BACKGROUND
pseudomonas fluorescens f <dig> is a pgpr strain isolated from the sugar-beet rhizosphere in ireland  <cit> .
initially this strain was selected and studied because of its capacity to inhibit growth of a range of phytopathogenic bacteria, fungi, oomycetes and nematodes including pectobacterium caratovorum <cit> , fusarium oxysporum <cit> , pythium ultimum <cit>  and globodera spp.
<cit> .
this antimicrobial capacity is strongly linked to the production of a secondary metabolite,  <dig> -diacetylphloroglucinol   <cit> , and renders the strain a candidate bio control microorganism for agrobiotech applications.
the biosynthetic and regulatory genes required for the synthesis of this polyketide are located in an  <dig> kb cluster of nine protein-coding genes that are highly conserved in other dapg-producing p. fluorescens but completely absent from non-producing strains  <cit> .
in addition, p. fluorescens f <dig> is an excellent rhizosphere coloniser of different plant species including wheat  <cit> , alfalfa  <cit> , and willow  <cit> .
for that reason, f <dig> is widely used as a model for studying rhizosphere colonization  <cit> .
several studies have sought to take advantage of this colonising ability by developing novel genetically modified derivatives with biotechnological traits, for example the capacity to degrade polychlorinated biphenyls and other environmental pollutants  <cit> .
the pseudomonas genus is composed of more than  <dig> species, which have been divided by multilocus sequence analysis  into nine major groups: p. fluorescens, p. syringae, p. lutea, p. putida, p. anguilliseptica, p. straminea, p. aeruginosa, p. oleovorans and p. stutzeri <cit> .
some of these groups are themselves composed of different subgroups containing multiple species.
for instance, the p. fluorescens group can be further divided into nine subgroups  <cit> .
the number of species present in each of these subgroups is somewhat difficult to assess since novel formal species, such as p. protegens <cit> , are frequently described.
a number of genome sequences for strains belonging to the p. fluorescens group have been recently obtained .
the initial comparative genomic analysis of the three first complete genome sequences of strains belonging to the p. fluorescens group has highlighted a large number of strain-specific genes  <cit> .
therefore, it has been proposed that the sequenced strains belong to a complex of species rather than to a single species  <cit> .
more recently, a comparative genomics analysis performed on  <dig> genome sequences of strains belonging to the p. fluorescens group has highlighted three main subclades  <cit> .
here we present an analysis of the genome sequence of p. fluorescens f <dig>  <cit>  compared to  <dig> other complete and draft genomes of strains classified as pseudomonas spp., p. brassicacearum, p. fluorescens, p. protegens, p. mandelii, p. chlororaphis, p. tolaasii and p. extremaustralis.
the genome of p. fluorescens f <dig>  <cit>  is composed of a single circular chromosome of  <dig> , <dig> bp with an average g + c content of  <dig> % and an overall coding density of  <dig> % .
following automatic annotation and subsequent manual curation, a total of  <dig> protein-coding sequences, nine ncrnas, five rrna operons and  <dig> trna loci were detected in the genome of this strain.
the f <dig> genome contains a variety of protein-coding genes that appear relevant for thriving in the rhizosphere environment.
this includes unusual metabolic adaptations within the p. fluorescens species, protein-coding genes related to motility, genes encoding putative toxins targeted to diverse organisms and a very large number of protein-coding genes involved in the assembly of different secretion systems.
the distribution of these traits in other strains belonging to the p. fluorescens group as well as the functionality of several of these genes are described in this manuscript.
RESULTS
p.fluorescens f <dig> phylogenomics comparison
in order to determine the phylogenetic relationship of f <dig> to other pseudomonads, a phylogenomic analysis of  <dig> pseudomonas genomes and draft genomes available at the time of this writing was performed with the composition vector method  <cit> .
the phylogenomics tree generated with such approach  is mostly congruent with previous concatenated sequence trees obtained in different mlsa analyses  <cit> ; and clearly highlighted the need to re-evaluate the taxonomic status of species belonging to the p. fluorescens group  <cit> .
compared to other trees, the major difference is the phylogenetic position of p. putida and p. syringae with respect to the p. fluorescens group.
however the distances shown here for this two species to the parent node are very small and differences could be due to the different methods used.
fifty strains  previously classified as p. fluorescens , p. brassicacearum, p. protegens, p. mandelii, p. chlororaphis, p. tolaasii, p. extremaustralis and pseudomonas spp.
formed a single cluster which branched significantly deeper than other clusters that represent defined species such as p. aeruginosa or p. syringae.
detailed phylogenomic analysis of the p. fluorescens group revealed the presence of at least five subgroups with strains previously classified as p. fluorescens, interspersed with strains classified in other species .
the analysis of the whole  <dig> strains orfome, represented as a vector of 6mer peptide frecuencies, has allowed us to increase the number of p. fluorescens subgroups from three  <cit>  to five.
it is likely that some of these subclusters may represent different species, especially considering that all of these subclusters branch above a  <dig>  dissimilarity threshold, a value enough to discriminate recognized species within the genus pseudomonas.
.
subgroup i contains f <dig> together with strains classified as pseudomonas brassicacearum, including the complete genome sequence of nfm <dig>  <cit> .
subgroup ii contains the sequenced strain p. fluorescens pf0- <dig> and other strains classified either as p. fluorescens or pseudomonas spp.
although, these two subgroups were initially described as one subclade by loper et al.
<cit> , the separation between these two subgroups is supported by a branching above  <dig>  dissimilarity.
subgroup iii includes p. fluorescens ncimb  <dig>  p. mandelii jr- <dig> and several unclassified pseudomonads, which were not analysed in previous studies.
subgroup iv contains p. protegens pf- <dig>  all the p. chlororaphis strains, p. fluorescens strains wayne <dig> and nz <dig> and one unclassified pseudomonad.
this subgroup is congruent with subcluster  <dig>  <cit> .
finally, subgroup v contains p. fluorescens strains sbw <dig>  wh <dig>  a <dig>  ss <dig> nz <dig> and nz <dig>  together with p. tolaasii strains, p. extremaustralis and four unclassified pseudomonads.
this subgroup corresponds to subcluster  <dig>  <cit> .
the genome sequence of strain f <dig> was then aligned with its closest fully sequenced relatives, p. brassiccacearum nfm <dig> and p. fluorescens pf0- <dig> .
this alignment shows that f <dig> genome is much more similar to the nfm <dig> than to the pf0- <dig> genome, supporting the subclustering described above.
it also shows that the major difference between the f <dig> and nfm <dig> genomes is a large inversion in the distal part of the genome, opposite to the replication origin.
it is interesting to note that both strains were isolated from a similar ecosystem, the rhizosphere of a crop although in different hemispheres and that both strains share similarities such as the occurrence of phase variation during rhizosphere colonization, the presence of denitrification genes  and the presence of three chemotaxis gene clusters .
the presence of the f <dig> orthologous coding sequences  in the  <dig> genomes sequences from the p. fluorescens group was assessed using orthomcl  <cit> .
following this analysis, the core genome of the p. fluorescens group is composed of  <dig> orthologous cdss present in all the fifty strains examined.
unsurprisingly, the size of the core genome is smaller in comparison to previous analysis performed with  <dig>  and  <dig>  genome sequences  <cit> .
the core genome of the p. fluorescens subgroup i is composed of  <dig> orthologous cds , which represents approximately 76% of the whole predicted proteome.
in addition  <dig> orthologous cdss  found in the f <dig> genome are not present in any other genome of strains within this subgroup.
these  <dig> cdss were subjected to blastp analysis against the non-redundant  ncbi database .
closest relatives of these unique protein-coding genes are mostly found in other gamma-proteobacterial genomes, including other pseudomonas species, and could be involved in important functions relevant for rhizosphere fitness .
the f <dig> genome encodes important traits involved in rhizocompetence
a number of plant-associated bacteria, including pseudomonas spp.
<cit>  and azospirillum brasilense <cit> , are able to use nitrogen oxides as alternative electrons acceptors under oxygen limiting conditions, a process called denitrification.
denitrification has been shown to play a role in rhizosphere colonization, since mutants of p. fluorescens impaired in nitrate or nitrite reductases are deficient in the colonization of the rhizosphere  <cit> .
furthermore, it has been shown that denitrification is statistically associated with rhizosphere competence in rhizosphere isolated fluorescent pseudomonads  <cit> .
the f <dig> genome encodes approximately  <dig> proteins involved in denitrification.
these genes are organized in four clusters on the chromosome .
the first cluster  contains the nark1k2ghji genes, which encode the membrane-bound nitrate reductase.
the sensor-response regulator narl-narx and the transcriptional regulator dnrs are encoded upstream of the nar genes.
the periplasmic  and the assimilatory nitrate reductase  are also present in the genome of f <dig> .
the second cluster  is composed of the nir and nor <dig> genes encoding nitrite and nitric oxide reductase, respectively.
the third cluster  contains the nos <dig> genes that encode the nitrous oxide reductase.
all these genes showed highest homology with denitrification genes in different pseudomonads .
finally the last cluster  contains the nos <dig> and nor <dig> genes.
although the nos <dig> genes showed homology  with the nos <dig> genes, the nor <dig> genes are quite divergent to nor <dig> genes and showed higher homology with genes encoded in acidovorax and several enterobacteriaceae.
it is interesting to note that the σ54-transcriptional activator norr <dig>  is encoded in the downstream region of the nos2nor <dig> cluster, which suggests that nos1nor <dig> and nos2nor <dig> genes might be independently regulated.
in order to test the functionality of these denitrification genes, the growth of f <dig> under anaerobic conditions was compared to the growth of pf0- <dig>  which does not contain denitrification genes.
while pf0- <dig> did not grow under these conditions, f <dig> was able to grow anaerobically using nitrate and nitrite as electron acceptors .
furthermore, a total depletion of nitrite was observed after  <dig> h of growth .
these results together with the presence of two copies of the nos genes, strongly indicate that f <dig> is capable of full denitrification.
the duplication of genes encoding nitric oxide reductase and nitrous oxide reductase might be related to high denitrification rates, as has been shown for a bacillus strain that contains two different copies of the nosz gene and showed high amounts of n <dig> production  <cit> .
the presence of denitrification genes was analyzed in the fifty strains forming the p. fluorescens group .
while all strains of subgroup i contained a complete denitrification pathway, none of the strains of subgroup ii possessed denitrification genes, which further support the distinction between subgroup i and ii.
interestingly some denitrification genes are present in few strains belonging to subgroups iii, iv and v, which might indicate horizontal gene transfer.
whereas nor <dig> genes were only present f <dig> and nfm <dig>  nos <dig> genes were systematically found in strains harbouring the nos <dig> genes.
another metabolic adaption is the potential degradation of aromatic diterpenes encoded in the f <dig> genome.
aromatic diterpenes are tricyclic resin acids naturally produced by trees and include abietic, dehydroabietic and palustric acids.
bacteria that harbor the dit genes can grow in these substrates as the sole carbon and energy source  <cit> .
these genes have been found in several proteobacteria such as burkholderia, cupriavidus and pseudomonas <cit> .
among pseudomonas, dit genes have been found in p. abietaniphila <cit> , p. diterpeniphila <cit>  and in p. aeruginosa strain pa <dig>  <cit> .
f <dig> chromosome harbors a  <dig> kb genetic island containing the dit genes .
this region is contiguous to the nos2-nor <dig> cluster and has a gene organization similar to the genetic island found in p. aeruginosa pa <dig>  although in this strain certain dit genes are duplicated and the genomic island is  <dig> kb long  <cit> .
growth of f <dig> on abietic acid was compared with growth of pf0- <dig>  after twenty days, growth was observed for f <dig> but not for pf0- <dig>  suggesting that dit genes in f <dig> are functional.
plant growth-promoting traits
previous studies have shown that p. fluorescens f <dig> is capable of inorganic phosphate solubilisation through the extracellular production of gluconic acid  <cit> .
moreover, f <dig> seems capable of mineralizing insoluble organic phosphates pool by the production of specific enzymes, such as the homologues of the alkaline phosphatases phod  and phox  of p. fluorescens pf0- <dig>  <cit>  and a beta-propeller phytase   <cit> .
altogether, these traits could contribute to mobilization of insoluble soil phosphate into bioavailable forms that can be taken up by the plant root.
acc deaminase activity in f <dig> has been reported previously  <cit>  and analysis of the genome sequence indicates that psf113_ <dig>  encodes a 1-aminocyclopropane-carboxylate  deaminase.
acc deaminase  catalyses the degradation of the ethylene precursor, acc, into ammonium and α-ketobutyrate  and has been linked to plant growth promotion activity in rhizosphere microorganisms  <cit> .
the activity of the acds enzyme in f <dig> is  <dig>  μm mg- <dig> protein hr- <dig>  almost twice the activity of enterobacter uw <dig>  <cit> .
the expression of acds is probably regulated by the lrp-like transcriptional regulator acdr, which is located upstream of acds.
motility and chemotaxis
like all pseudomonas strains, f <dig> genome encodes genes for the synthesis of polar flagella distributed in three clusters.
several of these genes in f <dig> have been previously analyzed  <cit>  and regulation of flagella synthesis requires the master regulatory gene fleq <cit> .
besides these genes, f <dig> chromosome harbors another  <dig> kb flagella locus containing  <dig> cdss , which is not present in any other pseudomonas genome, with the exception of p. extremaustralis .
conversely to the operons encoding flagellar genes in pseudomonads, this region contains an flhdc operon, encoding master regulatory proteins of flagella synthesis in other gamma-proteobacteria such as enterobacteria and azotobacter.
the  <dig> orfs showed high homology to flagellar genes of azotobacter vinelandii, a soil bacterium phylogenetically related to the genus pseudomonas, but producing a different type of flagella, which are peritrichous, instead of the typical single or double polar flagella produced by pseudomonads.
the region also showed synteny with the flagellar genes of a. vinelandii .
the a. vinelandii chromosome harbors flagellar genes in two clusters, i and ii.
genes in cluster i are conserved in the same order in the  <dig> kb region in the f <dig> chromosome.
cluster ii in a. vinelandii is located  <dig> kb downstream of cluster i. a reduced version of this region and lacking  <dig> orfs is located in an inverted orientation adjacent to cluster i in the f <dig> chromosome.
all the genes present in a. vinelandii clusters, but absent in the f <dig> genome, encode chemotaxis proteins such as chez, chey, and cheb or proteins that are not essential for flagella synthesis, such as rfbc, rfbg and rfbf.
ectopic expression of flhdc in f <dig> increased swimming motility  and resulted in hyperflagellation with multiple polar flagella .
these results indicate that f <dig> can produce the second type flagella, and that these flagella are functional.
in rhodobacter sphaeroides, a second acquired flagellum is also functional and coexists with the endogenous flagellar system  <cit> .
in this case, as in f <dig>  both flagella were polar.
although flagella whose synthesis is regulated by the master regulatory genes flhdc are generally peritrichous, regulation of polar flagella by this system has also been observed in burkholderia glumae <cit> , indicating that flhdc regulation does not imply peritrichous flagellation.
the f <dig> genome encodes five chemotaxis-like systems: wsp, chp, che <dig>  che <dig> and che <dig> .
the wsp system  is implicated in regulation of motility and biofilm formation in f <dig>  <cit>  and other p. fluorescens strains  <cit> .
the chp system  is located in the immediate vicinity of to pil genes and has been shown to control twitching motility in pseudomonas aeruginosa <cit> .
besides, the f <dig> genome encodes three che systems .
che <dig>  is located in the vicinity of protein-coding genes involved in the assembly of the endogenous flagellum.
mutation of the chea <dig> gene in f <dig> resulted in impaired motility and rhizosphere colonization  <cit>  indicating that this system participates in chemotactic motility.
it is interesting to note that no genes encoding methyl accepting chemotactic sensors  are genetically linked to this system, although more than thirty orfs predicted to encode mcp-like proteins are scattered in the f <dig> genome.
the che <dig>  and che <dig>  systems are present in a restricted number of strains of the p. fluorescens group .
the che  <dig> genes are only present in all strains of subgroup i and three strains of the subgroup v. the che  <dig> genes are present only in three strains of subgroup i: f <dig>  nfm <dig> and wood  <dig>  both che  <dig> and che  <dig> systems harbor two genes putatively encoding mcps, suggesting that these systems might be implicated in specialized functions.
this diversity of chemotaxis systems, as well as the production of a second type flagellum, highlights the importance of motility for f <dig>  it is important to note that chemotaxis and motility mutants are among the most affected mutants in competitive rhizosphere colonization  <cit>  and that the rhizospheric environment selects for hypermotile phenotypic variants  <cit>  that are frequently hypercompetitive  <cit> .
it also raises interesting questions about which chemotaxis systems are used by the second type flagellum and about motility under different physiological condition, for example anaerobic growth using nitrate or nitrite as electron acceptors.
protein and macromolecule secretion
gram negative bacteria rely on several secretion systems to influence their environment by translocating protein and dna into neighbouring cells and the extracellular milieu.
these secretion systems can range from simple transporters to multi-component complexes and have been classified into six types: from type i through type vi secretion system   <cit> .
the genome of f <dig> contains a wide variety of secretion systems, which include six t1sss, two t2sss, two t3sss, one t4ss, five t5asss, four t5bss, one t5dss and three t6ssss .
t2sss
t2ss is the most ubiquitous secretion system used by gram negative bacteria to secrete extracellular protein.
therefore, t2ss is well-conserved and involves a set of  <dig> to  <dig> proteins  <cit> .
according to xcpr phylogenetic analysis, pseudomonas t2sss could be divided in two main clusters: xcp , and hpx  .
two complete t2sss related to the xcp cluster  and to the hxc cluster  are present in the genome of f <dig> .
the genetic organization of the hxc cluster of f <dig> is highly similar with the hxc cluster of p. aeruginosa pao <dig> and p. fluorescens sbw <dig>  <cit> .
moreover, a predicted orf sharing 74% and 72% identity at the amino acid level with the hxc substrate psp of p. fluorescens sbw <dig>  <cit>  and ding of p. aeruginosa mdr <dig>  <cit>  is located immediately downstream of the hxc locus, which suggest that this protein could possibly be secreted by the hxc secretion system of f <dig>  the second t2ss locus of f <dig> is identical to the second xcp of p. aeruginosa pa <dig>  which is present on the genomic island rgp <dig>   <cit> .
based on homologies with known xcp effectors, it seems that at least four predicted f <dig> proteins could potentially be xcp substrates .
t3sss
t3ss is a nanomachine composed of approximately  <dig> proteins  <cit> , which has evolved into seven different families: ysc, hrp <dig>  hrp <dig>  spi- <dig>  spi- <dig>  rhizobiaceae and chamydia <cit> .
the distribution of t3ss loci in complete or draft pseudomonas genome sequences was investigated using the following orfs pa <dig>  pspto_ <dig>  pspph_ <dig> and psf113_ <dig>  as queries in sequential blastp and tblastn searches.
four t3ss phylogenetic families, ysc, spi- <dig>  hrp <dig> and rhizobia are encoded in the different pseudomonas chromosomes .
while ysc is only found in p. aeruginosa, the other t3ss phylogenetic families are distributed across different pseudomonas groups.
for example, strains belonging to the p. fluorescens group can contain spi- <dig>  hrp <dig> or rhizobia t3ss.
although the hrp <dig> locus is present in approximately 40% of the p. fluorescens genomes , two distinct sub-families are found in different chromosomal locations  <cit> .
the hrp <dig> locus  encoded in the f <dig> chromosome is, perhaps unsurprisingly, closely related to hrp <dig> of p. fluorescens q8r1- <dig>  <cit> , q2- <dig>  <cit> , wood1r  <cit>  and p. brassicacearum nfm <dig>  <cit> .
the predicted t3es ropaa <dig>  ropb and ropm reported by mavrodi et al.
<cit>  are also encoded in the genome of f <dig>  q2- <dig>  wood1r and nfm <dig>  in addition a putative paralogue of ropaa <dig>  called here ropaa <dig> , is found in all these strains.
in comparison to hrp <dig>  the spi- <dig> phylogenetic family is less abundant in the pseudomonas genome sequences .
comparative genomics analysis of spi- <dig> clusters belonging to strains belonging to the p. fluorescens group, revealed that these loci are found in the same chromosomal location, flanked by the genes nast and pyrd.
in f <dig>  however, the spi- <dig> cluster is flanked by nast  and soxr .
this is due to genome rearrangement, probably caused by is3-family transposases .
examination of other p. fluorescens genomes indicated that the genes invg, iagb and pseudogene related to hila are present at the same chromosomal location in strains pamc  <dig>  <cit> , ag <dig>  <cit> , 14– <dig>  <cit> , wh <dig>  <cit> , sbw <dig>  <cit> , r <dig>  <cit> , a <dig>  bg33r, ss <dig>  <cit> , nz <dig>  nz <dig> and pms <dig>  interestingly all these strains belong to subgroup v of the p. fluorescens group, which suggest that the spi- <dig> cluster was lost before speciation of this sub-clade.
while the spi- <dig> cluster is conserved across strains belonging to the sub-clade composed of upb <dig>  <cit> , cb <dig>  ncppb  <dig> and ncppb 2289; its random distribution across the other strains of the p. fluorescens group could suggest an acquisition by horizontal gene transfer.
investigation of the average gc content of the spi- <dig> clusters did, however, not show any shift in comparison to the average content of the genome examined .
this is not the case with the spi- <dig> cluster of two strains belonging to the p. putida group, which have an atypical gc content, therefore suggesting horizontal gene transfer acquisition.
t6sss
the type vi secretion machinery is the product of approximately  <dig> conserved genes which are generally found together inside a genomic locus  <cit> .
t6ss are widespread in proteobacteria, particularly among gamma-proteobacteria and have been classified into five distinct phylogenetic clusters   <cit> .
however a recent comparative genomic analysis has further divided the cluster  <dig> into 4a and 4b  <cit> .
p. aeruginosa possesses three different loci named hsi-i , ii  and iii , which perform different functions.
whereas hsi-i is be involved in inter-bacterial interactions through secretion of tse <dig>  tse <dig> and tse <dig>  <cit> , hsi-ii and iii could be linked to virulence towards animals and plants  <cit> .
to date, strains belonging to the p. fluorescens group possess between one to four t6sss per genome, which belong to cluster  <dig> to 4b .
f <dig> contains the three most common t6ss phylogenetic clusters, which are related to the hsi loci.
moreover, in this strain the two loci hsi-i  and hsi-ii  are located besides each other in a tandem arrangement, which is an uncommon feature of t6ss.
in addition to the t6sss loci, most bacterial strains encode additional vgrg and hcp genes that encode extracellular structural components of the secretion apparatus, which are encoded elsewhere in the genome  <cit> .
from the eight vgrgs and three hcps encoded in the f <dig> genome, five vgrgs and one hcp proteins are located outside t6ss loci  <cit> .
other virulence and niche adaption traits
different strains of p. fluorescens produce a set of secondary metabolites with antifungal and antibacterial properties  <cit> .
these metabolites are important for competition and survival in the rhizosphere and are the basis of biocontrol activities.
f <dig> genome carries gene responsible for siderophores synthesis, including pyoverdine  <cit>  and pyridine- <dig> -bis-thiocarboxylic acid  <cit> .
moreover f <dig> produces antimicrobial compounds unrelated to siderophores such as hydrogen cyanide  <cit>  and dapg  <cit>  .
besides the genes encoding the enzymes required for the biosynthesis of these compounds, the f <dig> genome contains a cluster of genes  that is likely to encode genes for the synthesis of an unknown antibiotic.
this  <dig> kb cluster encodes four polyketide synthase modules, one dehydratase, one transacylase, one transferase, one isochorismatase and one transport protein .
there are not homologous genes to these in the genus pseudomonas and the closest homologues  are found in genes responsible of the synthesis of macrolide antibiotics from different streptomyces species, including the lankacidin production cluster of streptomyces rochei <cit> .
the lankacidin  gene cluster of s. rochei is composed of  <dig> orfs , which encode a polyketide synthase /nonribosomal peptide synthetase  hybrid gene , type i pks genes, and the essential cluster for the lankacidin production, pyrroloquinoline quinone  biosynthetic genes   <cit> .
the presence of this cluster in other genomes was therefore investigated by comparative genomic analysis.
besides streptomyces rochei plasmid psla2-l and p. fluorescens f <dig> the lkc cluster was only found in one additional genome: hahella chejuensis kctc  <dig>  in p. fluorescens f <dig>  the pqq genes, which are crucial for lankacidin production in streptomyces rochei <cit>  are found outside the lkc gene cluster  <cit> .
the possible production of this unknown antibiotic is likely to contribute to rhizosphere and soil competence.
it has been shown that p. fluorescens strains can kill nematodes  <cit>  and are pathogenic to insects  <cit> .
preliminary results show that f <dig> is toxic towards the model nematode caenorhabditis elegans and the protozoan acanthamoeba polyphaga.
f <dig> repels these bacteriovores and/or prevents their growth feeding on this strain .
in addition, f <dig> kills the model insect galleria mellonella, after  <dig> h of infection with a dose of  <dig> bacteria per larvae .
these features could be explained by the production of putative insecticidal toxins , which are also found in pseudomonas strains nfm <dig> and pf0- <dig> and in other bacterial species such as yersinia enterocolitica <cit> .
in addition, a number of other putative toxins and virulence factors are present in p. fluorescens f <dig> genome including  <dig> predicted hemolysin/haemagglutinins, nine adhesin or agglutination proteins, two rtx toxins, seven rhs-family proteins and eight yd-repeat-containing proteins.
ice and prophage elements
analysis of the f <dig> genome has revealed the presence of one putative integrative conjugative element  of  <dig> kb containing  <dig> orfs located downstream a trna-glycine .
this putative bacteriophage-derived mobile genetic element is surrounded by attf and attr sites  and possesses protein-coding genes required for excision and integration .
the mobilization of the ice could be mediated by a gi-t4ss , which is responsible for the formation of the conjugative pilus and the resulting conjugative transfer  <cit> .
however, it remains to be determined whether this gi-t4ss is functional since a conserved t4ss component is disrupted by a is <dig> transposase .
in addition to protein-coding genes involved in the integration and transfer of this hypothetical ice, the cargo genes are related to t1ss  dit cluster  and nos2nor <dig> , described above.
f <dig> also carry two prophage elements .
prophage p <dig>  is, like the prophages of other p. fluorescens strains  <cit> , inserted between two highly conserved genes: muts  and cina .
prophage p <dig> closely resembles to prophage pp <dig> of p. fluorescens sbw <dig>  <cit>  and carries conserved tail synthesis genes of phage ctx but lacks integrase and head morphogenesis genes.
therefore this genomic region probably encodes a bacteriocin belonging to the r-type pyocins.
the second prophage of p. fluorescens f <dig>  is related to prophage  <dig> of p. fluorescens pf- <dig>  <cit> .
this genomic region contains protein-coding genes involved in tail assembly and head morphogenesis and therefore could correspond to the genome of a bacteriophage.
CONCLUSIONS
genome analysis of strain f <dig> has shown that this bacterium belongs to the pseudomonas fluorescens complex.
however, as indicated earlier by silby et al.
<cit>  and more recently by loper et al.
<cit> , the taxonomy of p. fluorescens requires further study, since significant differences are found in the genomic complement of different strains.
furthermore, the phylogenomic analysis has shown that the p. fluorescens group can be subdivided into at least, five subgroups.
indeed, while f <dig> shared only 35% of its genome with all the other sequenced strains within the p. fluorescens group, this value increases to 76% when the f <dig> genome is compared to the four closest relatives .
the fact that these strains have common unusual traits such as denitrification, phenotypic variation during rhizosphere colonization  <cit>  and highly conserved and unique chemotaxis systems, might indicate that these strains belong to the same species.
in addition f <dig> genome contains  <dig> genes that are not found in q2- <dig>  q8r1- <dig>  woodr <dig> and nfm <dig>  which encode proteins likely to be involved in the production of an unknown polyketide and in the assembly of a second flagella.
these traits could explain in part the excellent rhizocompetence ability of f <dig> and are currently under study.

 pseudomonas fluorescens f is a pgpr strain isolated from the sugar-beet rhizosphere in ireland. initially this strain was selected and studied because of its capacity to inhibit growth of a range of phytopathogenic bacteria, fungi, oomycetes and nematodes including pectobacterium caratovorum, fusarium oxysporum, pythium ultimum and globodera spp.. this antimicrobial capacity is strongly linked to the production of a secondary metabolite, -diacetylphloroglucinol, and renders the strain a candidate bio control microorganism for agrobiotech applications. the biosynthetic and regulatory genes required for the synthesis of this polyketide are located in an kb cluster of nine protein-coding genes that are highly conserved in other dapg-producing p. fluorescens but completely absent from non-producing strains. in addition, p. fluorescens f is an excellent rhizosphere coloniser of different plant species including wheat, alfalfa, and willow. for that reason, f is widely used as a model for studying rhizosphere colonization. several studies have sought to take advantage of this colonising ability by developing novel genetically modified derivatives with biotechnological traits, for example the capacity to degrade polychlorinated biphenyls and other environmental pollutants. the pseudomonas genus is composed of more than species, which have been divided by multilocus sequence analysis into nine major groups: p. fluorescens, p. syringae, p. lutea, p. putida, p. anguilliseptica, p. straminea, p. aeruginosa, p. oleovorans and p. stutzeri. some of these groups are themselves composed of different subgroups containing multiple species. for instance, the p. fluorescens group can be further divided into nine subgroups. the number of species present in each of these subgroups is somewhat difficult to assess since novel formal species, such as p. protegens, are frequently described. a number of genome sequences for strains belonging to the p. fluorescens group have been recently obtained. the initial comparative genomic analysis of the three first complete genome sequences of strains belonging to the p. fluorescens group has highlighted a large number of strain-specific genes. therefore, it has been proposed that the sequenced strains belong to a complex of species rather than to a single species. more recently, a comparative genomics analysis performed on genome sequences of strains belonging to the p. fluorescens group has highlighted three main subclades. here we present an analysis of the genome sequence of p. fluorescens f compared to other complete and draft genomes of strains classified as pseudomonas spp., p. brassicacearum, p. fluorescens, p. protegens, p. mandelii, p. chlororaphis, p. tolaasii and p. extremaustralis. the genome of p. fluorescens f is composed of a single circular chromosome of, bp with an average g + c content of % and an overall coding density of %. following automatic annotation and subsequent manual curation, a total of protein-coding sequences, nine ncrnas, five rrna operons and trna loci were detected in the genome of this strain. the f genome contains a variety of protein-coding genes that appear relevant for thriving in the rhizosphere environment. this includes unusual metabolic adaptations within the p. fluorescens species, protein-coding genes related to motility, genes encoding putative toxins targeted to diverse organisms and a very large number of protein-coding genes involved in the assembly of different secretion systems. the distribution of these traits in other strains belonging to the p. fluorescens group as well as the functionality of several of these genes are described in this manuscript.  p.fluorescens f phylogenomics comparison in order to determine the phylogenetic relationship of f to other pseudomonads, a phylogenomic analysis of pseudomonas genomes and draft genomes available at the time of this writing was performed with the composition vector method. the phylogenomics tree generated with such approach is mostly congruent with previous concatenated sequence trees obtained in different mlsa analyses; and clearly highlighted the need to re-evaluate the taxonomic status of species belonging to the p. fluorescens group. compared to other trees, the major difference is the phylogenetic position of p. putida and p. syringae with respect to the p. fluorescens group. however the distances shown here for this two species to the parent node are very small and differences could be due to the different methods used. fifty strains previously classified as p. fluorescens, p. brassicacearum, p. protegens, p. mandelii, p. chlororaphis, p. tolaasii, p. extremaustralis and pseudomonas spp. formed a single cluster which branched significantly deeper than other clusters that represent defined species such as p. aeruginosa or p. syringae. detailed phylogenomic analysis of the p. fluorescens group revealed the presence of at least five subgroups with strains previously classified as p. fluorescens, interspersed with strains classified in other species. the analysis of the whole strains orfome, represented as a vector of 6mer peptide frecuencies, has allowed us to increase the number of p. fluorescens subgroups from three to five. it is likely that some of these subclusters may represent different species, especially considering that all of these subclusters branch above a dissimilarity threshold, a value enough to discriminate recognized species within the genus pseudomonas.. subgroup i contains f together with strains classified as pseudomonas brassicacearum, including the complete genome sequence of nfm. subgroup ii contains the sequenced strain p. fluorescens pf0- and other strains classified either as p. fluorescens or pseudomonas spp. although, these two subgroups were initially described as one subclade by loper et al., the separation between these two subgroups is supported by a branching above dissimilarity. subgroup iii includes p. fluorescens ncimb p. mandelii jr- and several unclassified pseudomonads, which were not analysed in previous studies. subgroup iv contains p. protegens pf- all the p. chlororaphis strains, p. fluorescens strains wayne and nz and one unclassified pseudomonad. this subgroup is congruent with subcluster. finally, subgroup v contains p. fluorescens strains sbw wh a ss nz and nz together with p. tolaasii strains, p. extremaustralis and four unclassified pseudomonads. this subgroup corresponds to subcluster. the genome sequence of strain f was then aligned with its closest fully sequenced relatives, p. brassiccacearum nfm and p. fluorescens pf0-. this alignment shows that f genome is much more similar to the nfm than to the pf0- genome, supporting the subclustering described above. it also shows that the major difference between the f and nfm genomes is a large inversion in the distal part of the genome, opposite to the replication origin. it is interesting to note that both strains were isolated from a similar ecosystem, the rhizosphere of a crop although in different hemispheres and that both strains share similarities such as the occurrence of phase variation during rhizosphere colonization, the presence of denitrification genes and the presence of three chemotaxis gene clusters. the presence of the f orthologous coding sequences in the genomes sequences from the p. fluorescens group was assessed using orthomcl. following this analysis, the core genome of the p. fluorescens group is composed of orthologous cdss present in all the fifty strains examined. unsurprisingly, the size of the core genome is smaller in comparison to previous analysis performed with and genome sequences. the core genome of the p. fluorescens subgroup i is composed of orthologous cds, which represents approximately 76% of the whole predicted proteome. in addition orthologous cdss found in the f genome are not present in any other genome of strains within this subgroup. these cdss were subjected to blastp analysis against the non-redundant ncbi database. closest relatives of these unique protein-coding genes are mostly found in other gamma-proteobacterial genomes, including other pseudomonas species, and could be involved in important functions relevant for rhizosphere fitness. the f genome encodes important traits involved in rhizocompetence a number of plant-associated bacteria, including pseudomonas spp. and azospirillum brasilense, are able to use nitrogen oxides as alternative electrons acceptors under oxygen limiting conditions, a process called denitrification. denitrification has been shown to play a role in rhizosphere colonization, since mutants of p. fluorescens impaired in nitrate or nitrite reductases are deficient in the colonization of the rhizosphere. furthermore, it has been shown that denitrification is statistically associated with rhizosphere competence in rhizosphere isolated fluorescent pseudomonads. the f genome encodes approximately proteins involved in denitrification. these genes are organized in four clusters on the chromosome. the first cluster contains the nark1k2ghji genes, which encode the membrane-bound nitrate reductase. the sensor-response regulator narl-narx and the transcriptional regulator dnrs are encoded upstream of the nar genes. the periplasmic and the assimilatory nitrate reductase are also present in the genome of f. the second cluster is composed of the nir and nor genes encoding nitrite and nitric oxide reductase, respectively. the third cluster contains the nos genes that encode the nitrous oxide reductase. all these genes showed highest homology with denitrification genes in different pseudomonads. finally the last cluster contains the nos and nor genes. although the nos genes showed homology with the nos genes, the nor genes are quite divergent to nor genes and showed higher homology with genes encoded in acidovorax and several enterobacteriaceae. it is interesting to note that the σ54-transcriptional activator norr is encoded in the downstream region of the nos2nor cluster, which suggests that nos1nor and nos2nor genes might be independently regulated. in order to test the functionality of these denitrification genes, the growth of f under anaerobic conditions was compared to the growth of pf0- which does not contain denitrification genes. while pf0- did not grow under these conditions, f was able to grow anaerobically using nitrate and nitrite as electron acceptors. furthermore, a total depletion of nitrite was observed after h of growth. these results together with the presence of two copies of the nos genes, strongly indicate that f is capable of full denitrification. the duplication of genes encoding nitric oxide reductase and nitrous oxide reductase might be related to high denitrification rates, as has been shown for a bacillus strain that contains two different copies of the nosz gene and showed high amounts of n production. the presence of denitrification genes was analyzed in the fifty strains forming the p. fluorescens group. while all strains of subgroup i contained a complete denitrification pathway, none of the strains of subgroup ii possessed denitrification genes, which further support the distinction between subgroup i and ii. interestingly some denitrification genes are present in few strains belonging to subgroups iii, iv and v, which might indicate horizontal gene transfer. whereas nor genes were only present f and nfm nos genes were systematically found in strains harbouring the nos genes. another metabolic adaption is the potential degradation of aromatic diterpenes encoded in the f genome. aromatic diterpenes are tricyclic resin acids naturally produced by trees and include abietic, dehydroabietic and palustric acids. bacteria that harbor the dit genes can grow in these substrates as the sole carbon and energy source. these genes have been found in several proteobacteria such as burkholderia, cupriavidus and pseudomonas. among pseudomonas, dit genes have been found in p. abietaniphila, p. diterpeniphila and in p. aeruginosa strain pa. f chromosome harbors a kb genetic island containing the dit genes. this region is contiguous to the nos2-nor cluster and has a gene organization similar to the genetic island found in p. aeruginosa pa although in this strain certain dit genes are duplicated and the genomic island is kb long. growth of f on abietic acid was compared with growth of pf0- after twenty days, growth was observed for f but not for pf0- suggesting that dit genes in f are functional. plant growth-promoting traits previous studies have shown that p. fluorescens f is capable of inorganic phosphate solubilisation through the extracellular production of gluconic acid. moreover, f seems capable of mineralizing insoluble organic phosphates pool by the production of specific enzymes, such as the homologues of the alkaline phosphatases phod and phox of p. fluorescens pf0- and a beta-propeller phytase. altogether, these traits could contribute to mobilization of insoluble soil phosphate into bioavailable forms that can be taken up by the plant root. acc deaminase activity in f has been reported previously and analysis of the genome sequence indicates that psf113_ encodes a 1-aminocyclopropane-carboxylate deaminase. acc deaminase catalyses the degradation of the ethylene precursor, acc, into ammonium and α-ketobutyrate and has been linked to plant growth promotion activity in rhizosphere microorganisms. the activity of the acds enzyme in f is μm mg- protein hr- almost twice the activity of enterobacter uw. the expression of acds is probably regulated by the lrp-like transcriptional regulator acdr, which is located upstream of acds. motility and chemotaxis like all pseudomonas strains, f genome encodes genes for the synthesis of polar flagella distributed in three clusters. several of these genes in f have been previously analyzed and regulation of flagella synthesis requires the master regulatory gene fleq. besides these genes, f chromosome harbors another kb flagella locus containing cdss, which is not present in any other pseudomonas genome, with the exception of p. extremaustralis. conversely to the operons encoding flagellar genes in pseudomonads, this region contains an flhdc operon, encoding master regulatory proteins of flagella synthesis in other gamma-proteobacteria such as enterobacteria and azotobacter. the orfs showed high homology to flagellar genes of azotobacter vinelandii, a soil bacterium phylogenetically related to the genus pseudomonas, but producing a different type of flagella, which are peritrichous, instead of the typical single or double polar flagella produced by pseudomonads. the region also showed synteny with the flagellar genes of a. vinelandii. the a. vinelandii chromosome harbors flagellar genes in two clusters, i and ii. genes in cluster i are conserved in the same order in the kb region in the f chromosome. cluster ii in a. vinelandii is located kb downstream of cluster i. a reduced version of this region and lacking orfs is located in an inverted orientation adjacent to cluster i in the f chromosome. all the genes present in a. vinelandii clusters, but absent in the f genome, encode chemotaxis proteins such as chez, chey, and cheb or proteins that are not essential for flagella synthesis, such as rfbc, rfbg and rfbf. ectopic expression of flhdc in f increased swimming motility and resulted in hyperflagellation with multiple polar flagella. these results indicate that f can produce the second type flagella, and that these flagella are functional. in rhodobacter sphaeroides, a second acquired flagellum is also functional and coexists with the endogenous flagellar system. in this case, as in f both flagella were polar. although flagella whose synthesis is regulated by the master regulatory genes flhdc are generally peritrichous, regulation of polar flagella by this system has also been observed in burkholderia glumae, indicating that flhdc regulation does not imply peritrichous flagellation. the f genome encodes five chemotaxis-like systems: wsp, chp, che che and che. the wsp system is implicated in regulation of motility and biofilm formation in f and other p. fluorescens strains. the chp system is located in the immediate vicinity of to pil genes and has been shown to control twitching motility in pseudomonas aeruginosa. besides, the f genome encodes three che systems. che is located in the vicinity of protein-coding genes involved in the assembly of the endogenous flagellum. mutation of the chea gene in f resulted in impaired motility and rhizosphere colonization indicating that this system participates in chemotactic motility. it is interesting to note that no genes encoding methyl accepting chemotactic sensors are genetically linked to this system, although more than thirty orfs predicted to encode mcp-like proteins are scattered in the f genome. the che and che systems are present in a restricted number of strains of the p. fluorescens group. the che genes are only present in all strains of subgroup i and three strains of the subgroup v. the che genes are present only in three strains of subgroup i: f nfm and wood both che and che systems harbor two genes putatively encoding mcps, suggesting that these systems might be implicated in specialized functions. this diversity of chemotaxis systems, as well as the production of a second type flagellum, highlights the importance of motility for f it is important to note that chemotaxis and motility mutants are among the most affected mutants in competitive rhizosphere colonization and that the rhizospheric environment selects for hypermotile phenotypic variants that are frequently hypercompetitive. it also raises interesting questions about which chemotaxis systems are used by the second type flagellum and about motility under different physiological condition, for example anaerobic growth using nitrate or nitrite as electron acceptors. protein and macromolecule secretion gram negative bacteria rely on several secretion systems to influence their environment by translocating protein and dna into neighbouring cells and the extracellular milieu. these secretion systems can range from simple transporters to multi-component complexes and have been classified into six types: from type i through type vi secretion system. the genome of f contains a wide variety of secretion systems, which include six t1sss, two t2sss, two t3sss, one t4ss, five t5asss, four t5bss, one t5dss and three t6ssss. t2sss t2ss is the most ubiquitous secretion system used by gram negative bacteria to secrete extracellular protein. therefore, t2ss is well-conserved and involves a set of to proteins. according to xcpr phylogenetic analysis, pseudomonas t2sss could be divided in two main clusters: xcp, and hpx. two complete t2sss related to the xcp cluster and to the hxc cluster are present in the genome of f. the genetic organization of the hxc cluster of f is highly similar with the hxc cluster of p. aeruginosa pao and p. fluorescens sbw. moreover, a predicted orf sharing 74% and 72% identity at the amino acid level with the hxc substrate psp of p. fluorescens sbw and ding of p. aeruginosa mdr is located immediately downstream of the hxc locus, which suggest that this protein could possibly be secreted by the hxc secretion system of f the second t2ss locus of f is identical to the second xcp of p. aeruginosa pa which is present on the genomic island rgp. based on homologies with known xcp effectors, it seems that at least four predicted f proteins could potentially be xcp substrates. t3sss t3ss is a nanomachine composed of approximately proteins, which has evolved into seven different families: ysc, hrp hrp spi- spi- rhizobiaceae and chamydia. the distribution of t3ss loci in complete or draft pseudomonas genome sequences was investigated using the following orfs pa pspto_ pspph_ and psf113_ as queries in sequential blastp and tblastn searches. four t3ss phylogenetic families, ysc, spi- hrp and rhizobia are encoded in the different pseudomonas chromosomes. while ysc is only found in p. aeruginosa, the other t3ss phylogenetic families are distributed across different pseudomonas groups. for example, strains belonging to the p. fluorescens group can contain spi- hrp or rhizobia t3ss. although the hrp locus is present in approximately 40% of the p. fluorescens genomes, two distinct sub-families are found in different chromosomal locations. the hrp locus encoded in the f chromosome is, perhaps unsurprisingly, closely related to hrp of p. fluorescens q8r1-, q2-, wood1r and p. brassicacearum nfm. the predicted t3es ropaa ropb and ropm reported by mavrodi et al. are also encoded in the genome of f q2- wood1r and nfm in addition a putative paralogue of ropaa called here ropaa, is found in all these strains. in comparison to hrp the spi- phylogenetic family is less abundant in the pseudomonas genome sequences. comparative genomics analysis of spi- clusters belonging to strains belonging to the p. fluorescens group, revealed that these loci are found in the same chromosomal location, flanked by the genes nast and pyrd. in f however, the spi- cluster is flanked by nast and soxr. this is due to genome rearrangement, probably caused by is3-family transposases. examination of other p. fluorescens genomes indicated that the genes invg, iagb and pseudogene related to hila are present at the same chromosomal location in strains pamc, ag, 14–, wh, sbw, r, a bg33r, ss, nz nz and pms interestingly all these strains belong to subgroup v of the p. fluorescens group, which suggest that the spi- cluster was lost before speciation of this sub-clade. while the spi- cluster is conserved across strains belonging to the sub-clade composed of upb, cb ncppb and ncppb 2289; its random distribution across the other strains of the p. fluorescens group could suggest an acquisition by horizontal gene transfer. investigation of the average gc content of the spi- clusters did, however, not show any shift in comparison to the average content of the genome examined. this is not the case with the spi- cluster of two strains belonging to the p. putida group, which have an atypical gc content, therefore suggesting horizontal gene transfer acquisition. t6sss the type vi secretion machinery is the product of approximately conserved genes which are generally found together inside a genomic locus. t6ss are widespread in proteobacteria, particularly among gamma-proteobacteria and have been classified into five distinct phylogenetic clusters. however a recent comparative genomic analysis has further divided the cluster into 4a and 4b. p. aeruginosa possesses three different loci named hsi-i, ii and iii, which perform different functions. whereas hsi-i is be involved in inter-bacterial interactions through secretion of tse tse and tse, hsi-ii and iii could be linked to virulence towards animals and plants. to date, strains belonging to the p. fluorescens group possess between one to four t6sss per genome, which belong to cluster to 4b. f contains the three most common t6ss phylogenetic clusters, which are related to the hsi loci. moreover, in this strain the two loci hsi-i and hsi-ii are located besides each other in a tandem arrangement, which is an uncommon feature of t6ss. in addition to the t6sss loci, most bacterial strains encode additional vgrg and hcp genes that encode extracellular structural components of the secretion apparatus, which are encoded elsewhere in the genome. from the eight vgrgs and three hcps encoded in the f genome, five vgrgs and one hcp proteins are located outside t6ss loci. other virulence and niche adaption traits different strains of p. fluorescens produce a set of secondary metabolites with antifungal and antibacterial properties. these metabolites are important for competition and survival in the rhizosphere and are the basis of biocontrol activities. f genome carries gene responsible for siderophores synthesis, including pyoverdine and pyridine- -bis-thiocarboxylic acid. moreover f produces antimicrobial compounds unrelated to siderophores such as hydrogen cyanide and dapg. besides the genes encoding the enzymes required for the biosynthesis of these compounds, the f genome contains a cluster of genes that is likely to encode genes for the synthesis of an unknown antibiotic. this kb cluster encodes four polyketide synthase modules, one dehydratase, one transacylase, one transferase, one isochorismatase and one transport protein. there are not homologous genes to these in the genus pseudomonas and the closest homologues are found in genes responsible of the synthesis of macrolide antibiotics from different streptomyces species, including the lankacidin production cluster of streptomyces rochei. the lankacidin gene cluster of s. rochei is composed of orfs, which encode a polyketide synthase /nonribosomal peptide synthetase hybrid gene, type i pks genes, and the essential cluster for the lankacidin production, pyrroloquinoline quinone biosynthetic genes. the presence of this cluster in other genomes was therefore investigated by comparative genomic analysis. besides streptomyces rochei plasmid psla2-l and p. fluorescens f the lkc cluster was only found in one additional genome: hahella chejuensis kctc in p. fluorescens f the pqq genes, which are crucial for lankacidin production in streptomyces rochei are found outside the lkc gene cluster. the possible production of this unknown antibiotic is likely to contribute to rhizosphere and soil competence. it has been shown that p. fluorescens strains can kill nematodes and are pathogenic to insects. preliminary results show that f is toxic towards the model nematode caenorhabditis elegans and the protozoan acanthamoeba polyphaga. f repels these bacteriovores and/or prevents their growth feeding on this strain. in addition, f kills the model insect galleria mellonella, after h of infection with a dose of bacteria per larvae. these features could be explained by the production of putative insecticidal toxins, which are also found in pseudomonas strains nfm and pf0- and in other bacterial species such as yersinia enterocolitica. in addition, a number of other putative toxins and virulence factors are present in p. fluorescens f genome including predicted hemolysin/haemagglutinins, nine adhesin or agglutination proteins, two rtx toxins, seven rhs-family proteins and eight yd-repeat-containing proteins. ice and prophage elements analysis of the f genome has revealed the presence of one putative integrative conjugative element of kb containing orfs located downstream a trna-glycine. this putative bacteriophage-derived mobile genetic element is surrounded by attf and attr sites and possesses protein-coding genes required for excision and integration. the mobilization of the ice could be mediated by a gi-t4ss, which is responsible for the formation of the conjugative pilus and the resulting conjugative transfer. however, it remains to be determined whether this gi-t4ss is functional since a conserved t4ss component is disrupted by a is transposase. in addition to protein-coding genes involved in the integration and transfer of this hypothetical ice, the cargo genes are related to t1ss dit cluster and nos2nor, described above. f also carry two prophage elements. prophage p is, like the prophages of other p. fluorescens strains, inserted between two highly conserved genes: muts and cina. prophage p closely resembles to prophage pp of p. fluorescens sbw and carries conserved tail synthesis genes of phage ctx but lacks integrase and head morphogenesis genes. therefore this genomic region probably encodes a bacteriocin belonging to the r-type pyocins. the second prophage of p. fluorescens f is related to prophage of p. fluorescens pf-. this genomic region contains protein-coding genes involved in tail assembly and head morphogenesis and therefore could correspond to the genome of a bacteriophage.  genome analysis of strain f has shown that this bacterium belongs to the pseudomonas fluorescens complex. however, as indicated earlier by silby et al. and more recently by loper et al., the taxonomy of p. fluorescens requires further study, since significant differences are found in the genomic complement of different strains. furthermore, the phylogenomic analysis has shown that the p. fluorescens group can be subdivided into at least, five subgroups. indeed, while f shared only 35% of its genome with all the other sequenced strains within the p. fluorescens group, this value increases to 76% when the f genome is compared to the four closest relatives. the fact that these strains have common unusual traits such as denitrification, phenotypic variation during rhizosphere colonization and highly conserved and unique chemotaxis systems, might indicate that these strains belong to the same species. in addition f genome contains genes that are not found in q2- q8r1- woodr and nfm which encode proteins likely to be involved in the production of an unknown polyketide and in the assembly of a second flagella. these traits could explain in part the excellent rhizocompetence ability of f and are currently under study. 
this includes genes encoding proteins involved in the production of a second flagellar apparatus, the use of abietic acid as a growth substrate, the complete denitrification pathway, the possible production of a macrolide antibiotic and the assembly of multiple protein secretion systems. pseudomonas fluorescens f is a plant growth-promoting rhizobacterium isolated from the sugar-beet rhizosphere. f belongs to subgroup i, which is mostly composed of strains classified as p. brassicacearum. comparative analysis of genome sequences of strains belonging to the p. fluorescens group has revealed the existence of five distinct subgroups. this bacterium has been extensively studied as a model strain for genetic regulation of secondary metabolite production in p. fluorescens, as a candidate biocontrol agent against phytopathogens, and as a heterologous host for expression of genes with biotechnological application. the genome of p. fluorescens f is composed of numerous protein-coding genes, not usually found together in previously sequenced genomes, which are potentially decisive during the colonisation of the rhizosphere and/or interaction with other soil organisms. despite this strong conservation, f also contains a large number of unique protein-coding genes that encode traits potentially involved in the rhizocompetence of this strain. these features include protein coding genes required for denitrification, diterpenoids catabolism, motility and chemotaxis, protein secretion and production of antimicrobial compounds and insect toxins. the core genome of these five strains is highly conserved and represents approximately 76% of the protein-coding genes in any given genome.
 pseudomonas fluorescens f is a plant growth-promoting rhizobacterium isolated from the sugar-beet rhizosphere. this bacterium has been extensively studied as a model strain for genetic regulation of secondary metabolite production in p. fluorescens, as a candidate biocontrol agent against phytopathogens, and as a heterologous host for expression of genes with biotechnological application. the f genome sequence and annotation has been recently reported.  comparative analysis of genome sequences of strains belonging to the p. fluorescens group has revealed the existence of five distinct subgroups. f belongs to subgroup i, which is mostly composed of strains classified as p. brassicacearum. the core genome of these five strains is highly conserved and represents approximately 76% of the protein-coding genes in any given genome. despite this strong conservation, f also contains a large number of unique protein-coding genes that encode traits potentially involved in the rhizocompetence of this strain. these features include protein coding genes required for denitrification, diterpenoids catabolism, motility and chemotaxis, protein secretion and production of antimicrobial compounds and insect toxins.  the genome of p. fluorescens f is composed of numerous protein-coding genes, not usually found together in previously sequenced genomes, which are potentially decisive during the colonisation of the rhizosphere and/or interaction with other soil organisms. this includes genes encoding proteins involved in the production of a second flagellar apparatus, the use of abietic acid as a growth substrate, the complete denitrification pathway, the possible production of a macrolide antibiotic and the assembly of multiple protein secretion systems. 