BACKGROUND
the genus pseudoalteromonas was established in  <dig> and was found to be ubiquitous in the marine environment
<cit> .
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
<cit> .
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
<cit> .
p. tunicata d <dig>  as a model organism to study, was demonstrated to synthesize a range of inhibitory substances, including a toxic antibiotic protein and two pigments
<cit> .
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
<cit> .
p. flavipulchra jg <dig> was isolated from rearing water of healthy turbot  in qingdao, china.
strain jg <dig> 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
<cit> .
furthermore, strain jg <dig> was shown to synthesize the putative l-amino acid oxidase named pseudoalteromonas flavipulchra antibacterial protein  and  <dig> 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 <cit> .
the inhibitory properties of p. flavipulchra jg <dig> against fish pathogens indicate that the strain or its products could be utilized as biocontrol agent in aquaculture.
to obtain a better understanding of the genetic potential of p. flavipulchra jg <dig> 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.
RESULTS
genome features and comparison with other pseudoalteromonas genomes
the p. flavipulchra jg <dig> genome is composed of  <dig>  <dig>  <dig> bp and the calculated g + c content is  <dig> %.
a total of  <dig>  <dig> open reading frames  are identified within the p. flavipulchra jg <dig> genome .
among the predicted genes,  <dig>  <dig>  are not found in cog categories, and  <dig>  <dig> ,  <dig> ,  <dig>  <dig>  and  <dig>  genes are not applicable within the kegg, nr, swissprot and trembl databases, respectively
<cit> .
jg <dig> contains  <dig> tandem repeat regions,  <dig> transposons, and  <dig> iss  that account for about  <dig> % of the genome.
the p. flavipulchra jg <dig> genome is larger than that of the five other pseudoalteromonas strains whose genomic sequences have been published in the img database: p. tunicata d <dig> , p. haloplanktis tac <dig> , p. atlantica t6c  and two pseudoalteromonas strains tw- <dig>  and sm <dig> .
d <dig> is an anti-biofouling bacterium, both t6c and tw- <dig> are mesophile bacteria, and tac <dig> and sm <dig> are psychrotolerant adapting to cold aqueous and deep-sea sediment environment, respectively.
general features of these genomes were given in table
<dig> and their phylogenetic relationship based on the 16s rrna gene  indicated that p. flavipulchra jg <dig> formed a cluster with p. tunicata d <dig>  comparing with other sequenced pseudoalteromonas strains, p. tunicata d2
<cit>  also exhibited great inhibitory activities against several specific organisms as in p. flavipulchra jg1
<cit> , indicating that p. flavipulchra was functionally closely related to p. tunicata d <dig>
p. flavipulchra jg <dig>  p. tunicata d <dig> and p. haloplanktis tac <dig> share  <dig> orthologous genes, accounting for  <dig> %,  <dig> % and  <dig> % of all genes of jg <dig>  d <dig> and tac <dig>  respectively.
in addition, jg <dig> contains more specific genes  than d <dig>  and tac <dig> , and there are more common genes within jg <dig> and d <dig> .
we have also conducted a cog-based analysis among the specific and orthologous genes of these three strains.
as shown in figure
2b, a larger proportion of jg <dig> and d <dig> specific genes belong to signal transduction mechanisms , while the genome of tac <dig> harbors more specific genes belonging to lipid transport and metabolism .
genes involved in signal transduction were considered to allow p. tunicata d <dig> to generate phenotypic variation and provide the capacity of niche adaptation
<cit> , and genes assigned to the same cog category might also play a similar role in p. flavipulchra jg <dig> in that both d <dig> and jg <dig> exhibited competitive activities in their surrounding environments.
contrasting, in order to adapt cold condition, tac <dig> needs more genes involved in lipid transport and metabolism such as genes coding for lipid desaturase, which could increase membrane fluidity at low temperature
<cit> .
according to the genomic comparison results, jg <dig> and d <dig> share more functional similarities than to tac <dig>
significantly, p. flavipulchra contains the highest proportion of specific genes belonging to the cog categories defense mechanisms  and secondary metabolites biosynthesis, transport and catabolism , which account for  <dig> % and  <dig> %, respectively, among other bacteria.
.
this mainly attributes to more genes involved in abc-type antimicrobial peptide transport system , beta-lactamase class c and other penicillin binding proteins , cation/multidrug efflux pump , abc-type siderophore export system  and those related to the synthesis of potential bioactive compounds, such as nonribosomal peptide synthetase  modules  and polyketide synthases .
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
<cit>  and generate survival advantages in marine environments
<cit> .
biosynthesis of antimicrobial metabolites
p. flavipulchra jg <dig> was demonstrated to synthesize the antibacterial protein pfap and  <dig> known small molecular compounds with antibacterial activity against v. anguillarum <cit> .
the antibacterial protein pfap is a homologue of l-amino acid oxidase with high similarity to the l-lysine oxidase alpp of p. tunicata d <dig>
<cit>  and marinocine antimicrobial protein of marinomonas mediterranea mmb- <dig>
<cit> .
the sequence identities of protein pfap and these two antimicrobial proteins were 58% and 54%, with coverages of 95% and 92%, respectively.
pfap is a secreted protein that might catalyze the oxidative deamination of specific amino acids to the respective α-keto acids with concomitant release of ammonium and hydrogen peroxide
<cit> .
the antibacterial activity of extracellular proteins of jg <dig> could be abolished in the presence of catalase, suggesting that the inhibitory effect was directly mediated by the action of hydrogen peroxide .
the gene located downstream of pfap  codes a catalase  which responds to oxidative stress and causes decomposition of hydrogen peroxide.
the catalase activity might thus immediately protect jg <dig> from the damage of the hydrogen peroxide generated by pfap.
we also identified  <dig> further genes encoding catalases and several genes capable of hydrogen peroxide degradation in the genome.
thus, jg <dig> produces the antimicrobial protein pfap and simultaneously possesses several genes related to potential self-protection mechanisms.
the compounds p-hydroxybenzoic acid, trans-cinnamic acid, and 6-bromoindolyl-3-acetic acid isolated from p. flavipulchra jg <dig> were demonstrated to inhibit the growth of several pathogens
<cit> .
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 .
as an organic acid, it has also been shown to inhibit the growth of ethanologenic escherichia coli ly <dig> and ethanol synthesis
<cit> .
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  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
<cit> .
ubiquinone is not only an essential component of the aerobic respiratory chain, but functions in the reduced form  as an antioxidant, significantly reducing oxidative stress, as for example generated by hydrogen peroxide
<cit> .
we also identified all other genes in jg <dig> 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  directly
<cit> , or cinnamic acid could get hydroxylated after its formation by phenylalanine ammonia lyase
<cit> .
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 <cit>  and rhodobacter capsulatus <cit> .
however, in the genomic data of p. flavipulchra jg <dig> only two homologs of tal  can be identified.
therefore, this compound is most probably derived from tyrosine in jg <dig>  trans-cinnamic acid is often found in plants.
it has been used to augment the activity of various antibiotics against mycobacterium avium <cit>  and exhibited synergistic effects with several anti-tuberculosis drugs active against m. tuberculosis <cit> .
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
<cit> .
compound 6-bromoindolyl-3-acetic acid was shown to exhibit the strongest antibacterial activity among the small molecular compounds so far isolated from p. flavipulchra jg <dig>  the metabolite can be formed by halogenation of tryptophan followed by oxidative deamination and decarboxylation.
the indole-3-acetic acid  portion can be formed in bacteria following several iaa biosynthesis pathways, of which a single bacterial strain sometimes contains more than one
<cit> .
iaa can be converted from indole-3-acetamide and indole-3-acetaldehyde via the indole-3-pyruvic acid pathway or the tryptamine pathway in jg <dig>  the key enzymes involved in both of these pathways are present in the genome of jg <dig>  including amino acid monooxygenase, indoleacetamide hydrolase, indole-3-pyruvate decarboxylase and monoamine oxidase.
however, the tryptophan aminotransferase which transforms tryptophan into indole-3-pyruvate has not been detected in jg <dig>  alternatively, the reaction could be mediated by an l-amino acid oxidase and the pfap protein might be a candidate.
in addition, genes encoding tryptophan halogenases are found in the genome of jg <dig>  one of which  is located just downstream of pfap.
a tryptophan halogenase would catalyze bromination of tryptophan, which subsequently would be converted into 6-bromoindolyl-3-acetic acid via one of the above mentioned biosynthetic pathways .
the pfap protein, which is considered as a multifunctional protein, might be the most important antibacterial factor of p. flavipulchra thus far identified, as it also might play a role in the biosynthesis of 6-bromoindolyl-3-acetic acid.
to assess the potential of p. flavipulchra jg <dig> to produce secondary metabolites that have not yet been isolated and chemically characterized, we analyzed its genome using antismash
<cit> .
this allowed for the identification of four different bacteriocin-type gene clusters, lantipeptide biosynthesis genes, four type i polyketide/non-ribosomal peptide  hybrid clusters, three nrps gene clusters , as well as a high number of further nrps-related peptidyl-carrier proteins , condensation , adenylation , epimerization , and thioesterase  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 .
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
<cit> .
given the apparent genetic potential of p. flavipulchra jg <dig> 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 jg <dig> was shown great chitinase activity on chitin agar.
chitinase can catalyze the hydrolysis of β- <dig>   <dig> 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
<cit> .
moreover, chitinase can be classified into two families as glycoside hydrolases   <dig> and  <dig> based on the amino acid sequence similarity
<cit> .
p. flavipulchra jg <dig> harbored  <dig> genes  coding for this enzyme, four of which belong to gh <dig>  in the genomes of p. tunicata d <dig> and pseudoalteromonas strains sm <dig>  by comparison, only two genes are annotated as chitinases, while no genes encoding this enzyme are found in p. haloplanktis tac <dig> and p. atlantica t6c.
notably, chitinase has been confirmed to show antifungal activities in b. cereus <cit>  and streptomyces sp.
mg3
<cit>  as chitin is a component of the cell walls of fungi.
two extracellular chitinases purified from pseudomonas aeruginosa k- <dig> also showed lysozyme activities against both gram-positive and gram-negative bacteria
<cit> .
the chitinases identified in jg <dig> 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
<cit> .
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
<cit> .
the quorum sensing regulatory system qseb/qsec detected in the genome of jg <dig> 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
<cit> .
however, the major genes encoding autoinducers responsible for quorum sensing have not been detected in jg <dig>  the system may thus only regulate genes encoding the expression and assembly of flagella, motility and chemotaxis in jg <dig>  as a protein responsible for biogenesis of type iv pilus is just downstream to the qseb/qsec system.
quorum quenching  describes all processes that interfere with quorum sensing
<cit> .
qq enzymes, including ahl lactonase, acylase, oxidase and reductase, have been discovered in many bacterial genuses, such as bacillus, rhodococcus, pseudomonas and ralstonia <cit> .
extra-and intra-cellular products of strain jg <dig> 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 , 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 jg <dig>  two genes of jg <dig>  encoding penicillin acylase show high homology with pvdq
<cit>  and quip
<cit> , which in pseudomonas aeruginosa pao <dig> 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 jg <dig>  these potential ahl acylases therefore equip strain jg <dig> 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 jg <dig> is controlled by the counterparts of phob/phor and phou
<cit>  as well as several phosphate transport systems
<cit>  and inorganic phosphate  transport systems
<cit> .
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
<cit> .
homologs to the two component regulatory system phor/phob found in jg <dig> 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
<cit> .
jg <dig> might therefore up-regulate the production of defensive primary and secondary metabolites in the oligotrophic marine environment, thereby even more efficiently combating competing  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  and phage shock proteins  that are involved in controlling carbon and nutrient starvation
<cit>  are present in p. flavipulchra jg <dig>  furthermore, the genome of jg <dig> 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, jg <dig> has two proteins of the ahpf/tr family identified as ahpf and thioredoxin reductase
<cit> .
these antioxidant enzymes protect jg <dig> against peroxide derived dna damage as well as oxidative membrane or lipids destruction.
key regulators of the oxidative stress response are also present .
unlike p. tunicata d <dig> and p. haloplanktis tac125
<cit> , the ubiquitous molybdopterin metabolism might be present, since dinucleotide-utilizing enzymes involved in molybdopterin biosynthesis can be found in jg <dig>  however, genes coding for enzymes using cofactors, such as xanthine oxidase, biotin sulfoxide reductase
<cit> , have not been detected.
five putative dioxygenases  might also help jg <dig> to protect its metabolism against oxidative stress.
several genes involved in heavy metal detoxification were discovered in the genome of jg <dig>  including periplasmic divalent cation tolerance protein  and copper homeostasis protein
<cit> .
experiments proved that intracellular copper accumulation in e. coli could increase without cuta <cit> .
moreover, cuta affects not only copper tolerance but also tolerance levels to zinc, nickel, cobalt and cadmium.
motility and secretion
flagellum formation is an important response to environmental stress.
the swimming motility of p. flavipulchra jg <dig> observed by swimming plate method showed a distinct motility halo , while the swarming ability was not detected .
genes for synthesis of flagellum are present in p. flavipulchra jg <dig>  and the resulting polar flagellum is visible under the transmission electron microscope .
thirty three genes are involved in flagellar assembly in the p. flavipulchra jg <dig> genome.
the class iii flagellar operons which are controlled negatively by flgm are present
<cit> .
flgm is an inhibitory factor to sense the integrity of the bacterial flagellar structure and regulate flagellar synthesis by binding the transcription factor σ <dig> <cit> .
however, the transcriptional activators flhc and flhd protein
<cit>  were missing.
flhd alone also regulates the cell division rate, limiting cell division of bacterial communities entering the stationary phase, as shown for e. coli <cit> .
the deletion of flhd may help jg <dig> to maintain a stable division rate to more quickly respond to environmental changes thus giving competitive advantages.
p. flavipulchra jg <dig> 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
<cit> .
pili mediate attachment to both living and artificial surfaces and are involved in bacteriophage adsorption, dna uptake, biofilm initiation and development, and twitching motility
<cit> .
one of the pili gene clusters and its upstream region is highly conserved in p. tunicata, p. haloplanktis and pseudoalteromonas sp.
tw- <dig> and sm <dig> contains homologs to the two component response regulator system algz/algr, which is involved in the regulation of alginate synthesis and pili-mediated, twitching motility in pseudomonas aeruginosa <cit> .
the alginate biosynthesis cluster is absent in jg <dig> 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, jg <dig> might be able to rapidly respond to environmental stresses and exceed other microorganisms.
there are several secretion systems functional in jg <dig>  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  <dig> genes involved in the type vi secretion system .
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
<cit> .
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
<cit> .
t6ss may be one of antibacterial mechanisms of jg <dig> 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
<cit> .
jg <dig> 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
<cit> .
penicillin metabolism in jg <dig> could be catalyzed by penicillin amidase yielding 6-aminopenicillanic acid, and by beta-lactamase  to give penicilloic acid.
the penicillin amidase can also be found in tac <dig>  but not in other compared pseudoalteromonas strains.
jg <dig> also possesses d-amino-acid oxidase to convert cephalosporin c into -7--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
<cit> .
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
<cit> .
jg <dig> possesses many genes involved in defense mechanisms due to abc-type antimicrobial peptide transport system , abc-type multidrug transport system , cation/multidrug efflux pump  and na+-driven multidrug efflux pump .
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 jg <dig> 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
<cit> .
CONCLUSIONS
the genome of p. flavipulchra jg <dig> 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 .
both the macromolecule and small molecules contribute to the antibacterial activities of jg <dig>  besides these already identified chemical structures produced by strain jg <dig>  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 jg <dig> and could serve as therapeutic strategies against aquaculture pathogens.
furthermore, jg <dig> also evolves a range of mechanisms adapting the adverse marine environment or multidrug rearing conditions.
the analysis of the genome of p. flavipulchra jg <dig> presented here provides a better understanding of its competitive properties and also an extensive application prospect.

 the genus pseudoalteromonas was established in 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. 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 d as a model organism to study, was demonstrated to synthesize a range of inhibitory substances, including a toxic antibiotic protein and two pigments. 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 jg was isolated from rearing water of healthy turbot in qingdao, china. strain jg 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 jg was shown to synthesize the putative l-amino acid oxidase named pseudoalteromonas flavipulchra antibacterial protein and 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 jg against fish pathogens indicate that the strain or its products could be utilized as biocontrol agent in aquaculture. to obtain a better understanding of the genetic potential of p. flavipulchra jg 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 the p. flavipulchra jg genome is composed of bp and the calculated g + c content is %. a total of open reading frames are identified within the p. flavipulchra jg genome. among the predicted genes, are not found in cog categories, and,, and genes are not applicable within the kegg, nr, swissprot and trembl databases, respectively. jg contains tandem repeat regions, transposons, and iss that account for about % of the genome. the p. flavipulchra jg genome is larger than that of the five other pseudoalteromonas strains whose genomic sequences have been published in the img database: p. tunicata d, p. haloplanktis tac, p. atlantica t6c and two pseudoalteromonas strains tw- and sm. d is an anti-biofouling bacterium, both t6c and tw- are mesophile bacteria, and tac and sm are psychrotolerant adapting to cold aqueous and deep-sea sediment environment, respectively. general features of these genomes were given in table  and their phylogenetic relationship based on the 16s rrna gene indicated that p. flavipulchra jg formed a cluster with p. tunicata d comparing with other sequenced pseudoalteromonas strains, p. tunicata d2 also exhibited great inhibitory activities against several specific organisms as in p. flavipulchra jg1, indicating that p. flavipulchra was functionally closely related to p. tunicata d p. flavipulchra jg p. tunicata d and p. haloplanktis tac share orthologous genes, accounting for %, % and % of all genes of jg d and tac respectively. in addition, jg contains more specific genes than d and tac, and there are more common genes within jg and d. we have also conducted a cog-based analysis among the specific and orthologous genes of these three strains. as shown in figure  2b, a larger proportion of jg and d specific genes belong to signal transduction mechanisms, while the genome of tac harbors more specific genes belonging to lipid transport and metabolism. genes involved in signal transduction were considered to allow p. tunicata d to generate phenotypic variation and provide the capacity of niche adaptation, and genes assigned to the same cog category might also play a similar role in p. flavipulchra jg in that both d and jg exhibited competitive activities in their surrounding environments. contrasting, in order to adapt cold condition, tac needs more genes involved in lipid transport and metabolism such as genes coding for lipid desaturase, which could increase membrane fluidity at low temperature. according to the genomic comparison results, jg and d share more functional similarities than to tac significantly, p. flavipulchra contains the highest proportion of specific genes belonging to the cog categories defense mechanisms and secondary metabolites biosynthesis, transport and catabolism, which account for % and %, respectively, among other bacteria.. this mainly attributes to more genes involved in abc-type antimicrobial peptide transport system, beta-lactamase class c and other penicillin binding proteins, cation/multidrug efflux pump, abc-type siderophore export system and those related to the synthesis of potential bioactive compounds, such as nonribosomal peptide synthetase modules and polyketide synthases. 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 jg was demonstrated to synthesize the antibacterial protein pfap and known small molecular compounds with antibacterial activity against v. anguillarum. the antibacterial protein pfap is a homologue of l-amino acid oxidase with high similarity to the l-lysine oxidase alpp of p. tunicata d and marinocine antimicrobial protein of marinomonas mediterranea mmb-. the sequence identities of protein pfap and these two antimicrobial proteins were 58% and 54%, with coverages of 95% and 92%, respectively. pfap is a secreted protein that might catalyze the oxidative deamination of specific amino acids to the respective α-keto acids with concomitant release of ammonium and hydrogen peroxide. the antibacterial activity of extracellular proteins of jg could be abolished in the presence of catalase, suggesting that the inhibitory effect was directly mediated by the action of hydrogen peroxide. the gene located downstream of pfap codes a catalase which responds to oxidative stress and causes decomposition of hydrogen peroxide. the catalase activity might thus immediately protect jg from the damage of the hydrogen peroxide generated by pfap. we also identified further genes encoding catalases and several genes capable of hydrogen peroxide degradation in the genome. thus, jg produces the antimicrobial protein pfap and simultaneously possesses several genes related to potential self-protection mechanisms. the compounds p-hydroxybenzoic acid, trans-cinnamic acid, and 6-bromoindolyl-3-acetic acid isolated from p. flavipulchra jg 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. as an organic acid, it has also been shown to inhibit the growth of ethanologenic escherichia coli ly 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 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 as an antioxidant, significantly reducing oxidative stress, as for example generated by hydrogen peroxide. we also identified all other genes in jg 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 directly, or cinnamic acid could get hydroxylated after its formation by phenylalanine ammonia lyase. 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 jg only two homologs of tal can be identified. therefore, this compound is most probably derived from tyrosine in jg 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. compound 6-bromoindolyl-3-acetic acid was shown to exhibit the strongest antibacterial activity among the small molecular compounds so far isolated from p. flavipulchra jg the metabolite can be formed by halogenation of tryptophan followed by oxidative deamination and decarboxylation. the indole-3-acetic acid portion can be formed in bacteria following several iaa biosynthesis pathways, of which a single bacterial strain sometimes contains more than one. iaa can be converted from indole-3-acetamide and indole-3-acetaldehyde via the indole-3-pyruvic acid pathway or the tryptamine pathway in jg the key enzymes involved in both of these pathways are present in the genome of jg including amino acid monooxygenase, indoleacetamide hydrolase, indole-3-pyruvate decarboxylase and monoamine oxidase. however, the tryptophan aminotransferase which transforms tryptophan into indole-3-pyruvate has not been detected in jg alternatively, the reaction could be mediated by an l-amino acid oxidase and the pfap protein might be a candidate. in addition, genes encoding tryptophan halogenases are found in the genome of jg one of which is located just downstream of pfap. a tryptophan halogenase would catalyze bromination of tryptophan, which subsequently would be converted into 6-bromoindolyl-3-acetic acid via one of the above mentioned biosynthetic pathways. the pfap protein, which is considered as a multifunctional protein, might be the most important antibacterial factor of p. flavipulchra thus far identified, as it also might play a role in the biosynthesis of 6-bromoindolyl-3-acetic acid. to assess the potential of p. flavipulchra jg 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 hybrid clusters, three nrps gene clusters, as well as a high number of further nrps-related peptidyl-carrier proteins, condensation, adenylation, epimerization, and thioesterase 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. 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 jg 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 jg was shown great chitinase activity on chitin agar. chitinase can catalyze the hydrolysis of β- 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. moreover, chitinase can be classified into two families as glycoside hydrolases and based on the amino acid sequence similarity. p. flavipulchra jg harbored genes coding for this enzyme, four of which belong to gh in the genomes of p. tunicata d and pseudoalteromonas strains sm by comparison, only two genes are annotated as chitinases, while no genes encoding this enzyme are found in p. haloplanktis tac 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- also showed lysozyme activities against both gram-positive and gram-negative bacteria. the chitinases identified in jg 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. 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 jg 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 jg the system may thus only regulate genes encoding the expression and assembly of flagella, motility and chemotaxis in jg as a protein responsible for biogenesis of type iv pilus is just downstream to the qseb/qsec system. quorum quenching 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 jg 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, 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 jg two genes of jg encoding penicillin acylase show high homology with pvdq and quip, which in pseudomonas aeruginosa pao 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 jg these potential ahl acylases therefore equip strain jg 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 jg is controlled by the counterparts of phob/phor and phou as well as several phosphate transport systems and inorganic phosphate transport systems. 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 jg 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. jg might therefore up-regulate the production of defensive primary and secondary metabolites in the oligotrophic marine environment, thereby even more efficiently combating competing 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 and phage shock proteins that are involved in controlling carbon and nutrient starvation are present in p. flavipulchra jg furthermore, the genome of jg 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, jg has two proteins of the ahpf/tr family identified as ahpf and thioredoxin reductase. these antioxidant enzymes protect jg against peroxide derived dna damage as well as oxidative membrane or lipids destruction. key regulators of the oxidative stress response are also present. unlike p. tunicata d and p. haloplanktis tac125, the ubiquitous molybdopterin metabolism might be present, since dinucleotide-utilizing enzymes involved in molybdopterin biosynthesis can be found in jg however, genes coding for enzymes using cofactors, such as xanthine oxidase, biotin sulfoxide reductase, have not been detected. five putative dioxygenases might also help jg to protect its metabolism against oxidative stress. several genes involved in heavy metal detoxification were discovered in the genome of jg including periplasmic divalent cation tolerance protein and copper homeostasis protein. 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 flagellum formation is an important response to environmental stress. the swimming motility of p. flavipulchra jg observed by swimming plate method showed a distinct motility halo, while the swarming ability was not detected. genes for synthesis of flagellum are present in p. flavipulchra jg and the resulting polar flagellum is visible under the transmission electron microscope. thirty three genes are involved in flagellar assembly in the p. flavipulchra jg genome. the class iii flagellar operons which are controlled negatively by flgm are present. flgm is an inhibitory factor to sense the integrity of the bacterial flagellar structure and regulate flagellar synthesis by binding the transcription factor σ. however, the transcriptional activators flhc and flhd protein were missing. flhd alone also regulates the cell division rate, limiting cell division of bacterial communities entering the stationary phase, as shown for e. coli. the deletion of flhd may help jg to maintain a stable division rate to more quickly respond to environmental changes thus giving competitive advantages. p. flavipulchra jg 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- and sm contains homologs to the two component response regulator system algz/algr, which is involved in the regulation of alginate synthesis and pili-mediated, twitching motility in pseudomonas aeruginosa. the alginate biosynthesis cluster is absent in jg 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, jg might be able to rapidly respond to environmental stresses and exceed other microorganisms. there are several secretion systems functional in jg 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 genes involved in the type vi secretion system. 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 jg 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. jg 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 jg could be catalyzed by penicillin amidase yielding 6-aminopenicillanic acid, and by beta-lactamase to give penicilloic acid. the penicillin amidase can also be found in tac but not in other compared pseudoalteromonas strains. jg also possesses d-amino-acid oxidase to convert cephalosporin c into -7--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. jg possesses many genes involved in defense mechanisms due to abc-type antimicrobial peptide transport system, abc-type multidrug transport system, cation/multidrug efflux pump and na+-driven multidrug efflux pump. 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 jg 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 jg 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. both the macromolecule and small molecules contribute to the antibacterial activities of jg besides these already identified chemical structures produced by strain jg 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 jg and could serve as therapeutic strategies against aquaculture pathogens. furthermore, jg also evolves a range of mechanisms adapting the adverse marine environment or multidrug rearing conditions. the analysis of the genome of p. flavipulchra jg presented here provides a better understanding of its competitive properties and also an extensive application prospect. 
in our previous study, p. flavipulchra jg was found to synthesize a pseudoalteromonas flavipulchra antibacterial protein with l-amino acid oxidase activity and five small chemical compounds, which were the main competitive agents of the strain. chitinases and quorum quenching enzymes present in p. flavipulchra, which coincide with great chitinase and acyl homoserine lactones degrading activities of strain jg suggest other potential mechanisms contribute to antibacterial/antifungal activities. the genome of p. flavipulchra jg 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 jg and affords several admissible biocontrol procedures in aquaculture. the analysis of the genome of p. flavipulchra jg provides a better understanding of its competitive properties and also an extensive application prospect. furthermore, jg also evolves a range of mechanisms adapting the adverse marine environment or multidrug rearing conditions. 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. 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. moreover, movability and rapid response mechanisms to phosphorus starvation and other stresses, such as antibiotic, oxidative and heavy metal stress, enable jg to adapt to deleterious, fluctuating and oligotrophic marine environments. many strains of the genus pseudoalteromonas were confirmed to produce bioactive compounds that provide those advantages over their competitors.
 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 jg was found to synthesize a pseudoalteromonas flavipulchra antibacterial protein 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. 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 jg 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 jg to adapt to deleterious, fluctuating and oligotrophic marine environments.  the genome of p. flavipulchra jg 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 jg and affords several admissible biocontrol procedures in aquaculture. furthermore, jg also evolves a range of mechanisms adapting the adverse marine environment or multidrug rearing conditions. the analysis of the genome of p. flavipulchra jg provides a better understanding of its competitive properties and also an extensive application prospect. pseudoalteromonas flavipulchragenome analysisantibacterial metabolitesquorum quenchingsurvival advantages 