrients or colonization of surfaces. P. tunicata D2, as a model organism to study, was demonstrated to synthesize a range of inhibitory substances, including a toxic antibiotic protein and two pigments[6, 7]. Analysis of its complete genome sequence revealed that several genes and gene clusters were involved in the production of inhibitory compounds that were associated with its successful persistence and competition on marine surfaces.
P. flavipulchra JG1 was isolated from rearing water of healthy turbot (Scophthalmus maximus) in Qingdao, China. Strain JG1 is capable of adapting to the oligotrophic marine environment and reveals various advantageous survival abilities among competitive species. It was demonstrated to exhibit inhibitory activity against many Vibrio, Aeromonas and Bacillus strains and was nontoxic to zebra fish and mantis shrimp. Furthermore, Strain JG1 was shown to synthesize the putative L-amino acid oxidase named Pseudoalteromonas flavipulchra antibacterial Protein (PfaP) and 5 small molecular compounds with antibacterial activity. These compounds were identified as p-hydroxybenzoic acid, trans-cinnamic acid, 6-bromoindolyl-3-acetic acid, N-hydroxybenzoisoxazolone and 2′-deoxyadenosine. All of these metabolites have been observed to exhibit antibacterial activities against several pathogens, including V. anguillarum, V. harveyi, Photobacterium damselae subsp. damselae and A. hydrophila. The inhibitory properties of P. flavipulchra JG1 against fish pathogens indicate that the strain or its products could be utilized as biocontrol agent(s) in aquaculture.
To obtain a better understanding of the genetic potential of P. flavipulchra JG1 as a biocontrol organism, we have sequenced and analyzed its genome and compared it to the genomic data of closely related strains publicly available. We have found that the P. flavipulchra genome contains several genes and gene clusters that might be involved in the production of inhibitory compounds against pathogens and competitors in the marine environment. The analysis of P. flavipulchra genome also verifies excellent capabilities of this strain to adapt to environmental changes and challenges.
Genome features and comparison with other Pseudoalteromonas genomes
General features of JG1 and other Pseudoalteromonas genomes
P. flavipulchra JG1
P. tunicata D2
P. atlantica T6c
P. haloplanktis TAC125
Pseudoalteromonas sp. TW-7
Pseudoalteromonas sp. SM9913
5 505 361
4 982 425
5 187 005
3 850 272
4 104 952
4 037 671
4 828 917
4 421 622
4 507 935
3 404 858
3 673 686
3 569 827
G + C percentage
CDS assigned to COG
COG Cluster number
Significantly, P. flavipulchra contains the highest proportion of specific genes belonging to the COG categories defense mechanisms (V) and secondary metabolites biosynthesis, transport and catabolism (Q), which account for 4.56% and 5.40%, respectively, among other bacteria. (Additional file1: Figure S1). This mainly attributes to more genes involved in ABC-type antimicrobial peptide transport system (COG0577, COG1136), beta-lactamase class C and other penicillin binding proteins (COG1680), cation/multidrug efflux pump (COG0841), ABC-type siderophore export system (COG 4615) and those related to the synthesis of potential bioactive compounds, such as nonribosomal peptide synthetase (NRPS) modules (COG1020) and polyketide synthases (COG 3321). The abundance of genes involved in expression and transport of potential primary and secondary metabolites and defense compounds are consistent with the capability of P. flavipulchra to produce various antimicrobial compounds and generate survival advantages in marine environments.
Biosynthesis of antimicrobial metabolites
P. flavipulchra JG1 was demonstrated to synthesize the antibacterial protein PfaP and 5 known small molecular compounds with antibacterial activity against V. anguillarum.
The compounds p-hydroxybenzoic acid, trans-cinnamic acid, and 6-bromoindolyl-3-acetic acid isolated from P. flavipulchra JG1 were demonstrated to inhibit the growth of several pathogens. We now identified several genes most likely involved in the biosynthesis of these metabolites.
Compound p-hydroxybenzoic acid and its derivatives are widely applied as food preservatives and stabilizers (antioxidants). As an organic acid, it has also been shown to inhibit the growth of ethanologenic Escherichia coli LY01 and ethanol synthesis. The compound is probably derived from chorismate by action of a chorismate lyase, resembling the first step of ubiquinone biosynthesis. p-hydroxybenzoic acid is an important precursor of ubiquinones and P. flavipulchra harbors the ubiCA gene cluster (FaGL1386 and FaGL1387) responsible for the conversion of chorismate to p-hydroxybenzoic and then to 3-octaprenyl-4-hydroxybenzoate, which are the first two steps of the biosynthesis of ubiquinone. Ubiquinone is not only an essential component of the aerobic respiratory chain, but functions in the reduced form (ubiquinol) as an antioxidant, significantly reducing oxidative stress, as for example generated by hydrogen peroxide. We also identified all other genes in JG1 that are necessary for ubiquinone production.
For the biosynthesis of trans-cinnamic acid, two biosynthetic pathways can be taken into consideration: it can be derived from tyrosine by tyrosine ammonia lyase (TAL) directly, or cinnamic acid could get hydroxylated after its formation by phenylalanine ammonia lyase (PAL). Lyase-catalyzed reductive deamination releases ammonia thus forming the trans double bond found in the compound. This reaction sequence has been confirmed in Streptomyces maritimus and Rhodobacter capsulatus. However, in the genomic data of P. flavipulchra JG1 only two homologs of TAL (FaGL3502 and FaGL4409) can be identified. Therefore, this compound is most probably derived from tyrosine in JG1. trans-cinnamic acid is often found in plants. It has been used to augment the activity of various antibiotics against Mycobacterium avium and exhibited synergistic effects with several anti-tuberculosis drugs active against M. tuberculosis. In contrast to its wide occurrence in plants, trans-cinnamic acid is not very common in bacteria and only few reports on the biochemical characterization of TAL have been published.
To assess the potential of P. flavipulchra JG1 to produce secondary metabolites that have not yet been isolated and chemically characterized, we analyzed its genome using antiSMASH. This allowed for the identification of four different bacteriocin-type gene clusters, lantipeptide biosynthesis genes, four type I polyketide/non-ribosomal peptide (PKS/NRPS) hybrid clusters, three NRPS gene clusters (Additional file2: Table S1), as well as a high number of further NRPS-related peptidyl-carrier proteins (12), condensation (11), adenylation (12), epimerization (3), and thioesterase (2) domains distributed in the smaller scaffolds derived from our sequencing efforts. This strain thus appears to harbor tremendous potential to produce a large variety of peptide-based secondary metabolites. It is interesting to note that in terms of polyketide biosynthesis only type I PKS genes have been identified, all of these being part of larger NRPS biosynthetic loci. Among the biosynthetic gene clusters predicted by antiSMASH analysis, we identified a set of genes that can be correlated to polycyclic tetramic acid containing macrolactams (PTMs). This class of secondary metabolite gene clusters was recently found to be widespread among bacterial species and encodes the assembly of structurally complex, polycyclic secondary metabolites with a broad range of biological activities. Given the apparent genetic potential of P. flavipulchra JG1 to produce complex bioactive natural products, further studies to chemically unlock its secondary metabolite profile seem to be extremely promising.
Besides these secondary metabolites potential, P. flavipulchra JG1 was shown great chitinase activity on chitin agar. Chitinase can catalyze the hydrolysis of β-1, 4 glycosidic bonds linking the N-acetylglucosamine subunits of chitin in a variety of organisms, and the enzyme has been found in all sorts of organisms including bacteria, fungi, plants and animals[28, 29]. Moreover, chitinase can be classified into two families as glycoside hydrolases (GH) 18 and 19 based on the amino acid sequence similarity[30, 31]. P. flavipulchra JG1 harbored 5 genes (faGL1724, faGL2271, faGL2273, faGL2526 and faGL4793) coding for this enzyme, four of which belong to GH18. In the genomes of P. tunicata D2 and Pseudoalteromonas strains SM9913, by comparison, only two genes are annotated as chitinases, while no genes encoding this enzyme are found in P. haloplanktis TAC125 and P. atlantica T6c. Notably, chitinase has been confirmed to show antifungal activities in B. cereus and Streptomyces sp. MG3 as chitin is a component of the cell walls of fungi. Two extracellular chitinases purified from Pseudomonas aeruginosa K-187 also showed lysozyme activities against both gram-positive and gram-negative bacteria. The chitinases identified in JG1 can thus be considered as antibacterial/antifungal proteins with a different antagonizing mechanism.
Quorum sensing and quorum quenching
Quorum sensing is a process that has been developed by bacteria to allow communication with each other and to facilitate monitoring of their cell density by measuring the concentration of small secreted signal molecules, such as acyl homoserine lactone (AHL). Diverse functions of bacteria are regulated via quorum sensing systems, including antibiotic biosynthesis, motility patterns and expression of many virulence-related genes in some animal and plant pathogens. The quorum sensing regulatory system QseB/QseC detected in the genome of JG1 is organized in an operon with qseB encoding the response regulator and qseC the sensor histidine kinase. QseB/QseC belongs to two-component signal transduction systems which enable bacteria to sense, respond, and adapt to changes in their environment or in their intracellular state. However, the major genes encoding autoinducers responsible for quorum sensing have not been detected in JG1. The system may thus only regulate genes encoding the expression and assembly of flagella, motility and chemotaxis in JG1, as a protein responsible for biogenesis of type IV pilus is just downstream to the QseB/QseC system.
Quorum quenching (QQ) describes all processes that interfere with quorum sensing. QQ enzymes, including AHL lactonase, acylase, oxidase and reductase, have been discovered in many bacterial genuses, such as Bacillus, Rhodococcus, Pseudomonas and Ralstonia. Extra-and intra-cellular products of strain JG1 were shown to degrade long-chain AHLs, such as C10-HSL, C12-HSL, C14-HSL, 3-oxo-C12-HSL and 3-oxo-C14-HSL, but not short-chain AHLs (shorter than 10 carbons in the acyl chain), and the enzymatic activity could not be reversed by acidification. This suggested that there might be genes encoding AHL acylases in the genome of strain JG1. Two genes of JG1 (FaGL1422 and FaGL2554) encoding penicillin acylase show high homology with PvdQ and QuiP, which in Pseudomonas aeruginosa PAO1 were demonstrated to degrade long chain AHLs. Moreover, QuiP could utilize long chain AHLs as sole sources of carbon and energy. The catalytically active serine residue of N-terminal nucleophilic hydrolases, as found in the functionally verified AHL acylases PvdQ and QuiP, is present in both homologs found in JG1. These potential AHL acylases therefore equip strain JG1 with another mechanism to reduce the detrimental effects of pathogens and prevent or limit the impact of bacterial diseases in rearing animals.
Acquisition of phosphorus
Efficient uptake of phosphorus is important for marine microorganisms due to the low phosphorus level in the marine environment. The phosphate input in the metabolism of P. flavipulchra JG1 is controlled by the counterparts of PhoB/PhoR and PhoU as well as several phosphate transport systems (PstABC and PstS) and inorganic phosphate (Pi) transport systems (Pit). The Pst system, which is derepressed under conditions of Pi starvation, also regulates synthesis of alkaline phosphatase, a periplasmic protein produced in greatest quantity during Pi starvation. Homologs to the two component regulatory system PhoR/PhoB found in JG1 are responsible to control phosphate starvation and the system may also serve as a general transduction system for the expression of genes involved in secondary metabolism. Significantly, Pi starvation may stimulate bacteria to produce various secondary metabolites. For example, antibiotics biosynthesis in Streptomyces lividans is negatively regulated by phosphate via the PhoR/PhoB system. JG1 might therefore up-regulate the production of defensive primary and secondary metabolites in the oligotrophic marine environment, thereby even more efficiently combating competing (micro) organisms.
General stress response
As the natural habitats of bacteria are constantly subjected to deleterious and fluctuating conditions that can be harmful, bacteria all have evolved their abilities to sense and respond to these environmental changes. RpoS, RelA, universal stress protein A, starvation stringent protein (SspB) and phage shock proteins (PspA-E) that are involved in controlling carbon and nutrient starvation are present in P. flavipulchra JG1. Furthermore, the genome of JG1 also encodes for a large number of proteins involved in oxidative stress and metal homeostasis.
Besides three antioxidative proteins of the AhpC/Tsa family, including catalase, superoxide dismutase and an alkyl hydroperoxide reductase, JG1 has two proteins of the AhpF/TR family identified as AhpF and thioredoxin reductase. These antioxidant enzymes protect JG1 against peroxide derived DNA damage as well as oxidative membrane or lipids destruction. Key regulators of the oxidative stress response are also present (such as SoxR). Unlike P. tunicata D2 and P. haloplanktis TAC125, the ubiquitous molybdopterin metabolism might be present, since dinucleotide-utilizing enzymes involved in molybdopterin biosynthesis can be found in JG1. However, genes coding for enzymes using cofactors, such as xanthine oxidase, biotin sulfoxide reductase, have not been detected. Five putative dioxygenases (FaGL2063, FaGL3237, FaGL2992, FaGL1118 and FaGL4206) might also help JG1 to protect its metabolism against oxidative stress.
Several genes involved in heavy metal detoxification were discovered in the genome of JG1, including periplasmic divalent cation tolerance protein (CutA) and copper homeostasis protein (CutC). Experiments proved that intracellular copper accumulation in E. coli could increase without cutA. Moreover, cutA affects not only copper tolerance but also tolerance levels to zinc, nickel, cobalt and cadmium.
Motility and secretion
P. flavipulchra JG1 possesses three gene clusters for the biosynthesis of type IV pili including PilM/N/O/P/Q and PilF which interact to allow pilus assembly to occur, and also PilA, which is the most important component of type IV pili. Pili mediate attachment to both living and artificial surfaces and are involved in bacteriophage adsorption, DNA uptake, biofilm initiation and development, and twitching motility. One of the pili gene clusters and its upstream region is highly conserved in P. tunicata, P. haloplanktis and Pseudoalteromonas sp. TW-7 and SM9913 contains homologs to the two component response regulator system alg Z/alg R, which is involved in the regulation of alginate synthesis and pili-mediated, twitching motility in Pseudomonas aeruginosa. The alginate biosynthesis cluster is absent in JG1 as in P. tunicata, therefore, this regulatory system may only play a role in the expression of the pili cluster. With these appendages involved in motility, JG1 might be able to rapidly respond to environmental stresses and exceed other microorganisms.
There are several secretion systems functional in JG1, such as type I, II and VI secretion system, as well as the TAT and Sec-SRP export systems. In contrast, type III secretion system is absent. There are 15 genes involved in the type VI secretion system (T6SS). Previous studies indicated that many of these T6SS-containing bacteria are known pathogens and T6SS have been experimentally shown to play a role in virulence in several cases. However, recent studies suggest that T6SS may limit bacterial replication or virulence and instead be used for intraspecies microbial cooperation, such as mediating antagonistic interactions between bacteria. T6SS may be one of antibacterial mechanisms of JG1 that allows the bacterium inhibiting other microorganisms and affecting bacterial-host interactions.
Drugs resistance and transport
Microorganisms have developed various ways to resist the toxic effects of antibiotics and other drugs[59, 60]. JG1 was shown excellent resistant activities to several common antibiotics such as penicillin, kanamycin, cephalosporin, tetracycline and chloramphenicol. One of the mechanisms may be enzymes that inactivate antibiotics by hydrolysis or the formation of inactive derivatives. Penicillin metabolism in JG1 could be catalyzed by penicillin amidase yielding 6-aminopenicillanic acid, and by beta-lactamase (penicillinase) to give penicilloic acid. The penicillin amidase can also be found in TAC125, but not in other compared Pseudoalteromonas strains. JG1 also possesses D-amino-acid oxidase to convert cephalosporin C into (7R)-7-(5-carboxy-5-oxopentanoyl)-aminocephalosporinate, consequently disarming this bactericide.
A second mechanism of antibiotic resistance is the inhibition of drug entry into the cell. The low permeability of the outer membrane of gram-negative bacteria could reduce drug diffusion across the cell envelope. However, these barriers cannot entirely prevent all drugs from exerting their toxic action once they have entered the cell. The active efflux of drugs is thus essential to ensure the survival of the cell[63, 64]. JG1 possesses many genes involved in defense mechanisms due to ABC-type antimicrobial peptide transport system (34 genes), ABC-type multidrug transport system (16 genes), cation/multidrug efflux pump (12 genes) and Na+-driven multidrug efflux pump (7 genes). The ABC-type transport systems have genes encoding ATPase and permease components. These multidrug transporters recognize lipophilic drugs by their physic-chemical properties that allow them to intercalate into the lipid bilayer, and transport these agents from the lipid bilayer to the exterior. The five small molecular compounds with antibacterial activity produced by JG1 were all lipophilic substances and multidrug transporters could reduce the intracellular accumulation of these compounds and protect the bacterium from the toxic effect of these bacteriostats. The transporters mediate the excretion of specific antibiotics in Streptomyces strains also dedicated to ensure self-resistance to the antibiotics that they produce.
The genome of P. flavipulchra JG1 unveils significant genetic advantages against other microorganisms, encoding antimicrobial agents as well as abilities to adapt to various adverse environments. The antibacterial protein PfaP not only catalytically produces hydrogen peroxide as a bacteriostat but likely also participates in the biosynthesis of small molecular antibacterial compound (6-bromoindolyl-3-acetic acid). Both the macromolecule and small molecules contribute to the antibacterial activities of JG1. Besides these already identified chemical structures produced by strain JG1, a large number of peptide-based secondary metabolites encoded in the genome still awaits discovery. The identification of various antimicrobial enzymes enriches the antagonistic mechanisms of P. flavipulchra JG1 and could serve as therapeutic strategies against aquaculture pathogens. Furthermore, JG1 also evolves a range of mechanisms adapting the adverse marine environment or multidrug rearing conditions. The analysis of the genome of P. flavipulchra JG1 presented here provides a better understanding of its competitive properties and also an extensive application prospect.
Bacterial growth and DNA extraction
P. flavipulchra JG1 was isolated from rearing water of healthy turbot (Scophthalmus maximus) in Qingdao, China and was routinely grown on marine agar 2216 (MA; Difco) at 28°C. Genomic DNA was extracted from 5 ml overnight culture by standard methods. Genomic DNA was quantified on 1% agarose gel stained with ethidium bromide and assessed spectrophotometrically.
The biosensors Chromobacterium violaceum CV026 and VIR24 were used to detect the short-chain (C4-C8) and long-chain (C8-C14) acyl homoserine lactones (AHLs), respectively. Both of them were grown on Luria–Bertani (LB) agar at 28°C.
Genome sequencing, annotation and analysis
The genome sequence of P. flavipulchra JG1 was determined using the Illumina HiSeq2000 with a 500 bp paired-end library and achieved about 600 Mb data with 111.9-fold coverage. The reads were assembled using SOAPdenovo assembler software subsequently. A total of 122 contigs ranging from 128 bp to 264 447 bp (the N50 and N90 contig sizes were 107 608 bp and 34 132 bp, respectively) were obtained and combined into 61 scaffolds ranging from 500 bp to 879 239 bp (the N50 and N90 contig sizes were 338 061 bp and 74 731 bp, respectively). Putative protein-encoding genes were identified with GLIMMER, transposons were predicted with Repeat Masker and Repeat Protein Masker, and tandem repeat sequences were identified through Tandem Repeat Finder. Annotation was performed with BLASTALL 2.2.21 searching against protein databases KEGG (Kyoto encyclopedia of genes and genomes;http://www.genome.jp/kegg/), COG (http://www.ncbi.nlm.nih.gov/COG/), SwissProt and TrEMBL (http://www.uniprot.org/) and NR (NCBI non-redundant database;http://www.ncbi.nlm.nih.gov/RefSeq/). The criteria used to assign function to a CDS were a minimum cutoff of 30% identity and at least four best hits among the COG, KEGG, NR, SwissProt or TrEMBL protein databases. Phylogenetic analysis was performed by alignment of sequences using Clustal W and neighbor-joining trees were generated with 1 000 bootstraps. The prediction of signal peptides (SP) was performed using SignalP v 4.1. The conserved domains were predicted using Conserved Domain Database of NCBI.
Nucleotide sequence accession numbers
This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AJMP00000000. The version described in this paper is the first version, AJMP01000000.
According to the result of phylogenetic analysis, the genome sequences of P. tunicata D2 and P. haloplanktis TAC125 were retrieved from NCBI. Proteins from P. flavipulchra JG1 were compared with those of D2 and TAC125, using BLASTP with an E-value cutoff of 1e-5. Orthologous proteins are defined as reciprocal best hit proteins with a minimum 40% identity and 70% of the length of the query protein, calculated by the BLAST algorithm. Proteins without orthologs are considered to be specific proteins. The COG function category was analyzed by searching all predicted proteins against the COG database on the basis of the BLASTP.
Catalase effect on the antibacterial activity
An 8 mm disc loaded with 0.1 mg, 0.2 mg and 0.5 mg of catalase in 10 μl distilled water was used and placed neighboring to the circular wells loaded with 20 μl extracellular proteins of P. flavipulchra JG1, respectively. The catalase inhibition of antibacterial effect was observed by the eclipse of inhibition areas.
AHLs degradation bioassay
C6 to C14-HSL and 3-oxo-C6 to 3-oxo-C14-HSL were used for evaluating the AHL degradation activity of P. flavipulchra JG1. Briefly, extra- and intra-cellular products of strain JG1 were mixed with different acyl chains of AHLs, the final concentrations of these AHLs were 1 μM for C10-HSL, C12-HSL and C14-HSL, and 0.1 μM for the rest AHLs. The mixtures were incubated at 28°C for 24 hours and the residual AHLs were detected by Chromobacterium violaceum CV026 and VIR24 plate assay.
Electron microscopy and motility assay
An overnight culture of strain JG1 was negatively stained with 1% phosphotungstic acid (pH 7.4) on a Formvar carbon-coated grid and observed with a transmission electron microscope (TEM-1200EX, Japan). Swimming and swarming motilities were evaluated by point inoculating JG1 on MA plates containing 0.3% and 0.5% agar, respectively. The plates were analyzed after incubation at 28°C for about 24 h. The experiment was performed in triplicate.
Chitinase activity and antibiotic resistance
The chitinase activity of P. flavipulchra JG1 were observed using chitin agar following the method described by Hsu et al.. Resistance to antibiotics of strain JG1 was investigated by the agar diffusion method using the filter discs containing different antibiotics. Briefly, 100 μl aliquots of overnight broth culture of JG1 were spread onto the surface of MA plates, and different antibiotic discs were placed on the target plates, respectively, which were then incubated at 28°C for 24 h. Inhibition zones of the antibiotics-containing discs were observed.
We thank Dr. Robert J.C. McLean (Texas State University, USA) for biosensors C. violaceum CV026 and Dr. Tomohiro Morohoshi (Utusnomiya University, Japan) for C. violaceum VIR24. This work was supported by grants from the International Science and Technology Cooperation Programme of China (No. 2012DFG31990) and the National Natural Science Foundation of China (No. 40876067). Funding of the work in the laboratory of TAMG was provided by the DFG (GU 1233/2-1).
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