BACKGROUND
pseudomonas fluorescens is a diverse species of bacteria that is found throughout natural habitats and associated with plants.
contributing to their diverse lifestyles is their ability to produce an equally diverse array of secondary metabolites that affect interactions with hosts and other inhabitants of their ecosystems.
some isolates benefit plants by producing growth promoting hormones or antimicrobial compounds to control against pathogens  <cit> .
others are deleterious and have the capacity to synthesize and secrete novel compounds that negatively affect growth of plants  <cit> .
the physiological diversity of p. fluorescens is mirrored by its tremendous genetic diversity.
however, the genetic diversity may reflect the possibility that p. fluorescens is not a single species, but rather a complex of at least two lineages.
molecular phylogenetic studies of  <dig> isolates suggested p. fluorescens should be represented by the p. chlororaphis and p. fluorescens lineages  <cit> .
alternatively or additionally, p. fluorescens may have an open pan genome  <cit> .
finished genome sequences are available for the sbw <dig>  pf- <dig>  and pf0- <dig> isolates of p. fluorescens  <cit> .
their genomes exceed  <dig>  megabases and their relatively large sizes are not unexpected for free-living bacteria  <cit> .
comparative analyses of the three isolates of p. fluorescens revealed substantial variation in diversity of genome content and heterogeneity in genome organization  <cit> .
each genome has  <dig>  to nearly  <dig>  unique genes when compared to each other.
plant-associated isolates of p. fluorescens potentially have mechanisms for interacting with plants.
many gram-negative bacteria use a type iii secretion system  to interact with their hosts  <cit> .
the t3ss is the most complex of the bacterial secretion systems and is typically encoded by a large cluster of genes arranged as a single superoperon.
its function is to inject type iii effector proteins directly into host cells  <cit> .
these type iii effectors are important host-range determinants of plant pathogenic bacteria because they perturb and potentially elicit plant defenses  <cit> .
it is unclear as to how prevalent t3ss-encoding regions are in p. fluorescens.
nearly 60% of a surveyed collection of p. fluorescens strains had a homolog of rscn, which encodes the atpase of the t3ss  <cit> .
however, it is not known whether all genes necessary to complete the t3ss are present in these isolates.
of the three completed genomes, genes encoding the t3ss are present only in sbw <dig>  several important or typically conserved genes are missing or truncated in the t3ss-encoding locus of sbw <dig>  <cit> .
despite the cryptic appearance of the t3ss, when constitutively expressing the transcriptional regulator of the t3ss, sbw <dig> could deliver a heterologous type iii effector into plant cells, suggesting the t3ss may still be functional  <cit> .
the role of the t3ss for the lifestyle of p. fluorescens is still unclear.
in sbw <dig>  despite the cryptic t3ss, single mutants of some but not all the remaining t3ss-encoding genes were reduced in fitness in the rhizosphere of sugar beets  <cit> .
this is not unheard of, as mutants of seemingly cryptic t3ss in pathogens are compromised in virulence  <cit> .
however, in the case of sbw <dig>  the t3ss mutants were also compromised in growth in vitro  <cit> .
a t3ss mutant of the biocontrol isolate p. fluorescens kd was compromised in its ability to protect cucumbers against damping-off disease caused by pythium ultimum  <cit> .
this may be a result of kd requiring a functional t3ss to elicit host defenses, thereby indirectly protecting against p. ultimum or potentially as a direct mechanism against the pathogen.
we are interested in exploiting p. fluorescens for control of grassy weeds.
we have previously reported the selection, isolation, and characterization of five strains of p. fluorescens that inhibit germination of seeds of grassy weeds  <cit> .
further characterizations led to the identification of germination-arrest factor  produced by these isolates.
gaf is a small, extremely hydrophilic secreted herbicide that reacts with ninhydrin and possesses an acid group, suggestive of a small peptide or amino acid analog  <cit> .
in particular, the high specificity of gaf towards grasses and inhibitory activity at only certain developmental stages during seed germination provides promise for its potential as a natural herbicide for the control of grassy weeds in grass seed production and turf management settings.
we selected p. fluorescens wh <dig> as the archetypal gaf-producing isolate.
wh <dig> was extracted from the rhizosphere of poa sp.
and triticum aestivum at the hyslop research farm in benton county, oregon, usa  <cit> .
we sequenced and developed an improved high-quality draft sequence for wh <dig> using a hybrid illumina and 454-based sequencing approach.
this standard is considered sufficient for our purposes of assessing gene inventory and comparing genome organization  <cit> .
comparative genomic analysis showed a high number of orthologous genes and strong similarity in genome organization between wh <dig> and sbw <dig>  phylogenomic analysis supported this observation and placed wh <dig> in the same lineage as sbw <dig>  or the proposed p. fluorescens lineage.
the high similarity in orthology and genome organization is in contrast to previous observations of p. fluorescens and in comparisons of wh <dig> to pf- <dig> or pf0- <dig>  <cit> .
from a non-saturating tn5-mutagenesis screen of wh <dig>  we previously identified two mutants compromised in gaf activity .
mapping of dna sequences flanking the two mutants revealed genes encoding proteins with potential functions in regulation and biosynthesis of gaf.
finally, inspection of the wh <dig> genome revealed several candidate host-association mechanisms, including what appears to be a complete type iii and two complete type vi secretion systems.
RESULTS
sequencing and developing an improved, high-quality draft genome sequence
we used an illumina and a  <dig> flx gs lr <dig> to sequence the genome of wh <dig> .
the theoretical coverage using all filtered reads was estimated to be 316× assuming a genome size of approximately  <dig>  megabases.
we employed a number of steps to meet the standards of an improved, high-quality draft genome sequence of wh <dig> for comparative purposes.
we used velvet  <dig> .
<dig>  combinations of short-reads, and a variety of parameter settings to de novo assemble the short reads to generate approximately  <dig> different assemblies  <cit> .
we developed and used ad hoc perl scripts with an associated visualization tool to compare each of the different assemblies to each other.
this step enabled us to eliminate entire assemblies with large contigs not supported by any other assembly.
*based on approximated genome size of  <dig>  mb; ^highest confident draft assemblies using velvet  <dig> .
<dig>  <cit> ; ^improved, high quality draft including sanger reads.
n/a = not applicable; §based on sum total of all contigs >  <dig> bp in length.
we identified a single high-quality de novo assembly based on nearly  <dig> million reads from all three sequencing methods .
the velvet parameters were hash length of  <dig>  expected coverage of  <dig>  and a coverage cutoff of  <dig>  actual coverage of this assembly based on the total number of used reads was  <dig> ~ 120×, which was less than one-third the theoretical coverage.
this assembly had  <dig> contigs greater than one kb and a total of  <dig> contigs greater than  <dig> bp.
the largest contig was  <dig> kb and the n <dig> number and size were  <dig> contigs and  <dig> kb, respectively.
we used experimental and in silico approaches to improve the draft assembly by reducing the number of physical gaps.
of the  <dig> contigs greater than one kb,  <dig> contigs  had significant homology to a reference sequence shared by the end of another contig.
these  <dig> contigs potentially flanked  <dig> physical gaps .
we were able to amplify across  <dig>  of the gaps using pcr.
physical gaps were subsequently resolved by reassembling the nearly  <dig> million short-reads with the  <dig> sanger reads.
of the remaining scaffolds, we associated more based on in silico evidence.
some contigs shared long-range synteny to a reference genome  and their ends had fifteen or more basepairs of sequence with 100% overlap to each other.
this phenomenon is a result of velvet failing to extend the contig because of low coverage.
secondly, some contigs could be paired together because their ends had partial coding regions with homology to a common reference gene.
in total, nineteen more contigs were associated, resulting in a final assembly of  <dig> scaffolds greater than  <dig> bp.
the largest scaffold was  <dig> kb and the n <dig> number and size were  <dig> and  <dig> kb, respectively.
the improved, high-quality draft genome sequence had  <dig> sequence gaps totaling  <dig>  ns.
there were  <dig> large sequence gaps with more than  <dig> ns of which eight had more than  <dig>  ns each.
we presumed these were artifacts of the velvet assembly because the fragment size of our paired-end library was no larger than  <dig> bp.
we corrected the sizes for  <dig> gaps to their corresponding length found in homologous reference sequences.
in the other  <dig> cases, we simply reduced the number of ns in the region to  <dig> bp, to reflect the maximum size of our paired-end library.
both approaches to correct the size of sequence gaps were validated using pcr of randomly selected regions .
in total, we reduced the number of ns to  <dig>  or ~2% of the original number of ns.
the release of the finished genome sequence of sbw <dig> fortuitously coincided with our efforts of improving the draft genome sequence of wh <dig>  <cit> .
we noted that nearly 90% of the homologous sequences we found in the ncbi nt dataset using our blastn-based approaches were to p. fluorescens sbw <dig>  we therefore surmised that the genome of wh <dig> would be similar to the finished genome of p. fluorescens sbw <dig> and used it as a reference for mauve aligner to reorder the  <dig> wh <dig> scaffolds  <cit> .
the genome of wh <dig> is presumed to be a single circular chromosome .
a total of  <dig> scaffolds greater than one kb could be ordered using mauve aligner.
the remaining  <dig> contigs could not be reordered and were excluded from our circular representation of the genome.
these  <dig> contigs were all smaller than one kb and their sum total was only  <dig> kb.
attempts to use pf0- <dig> or pf- <dig> as a reference for mauve aligner were largely unsuccessful, supporting our observation that wh <dig> and sbw <dig> had higher synteny than previously detected in p. fluorescens and suggesting our wh <dig> de novo assembly was of high quality.
we found no evidence of plasmids in the genome of wh <dig>
this whole genome shotgun project has been deposited at ddbj/embl/genbank under the accession aeaz <dig>  the version described in this paper is the first version, aeaz <dig>  the wh <dig> genome sequence and its associated tools can also be accessed from our website at: http://changbugs.cgrb.oregonstate.edu/microbes/org_detail.html?org=wh6-g <dig>
one challenge with de novo assembly is dealing with repeated sequences  <cit> .
small repeated sequences are present in genomes of p. fluorescens but were not expected to have a large effect on our ability to assemble the wh <dig> genome because of the size of our paired-end fragments  <cit> .
larger repeats, however, could not be resolved.
we only observed one rrna operon in the genome of wh <dig>  we suspect that wh <dig> has five rrna operons similar to sbw <dig> and pf- <dig>  but they collapsed into one contig.
there was approximately 5× more coverage for the contig containing the one rrna operon of wh <dig> compared to the other contigs.
similarly, nonribosomal peptide synthases  are encoded by large genes with repeated modules  <cit> .
the modular domains either collapsed on one another in the assembly, or were assembled into short contigs that we could not extend.
a large fraction of these partial nrps-encoding genes were found in the small contigs that we could not reorder using mauve aligner.
here too, we noticed higher coverage than the other scaffolds.
comparative and phylogenomic analyses of p. fluorescens
at a large scale, the genome of wh <dig> was similar to the genomes of the other p. fluorescens isolates .
the size of the genome is slightly smaller, which may be a consequence of the draft nature of our genome assembly.
nonetheless, the  <dig> predicted coding sequences  and  <dig> % coding capacity were very similar.
*improved, high quality draft genome sequence ^ <cit> ; § <cit> .
previous analyses of p. fluorescens found sbw <dig>  pf- <dig>  and pf0- <dig> to be divergent, with only ~61% of the genes shared and little long-range synteny  <cit> .
we used hal to carry out similar analyses to determine the effect of the wh <dig> genome on the phylogenetic relationship of the p. fluorescens species and potential changes to the size of its pan genome  <cit> .
hal uses a markov clustering algorithm based on e-values from reciprocal all-by-all blastp analysis to create clusters of orthologs.
core sequences from each species are concatenated and the super alignment is used in phylogenomic analysis.
using a core of  <dig> translated sequences common to p. fluorescens, representative strains of p. syringae, and p. aeruginosa pao <dig>  hal clustered the different species of pseudomonas as expected  <cit> .
further, hal clearly defined two separate lineages for p. fluorescens, placing wh <dig> with sbw <dig> .
within the p. fluorescens species as presently defined,  <dig> genes formed the core and represented 53%,  <dig> %,  <dig> %, and  <dig> % of the genomes of wh <dig>  sbw <dig>  pf- <dig>  and pf0- <dig>  respectively .
this was nearly a 10% reduction relative to previous analysis of three genomes  <cit> .
a large fraction of the core genes was assigned to categories with general cellular processes such as energy production and conversion, amino acid transport and metabolism, translation, and transcription .
approximately 90% of the  <dig> core genes clustered with orthologs sharing identical cog designations suggesting our automated annotation pipeline was accurate.
there were some exceptions but their rarity and subtle differences did not warrant manual curation.
for example, one cluster of orthologs had genes annotated as "arabinose efflux permeases"  for genes from the published isolates of p. fluorescens but "permease of the major facilitator superfamily"  for the ortholog of wh <dig>
a total of  <dig> of the translated products of wh <dig> had an orthologous sequence in another isolate of p. fluorescens.
almost 69% of the wh <dig> genes had an orthologous sequence in sbw <dig>  as compared to pf- <dig> and pf0- <dig> with 62% and 59%, respectively .
we found similar levels of overlap using reciprocal blastp .
the 69% orthology between wh <dig> and sbw <dig> is much higher than previously observed between isolates of p. fluorescens  <cit> .
these levels were still lower than those between different pathovars of p. syringae, which had greater than 80% orthology  <cit> .
therefore, the generalization that p. fluorescens have highly variable genomes still holds true.
the genomes of wh <dig> and sbw <dig> also showed extensive long-range synteny .
this amount of synteny was unexpected given previous comparisons  <cit> .
when compared to pf- <dig> or pf0- <dig>  we found little long-range synteny, which tended to be near the origin of replication.
synteny rapidly degraded away from the origin with an increase in inversions between the genomes  <cit> .
taken together these lines of evidence all suggest wh <dig> and sbw <dig> to be similar and support, though perhaps prematurely, a redefinition of the p. fluorescens species  <cit> .
it could be argued that the high level of synteny we found with sbw <dig> was an artifact of using sbw <dig> to reorder the wh <dig> scaffolds.
though we cannot exclude this possibility, we highlight several points that suggest otherwise.
we used a de novo approach to assemble the genome of wh <dig>  the long-range synteny to the sbw <dig> genome was observed within each and across the de novo assembled scaffolds of wh <dig> .
furthermore, synteny with sbw <dig> was also supported by our ability to use sbw <dig> to successfully and substantially reduce the number of wh <dig> scaffolds and improve the wh <dig> genome sequence .
finally, analysis of gc skew gave higher confidence in the reordering of wh <dig> scaffolds .
genomes often have a bias of guanine in the leading strand  <cit> .
inversions of gc skew in regions distant from the replication origins and termini are indicative of a recent recombination event  <cit> .
barring these events, inversions of gc skew could also potentially indicate large-scale misassemblies or incorrect reordering of contigs.
for the most part, the genome of wh <dig> showed the expected bias of guanine in the leading strand; there are perhaps two small inversions in gc skew flanked by physical gaps between scaffolds near the terminator.
our use of sbw <dig> as a reference for reordering scaffolds is therefore acceptable and the observed synteny between wh <dig> and sbw <dig> appeared to reflect true similarities in genome organization.
more than 30% of the wh <dig> coding regions were unique .
examinations of their annotated functions suggested greater diversity in metabolic and host-association functions such as carbohydrate transport and metabolism, inorganic ion transport and metabolism, secondary metabolite biosynthesis, transport and catabolism, intracellular trafficking, secretion and vesicular transport, as well as defense mechanisms .
examples of cdss unique to wh <dig> and enriched in these functional categories include  <dig> candidate permeases of the major facilitator superfamily, a large and diverse superfamily of secondary active transporters that control movement of substrates across membranes .
wh <dig> also had  <dig> unique cdss that encode for putative tonb-dependent receptors, involved in uptake of iron and potentially other substrates .
restriction modification  systems are widespread defense mechanisms that protect prokaryotes from attack by foreign dna  <cit> .
rm systems are diverse and can vary dramatically in numbers.
wh <dig> has at least  <dig> cdss with annotated functions or domains common to proteins of rm systems.
pfwh6_5037- <dig>  for example, encode for a type i rm system that appears to be unique to wh <dig>  finally, other cdss unique to wh <dig> and of direct interest to us are described in the following sections.
the greater than  <dig> genes unique to wh <dig> were dispersed throughout its genome with only a slight bias in location closer to the terminators .
this bias was previously generalized for p. fluorescens  <cit> .
mapping gaf mutants
we previously identified two wh <dig> mutants from a non-saturating tn5-mutagenesis screen for those affected in arresting the germination of poa seeds  <cit> .
we cloned, sequenced and mapped their flanking sequences to identify the disrupted genes.
mutant wh6-3::tn <dig> had an insertion in pfwh6_ <dig>  this cds is annotated as a "predicted transmembrane transcriptional regulator ".
its closest homolog, with 94% similarity is prtr encoded by p. fluorescens ls107d <dig>  <cit> .
the tn <dig> element had inserted at nucleotide position  <dig> within codon asp <dig>  because loss of prtr led to a loss of gaf activity, prtr is likely an activator rather than a repressor, as was the case in p. fluorescens ls107d <dig>  <cit> .
just upstream of prtr in wh <dig> is prti, which encodes a candidate ecf σ <dig> factor.
this arrangement is reminiscent of many sigma-anti-sigma factor pairs and suggests that the genes are potentially co-regulated and both may have roles in regulating gaf gene expression  <cit> .
it is peculiar that we failed to identify an insertion in prti but one obvious explanation is that our screen was not saturating.
regardless, it will be important to examine the necessity of prti for gaf activity to resolve its role.
mutant wh6-2::tn <dig> had an insertion in pfwh6_ <dig>  a gene encoding a candidate aminotransferase class iii.
the identification of an aminotransferase as necessary for gaf supports our previous findings suggesting that gaf contains an amino group and may be a small peptide or amino acid analog  <cit> .
aminotransferases are pyridoxal phosphate -dependent enzymes that catalyze the transfer of an amino group from a donor group  to an acceptor molecule  <cit> .
the tn <dig> element had inserted at nucleotide position  <dig> within codon lys <dig>  based on comparisons to the acetyl ornithine aminotransferase family the insertion is distal to the conserved residues that compose pyridoxal 5'-phosphate binding sites, the conserved residues that compose inhibitor-cofactor binding pockets, and the catalytic residue  <cit> .
further characterization of wh6-2::tn <dig> is necessary to examine its enzymatic properties and role in biosynthesis of gaf.
regulators of gene expression
bacteria with large genomes tend to have complex regulatory networks to integrate and respond to a multitude of environmental signals.
the extracytoplasmic function  σ <dig> factors are a class of important transcriptional regulators of cell-surface signaling systems.
using a hidden markov model  for ecf-encoding genes, we found  <dig>   <dig>   <dig>  and  <dig> ecfs in wh <dig>  sbw <dig>  pf- <dig> and pf0- <dig>  respectively  <cit> .
of the  <dig> identified in wh <dig>  ten are part of the core set common to all four sequenced p. fluorescens isolates and included prti and prtr, which we identified as necessary for gaf activity.
because we had previously shown that pf- <dig> and pf0- <dig> do not have gaf activity, these results suggest that the putative prti/r-regulon may be different between the different isolates of p. fluorescens  <cit> .
four of the  <dig> ecf-encoding genes were exclusive to the plant-associated strains wh <dig>  sbw <dig>  or pf- <dig>  two of these were only shared with sbw <dig>  of which one was rspl .
the other two lacked sufficient annotations to speculate on their functions.
the remaining five ecfs were unique to wh <dig> and all are potentially co-expressed with genes encoding outer membrane receptors involved in iron perception or uptake .
virulence factors
pseudomonads produce a wide-range of secondary metabolites with potential benefit or detriment to plants and microbes  <cit> .
many are synthesized by non-ribosomal peptide synthases  or polyketide synthases  <cit> .
we found evidence for several nrps-encoding genes.
because of their modular architecture, most nrps-encoding genes of wh <dig> were fragmented and found on small contigs that failed to assemble or reorder.
therefore, it was not possible to determine the structure of the repeats or infer functions based on homology.
we were, however, able to identify several other candidate toxins and virulence factors .
*table derived from  <cit> ; candidates were identified using blastp .
there are  <dig> wh <dig> proteins with homology to candidate tat-secreted proteins of p. syringae pv tomato dc <dig>  <cit> ; ^no orthologs were detected in genomes of other p. fluorescens.
we identified several secretion systems in wh <dig> unique to host-associated bacteria and/or necessary for full virulence of pathogenic bacteria.
wh <dig> appears to encode a complete and functional type iii secretion system .
we named its genes according to the nomenclature first proposed for sbw <dig>  <cit> .
there is strong homology and synteny between the t3ss-encoding regions of wh <dig> and p. syringae, raising the possibility of a recent acquisition of the t3ss-encoding locus by wh <dig>  similar to kd  <cit> .
phylogenetic analyses of rscn, however, placed wh <dig> with the group  <dig> of biocontrol isolates of p. fluorescens .
additionally,  <dig> kb of sequences on either side of the t3ss-encoding region of wh <dig> were highly syntenic to regions flanking the t3ss-encoding region of sbw <dig> with the exception of the type iii effector gene, rope.
together, these data argue against a recent acquisition of the t3ss-encoding region by wh <dig>
there were some differences between t3ss-encoding regions of wh <dig> and p. syringae.
the rspr, rspz, and rspv genes of wh <dig> were not present and we failed to detect any homology between the rspf/hrpf, rspa/hrpa, and rspg/hrpg genes.
data, however suggest these differences likely have little to no effect on t3ss function.
hrpr and hrps are highly similar and are functionally redundant.
in some erwinia strains, hrps by itself is demonstrably sufficient for t3ss function  <cit> .
deletion mutants of hrpz are still functional and hrpv functions as a negative regulator of the t3ss  <cit> .
hrpf and hrpa are homologous to each other and are structural components of the t3ss.
they are the most polymorphic proteins encoded within the t3ss-cluster and the absence of significant homology between rspf and rspa to their counterparts of p. syringae was therefore not surprising  <cit> .
our automated annotation approach failed to identify rspg but upon visual inspection, we noted a small cds that encodes a potential product of  <dig> amino acids.
blast searches failed to detect homology to hrpg, but given its position in the t3ss-encoding region and similarity in size to the translated product of hrpg, we have annotated it as rspg.
in total, these data support the notion that wh <dig> encodes a complete and functional t3ss, although, its role in the lifestyle of wh <dig> remains unknown.
candidate type iii effectors of wh6
we used a homology-based approach to search for type iii effector genes in the genome of wh <dig>  our database of type iii effectors included those from t3ss-using phytopathogens and some mammal pathogens.
we only identified one translated sequence with homology to pipb from salmonella, and another with homology to hopi <dig> from p. syringae .
however, neither appeared to be strong candidates for a type iii effector.
we identified a homolog of pipb in the genome of pf- <dig>  which does not encode the t3ss.
hopi <dig> encodes a j domain and its homolog in wh <dig> was annotated as the molecular chaperone, dnaj  <cit> .
these results suggest that if wh <dig> does encode type iii effectors, they are very divergent in sequence.
sbw <dig>  in contrast, had at least five genes with homology to known type iii effectors, of which two were expressed to sufficiently high levels and delivered by a heterologous t3ss-encoding bacterium  <cit> .
computational approaches have been successfully used to identify candidate type iii effectors from p. syringae, based in part on identifying a cis-regulatory element upstream of their genes and also some genes of the t3ss  <cit> .
this so-called hrp-box is recognized by hrpl, an extracytoplasmic function  σ <dig> factor encoded within the t3ss-encoding region of p. syringae  <cit> .
we therefore used a hidden markov model  trained using  <dig> known hrpl-regulated genes of p. syringae pv tomato dc <dig> to mine the genome of wh <dig> for hrp-boxes  <cit> .
we found  <dig> hrp-boxes in the genome of wh <dig>  but only  <dig> were within  <dig> bp of a cds.
two were located upstream of rspf and rscr in the t3ss-encoding region, with bit scores of  <dig>  and  <dig> , respectively.
we also identified a hrp-box upstream of rspj but it had a lower bit score of  <dig> .
fifteen of the cdss downstream of candidate hrp-boxes had annotated functions not typically associated with type iii effectors and we did not list them as possible candidates .
the remaining eight cdss downstream of hrp-boxes were annotated as hypothetical proteins and the five with the highest bit scores for their corresponding hrp-boxes were not present in the genomes of pf- <dig> and pf0-1; all but pfwh6_ <dig> were unique to wh <dig> .
further investigation of their first amino-terminal residues indicated that three have characteristics suggestive of t3ss-dependent secretion  <cit> .
only coding sequences with no predicted homology or identifiable orthologs in pf- <dig> or pf0- <dig> are shown.
*pfwh6_# unless otherwise noted; ^n-terminal translocation signal scores were assigned based on "a"; >10% serine in first  <dig> amino acids, "b"; absence of aliphatic amino acids in position  <dig> or  <dig>  and/or "c"; absence of negatively-charged amino acids in the first  <dig> amino acids  <cit> .
our two computational approaches yielded very few candidate type iii effectors.
one possible explanation is that because rspl and hrpl have only 50% identity , they recognize slightly different cis-regulatory sequences and our hmm was not adequately trained for the cis-regulatory sequence recognized by rspl.
this is an unlikely scenario.
three sequences with strong similarity to the hrp-box of p. syringae were found in the t3ss-encoding regions for wh <dig> and sbw <dig>  <cit> .
additionally, it has been observed that all hrpl-dependent phytopathogenic bacteria share an identical motif in the hrp-box despite having as little as 52% similarity  <cit> .
furthermore, in σ <dig> factors, dna binding specificity is conferred by the helix-turn-helix domain  <dig>   <cit> .
domain  <dig>  of the wh <dig> rspl is highly similar  to the corresponding domain of hrpl.
an alternative explanation is that wh <dig> encodes very few type iii effectors with little homology to those that have been identified.
this is not unheard of.
p. aeruginosa for example, has only three type iii effectors  <cit> .
type vi secretion systems
the type vi secretion system  is another secretion apparatus that is common to host-associated bacteria.
computational approaches suggest the t6ss may also be in p. fluorescens  <cit> .
we found evidence for two complete and functional t6sss in wh <dig>  we have named these two systems t6ss- <dig>  and t6ss- <dig> .
it is not uncommon for organisms to possess multiple t6sss that are of different lineages and acquired independently  <cit> .
additionally, in other strains that have been characterized, different t6sss appear to be independently regulated, suggesting each t6ss may have functions specific to different aspects of the lifestyle of the bacterium  <cit> .
whether this is also the case with wh <dig> awaits further characterization.
t6ss- <dig> belongs to the group a lineage and shares homology and synteny to hsi-i of p. aeruginosa pao <dig>  <cit> .
we therefore named the corresponding genes in wh <dig> according to the nomenclature established for hsi-i .
synteny extended beyond the t6ss-encoding region and included the tagqrst genes bordering ppka.
we did not, however, find evidence for tagj <dig> in wh <dig>  <cit> .
t6ss- <dig> is a group b secretion system  <cit> .
less is understood about the group b secretion systems but t6ss- <dig> showed strong homology and synteny to a corresponding t6ss encoded in the genome of the phytopathogen p. syringae pv tomato dc <dig> .
there are few proteins that are demonstrable type vi effectors.
three homologs of vgrg and hcp have been shown to be secreted by the t6ss but both likely have functions for the t6ss itself  <cit> .
we found four vgrg genes, of which only one was associated with t6ss- <dig>  the other three genes were found elsewhere in the genome.
whether products from these latter three are secreted proteins of the t6ss or merely homologous in sequence is unknown.
both t6sss of wh <dig> had a homolog of hcp.
recently, three additional proteins from p. aeruginosa pao <dig> were shown to be secreted by the t6ss, but their orthologs were not found in wh <dig>  <cit> .
CONCLUSIONS
p. fluorescens is a genetically and physiologically diverse species found in many habitats.
we sequenced the genome of the isolate wh <dig> because it produces germination-arrest factor , an herbicide that specifically arrests seed germination of graminaceous species.
comparisons of the wh <dig> genome to genomes of sbw <dig>  pf- <dig>  and pf0- <dig> helped better define this species, with wh <dig> and sbw <dig> forming one lineage.
comparative studies revealing substantial similarity in gene inventory and synteny supported its placement and the argument of at least two major lineages of p. fluorescens  <cit> .
with the genome sequence, we were able to deduce potential functions for two genes necessary for gaf activity.
one encoded a candidate anti-sigma factor.
our previous results suggest that prtr is an activator and suggests it has a role in regulating expression of genes necessary for gaf.
the second gene encoded a candidate aminotransferase, which tentatively supports our previous speculation that gaf is a small peptide or amino acid analog.
further studies are required to confirm their functions.
a less labor-intensive and saturating screen will also be necessary for a fuller understanding of the pathway controlling gaf expression and biosynthesis.
the genome sequence will certainly facilitate such future endeavors.
we also identified a number of mechanisms that potentially affect plant health and some typically associated with host-associated bacteria.
one of the more extensively characterized mechanisms is the type iii secretion system.
wh <dig> appears to encode the necessary repertoire of genes for a complete and functional t3ss.
we also identified two t6sss in wh <dig>  further studies are necessary to identify the role these secretion systems and their effectors play in the lifestyle of wh <dig>

 pseudomonas fluorescens is a diverse species of bacteria that is found throughout natural habitats and associated with plants. contributing to their diverse lifestyles is their ability to produce an equally diverse array of secondary metabolites that affect interactions with hosts and other inhabitants of their ecosystems. some isolates benefit plants by producing growth promoting hormones or antimicrobial compounds to control against pathogens. others are deleterious and have the capacity to synthesize and secrete novel compounds that negatively affect growth of plants. the physiological diversity of p. fluorescens is mirrored by its tremendous genetic diversity. however, the genetic diversity may reflect the possibility that p. fluorescens is not a single species, but rather a complex of at least two lineages. molecular phylogenetic studies of isolates suggested p. fluorescens should be represented by the p. chlororaphis and p. fluorescens lineages. alternatively or additionally, p. fluorescens may have an open pan genome. finished genome sequences are available for the sbw pf- and pf0- isolates of p. fluorescens. their genomes exceed megabases and their relatively large sizes are not unexpected for free-living bacteria. comparative analyses of the three isolates of p. fluorescens revealed substantial variation in diversity of genome content and heterogeneity in genome organization. each genome has to nearly unique genes when compared to each other. plant-associated isolates of p. fluorescens potentially have mechanisms for interacting with plants. many gram-negative bacteria use a type iii secretion system to interact with their hosts. the t3ss is the most complex of the bacterial secretion systems and is typically encoded by a large cluster of genes arranged as a single superoperon. its function is to inject type iii effector proteins directly into host cells. these type iii effectors are important host-range determinants of plant pathogenic bacteria because they perturb and potentially elicit plant defenses. it is unclear as to how prevalent t3ss-encoding regions are in p. fluorescens. nearly 60% of a surveyed collection of p. fluorescens strains had a homolog of rscn, which encodes the atpase of the t3ss. however, it is not known whether all genes necessary to complete the t3ss are present in these isolates. of the three completed genomes, genes encoding the t3ss are present only in sbw several important or typically conserved genes are missing or truncated in the t3ss-encoding locus of sbw. despite the cryptic appearance of the t3ss, when constitutively expressing the transcriptional regulator of the t3ss, sbw could deliver a heterologous type iii effector into plant cells, suggesting the t3ss may still be functional. the role of the t3ss for the lifestyle of p. fluorescens is still unclear. in sbw despite the cryptic t3ss, single mutants of some but not all the remaining t3ss-encoding genes were reduced in fitness in the rhizosphere of sugar beets. this is not unheard of, as mutants of seemingly cryptic t3ss in pathogens are compromised in virulence. however, in the case of sbw the t3ss mutants were also compromised in growth in vitro. a t3ss mutant of the biocontrol isolate p. fluorescens kd was compromised in its ability to protect cucumbers against damping-off disease caused by pythium ultimum. this may be a result of kd requiring a functional t3ss to elicit host defenses, thereby indirectly protecting against p. ultimum or potentially as a direct mechanism against the pathogen. we are interested in exploiting p. fluorescens for control of grassy weeds. we have previously reported the selection, isolation, and characterization of five strains of p. fluorescens that inhibit germination of seeds of grassy weeds. further characterizations led to the identification of germination-arrest factor produced by these isolates. gaf is a small, extremely hydrophilic secreted herbicide that reacts with ninhydrin and possesses an acid group, suggestive of a small peptide or amino acid analog. in particular, the high specificity of gaf towards grasses and inhibitory activity at only certain developmental stages during seed germination provides promise for its potential as a natural herbicide for the control of grassy weeds in grass seed production and turf management settings. we selected p. fluorescens wh as the archetypal gaf-producing isolate. wh was extracted from the rhizosphere of poa sp. and triticum aestivum at the hyslop research farm in benton county, oregon, usa. we sequenced and developed an improved high-quality draft sequence for wh using a hybrid illumina and 454-based sequencing approach. this standard is considered sufficient for our purposes of assessing gene inventory and comparing genome organization. comparative genomic analysis showed a high number of orthologous genes and strong similarity in genome organization between wh and sbw phylogenomic analysis supported this observation and placed wh in the same lineage as sbw or the proposed p. fluorescens lineage. the high similarity in orthology and genome organization is in contrast to previous observations of p. fluorescens and in comparisons of wh to pf- or pf0-. from a non-saturating tn5-mutagenesis screen of wh we previously identified two mutants compromised in gaf activity. mapping of dna sequences flanking the two mutants revealed genes encoding proteins with potential functions in regulation and biosynthesis of gaf. finally, inspection of the wh genome revealed several candidate host-association mechanisms, including what appears to be a complete type iii and two complete type vi secretion systems.  sequencing and developing an improved, high-quality draft genome sequence we used an illumina and a flx gs lr to sequence the genome of wh. the theoretical coverage using all filtered reads was estimated to be 316× assuming a genome size of approximately megabases. we employed a number of steps to meet the standards of an improved, high-quality draft genome sequence of wh for comparative purposes. we used velvet. combinations of short-reads, and a variety of parameter settings to de novo assemble the short reads to generate approximately different assemblies. we developed and used ad hoc perl scripts with an associated visualization tool to compare each of the different assemblies to each other. this step enabled us to eliminate entire assemblies with large contigs not supported by any other assembly. *based on approximated genome size of mb; ^highest confident draft assemblies using velvet.; ^improved, high quality draft including sanger reads. n/a = not applicable; §based on sum total of all contigs > bp in length. we identified a single high-quality de novo assembly based on nearly million reads from all three sequencing methods. the velvet parameters were hash length of expected coverage of and a coverage cutoff of actual coverage of this assembly based on the total number of used reads was ~ 120×, which was less than one-third the theoretical coverage. this assembly had contigs greater than one kb and a total of contigs greater than bp. the largest contig was kb and the n number and size were contigs and kb, respectively. we used experimental and in silico approaches to improve the draft assembly by reducing the number of physical gaps. of the contigs greater than one kb, contigs had significant homology to a reference sequence shared by the end of another contig. these contigs potentially flanked physical gaps. we were able to amplify across of the gaps using pcr. physical gaps were subsequently resolved by reassembling the nearly million short-reads with the sanger reads. of the remaining scaffolds, we associated more based on in silico evidence. some contigs shared long-range synteny to a reference genome and their ends had fifteen or more basepairs of sequence with 100% overlap to each other. this phenomenon is a result of velvet failing to extend the contig because of low coverage. secondly, some contigs could be paired together because their ends had partial coding regions with homology to a common reference gene. in total, nineteen more contigs were associated, resulting in a final assembly of scaffolds greater than bp. the largest scaffold was kb and the n number and size were and kb, respectively. the improved, high-quality draft genome sequence had sequence gaps totaling ns. there were large sequence gaps with more than ns of which eight had more than ns each. we presumed these were artifacts of the velvet assembly because the fragment size of our paired-end library was no larger than bp. we corrected the sizes for gaps to their corresponding length found in homologous reference sequences. in the other cases, we simply reduced the number of ns in the region to bp, to reflect the maximum size of our paired-end library. both approaches to correct the size of sequence gaps were validated using pcr of randomly selected regions. in total, we reduced the number of ns to or ~2% of the original number of ns. the release of the finished genome sequence of sbw fortuitously coincided with our efforts of improving the draft genome sequence of wh. we noted that nearly 90% of the homologous sequences we found in the ncbi nt dataset using our blastn-based approaches were to p. fluorescens sbw we therefore surmised that the genome of wh would be similar to the finished genome of p. fluorescens sbw and used it as a reference for mauve aligner to reorder the wh scaffolds. the genome of wh is presumed to be a single circular chromosome. a total of scaffolds greater than one kb could be ordered using mauve aligner. the remaining contigs could not be reordered and were excluded from our circular representation of the genome. these contigs were all smaller than one kb and their sum total was only kb. attempts to use pf0- or pf- as a reference for mauve aligner were largely unsuccessful, supporting our observation that wh and sbw had higher synteny than previously detected in p. fluorescens and suggesting our wh de novo assembly was of high quality. we found no evidence of plasmids in the genome of wh this whole genome shotgun project has been deposited at ddbj/embl/genbank under the accession aeaz the version described in this paper is the first version, aeaz the wh genome sequence and its associated tools can also be accessed from our website at: http://changbugs.cgrb.oregonstate.edu/microbes/org_detail.html?org=wh6-g one challenge with de novo assembly is dealing with repeated sequences. small repeated sequences are present in genomes of p. fluorescens but were not expected to have a large effect on our ability to assemble the wh genome because of the size of our paired-end fragments. larger repeats, however, could not be resolved. we only observed one rrna operon in the genome of wh we suspect that wh has five rrna operons similar to sbw and pf- but they collapsed into one contig. there was approximately 5× more coverage for the contig containing the one rrna operon of wh compared to the other contigs. similarly, nonribosomal peptide synthases are encoded by large genes with repeated modules. the modular domains either collapsed on one another in the assembly, or were assembled into short contigs that we could not extend. a large fraction of these partial nrps-encoding genes were found in the small contigs that we could not reorder using mauve aligner. here too, we noticed higher coverage than the other scaffolds. comparative and phylogenomic analyses of p. fluorescens at a large scale, the genome of wh was similar to the genomes of the other p. fluorescens isolates. the size of the genome is slightly smaller, which may be a consequence of the draft nature of our genome assembly. nonetheless, the predicted coding sequences and % coding capacity were very similar. *improved, high quality draft genome sequence ^; §. previous analyses of p. fluorescens found sbw pf- and pf0- to be divergent, with only ~61% of the genes shared and little long-range synteny. we used hal to carry out similar analyses to determine the effect of the wh genome on the phylogenetic relationship of the p. fluorescens species and potential changes to the size of its pan genome. hal uses a markov clustering algorithm based on e-values from reciprocal all-by-all blastp analysis to create clusters of orthologs. core sequences from each species are concatenated and the super alignment is used in phylogenomic analysis. using a core of translated sequences common to p. fluorescens, representative strains of p. syringae, and p. aeruginosa pao hal clustered the different species of pseudomonas as expected. further, hal clearly defined two separate lineages for p. fluorescens, placing wh with sbw. within the p. fluorescens species as presently defined, genes formed the core and represented 53%, %, %, and % of the genomes of wh sbw pf- and pf0- respectively. this was nearly a 10% reduction relative to previous analysis of three genomes. a large fraction of the core genes was assigned to categories with general cellular processes such as energy production and conversion, amino acid transport and metabolism, translation, and transcription. approximately 90% of the core genes clustered with orthologs sharing identical cog designations suggesting our automated annotation pipeline was accurate. there were some exceptions but their rarity and subtle differences did not warrant manual curation. for example, one cluster of orthologs had genes annotated as "arabinose efflux permeases" for genes from the published isolates of p. fluorescens but "permease of the major facilitator superfamily" for the ortholog of wh a total of of the translated products of wh had an orthologous sequence in another isolate of p. fluorescens. almost 69% of the wh genes had an orthologous sequence in sbw as compared to pf- and pf0- with 62% and 59%, respectively. we found similar levels of overlap using reciprocal blastp. the 69% orthology between wh and sbw is much higher than previously observed between isolates of p. fluorescens. these levels were still lower than those between different pathovars of p. syringae, which had greater than 80% orthology. therefore, the generalization that p. fluorescens have highly variable genomes still holds true. the genomes of wh and sbw also showed extensive long-range synteny. this amount of synteny was unexpected given previous comparisons. when compared to pf- or pf0- we found little long-range synteny, which tended to be near the origin of replication. synteny rapidly degraded away from the origin with an increase in inversions between the genomes. taken together these lines of evidence all suggest wh and sbw to be similar and support, though perhaps prematurely, a redefinition of the p. fluorescens species. it could be argued that the high level of synteny we found with sbw was an artifact of using sbw to reorder the wh scaffolds. though we cannot exclude this possibility, we highlight several points that suggest otherwise. we used a de novo approach to assemble the genome of wh the long-range synteny to the sbw genome was observed within each and across the de novo assembled scaffolds of wh. furthermore, synteny with sbw was also supported by our ability to use sbw to successfully and substantially reduce the number of wh scaffolds and improve the wh genome sequence. finally, analysis of gc skew gave higher confidence in the reordering of wh scaffolds. genomes often have a bias of guanine in the leading strand. inversions of gc skew in regions distant from the replication origins and termini are indicative of a recent recombination event. barring these events, inversions of gc skew could also potentially indicate large-scale misassemblies or incorrect reordering of contigs. for the most part, the genome of wh showed the expected bias of guanine in the leading strand; there are perhaps two small inversions in gc skew flanked by physical gaps between scaffolds near the terminator. our use of sbw as a reference for reordering scaffolds is therefore acceptable and the observed synteny between wh and sbw appeared to reflect true similarities in genome organization. more than 30% of the wh coding regions were unique. examinations of their annotated functions suggested greater diversity in metabolic and host-association functions such as carbohydrate transport and metabolism, inorganic ion transport and metabolism, secondary metabolite biosynthesis, transport and catabolism, intracellular trafficking, secretion and vesicular transport, as well as defense mechanisms. examples of cdss unique to wh and enriched in these functional categories include candidate permeases of the major facilitator superfamily, a large and diverse superfamily of secondary active transporters that control movement of substrates across membranes. wh also had unique cdss that encode for putative tonb-dependent receptors, involved in uptake of iron and potentially other substrates. restriction modification systems are widespread defense mechanisms that protect prokaryotes from attack by foreign dna. rm systems are diverse and can vary dramatically in numbers. wh has at least cdss with annotated functions or domains common to proteins of rm systems. pfwh6_5037- for example, encode for a type i rm system that appears to be unique to wh finally, other cdss unique to wh and of direct interest to us are described in the following sections. the greater than genes unique to wh were dispersed throughout its genome with only a slight bias in location closer to the terminators. this bias was previously generalized for p. fluorescens. mapping gaf mutants we previously identified two wh mutants from a non-saturating tn5-mutagenesis screen for those affected in arresting the germination of poa seeds. we cloned, sequenced and mapped their flanking sequences to identify the disrupted genes. mutant wh6-3::tn had an insertion in pfwh6_ this cds is annotated as a "predicted transmembrane transcriptional regulator ". its closest homolog, with 94% similarity is prtr encoded by p. fluorescens ls107d. the tn element had inserted at nucleotide position within codon asp because loss of prtr led to a loss of gaf activity, prtr is likely an activator rather than a repressor, as was the case in p. fluorescens ls107d. just upstream of prtr in wh is prti, which encodes a candidate ecf σ factor. this arrangement is reminiscent of many sigma-anti-sigma factor pairs and suggests that the genes are potentially co-regulated and both may have roles in regulating gaf gene expression. it is peculiar that we failed to identify an insertion in prti but one obvious explanation is that our screen was not saturating. regardless, it will be important to examine the necessity of prti for gaf activity to resolve its role. mutant wh6-2::tn had an insertion in pfwh6_ a gene encoding a candidate aminotransferase class iii. the identification of an aminotransferase as necessary for gaf supports our previous findings suggesting that gaf contains an amino group and may be a small peptide or amino acid analog. aminotransferases are pyridoxal phosphate -dependent enzymes that catalyze the transfer of an amino group from a donor group to an acceptor molecule. the tn element had inserted at nucleotide position within codon lys based on comparisons to the acetyl ornithine aminotransferase family the insertion is distal to the conserved residues that compose pyridoxal 5'-phosphate binding sites, the conserved residues that compose inhibitor-cofactor binding pockets, and the catalytic residue. further characterization of wh6-2::tn is necessary to examine its enzymatic properties and role in biosynthesis of gaf. regulators of gene expression bacteria with large genomes tend to have complex regulatory networks to integrate and respond to a multitude of environmental signals. the extracytoplasmic function σ factors are a class of important transcriptional regulators of cell-surface signaling systems. using a hidden markov model for ecf-encoding genes, we found and ecfs in wh sbw pf- and pf0- respectively. of the identified in wh ten are part of the core set common to all four sequenced p. fluorescens isolates and included prti and prtr, which we identified as necessary for gaf activity. because we had previously shown that pf- and pf0- do not have gaf activity, these results suggest that the putative prti/r-regulon may be different between the different isolates of p. fluorescens. four of the ecf-encoding genes were exclusive to the plant-associated strains wh sbw or pf- two of these were only shared with sbw of which one was rspl. the other two lacked sufficient annotations to speculate on their functions. the remaining five ecfs were unique to wh and all are potentially co-expressed with genes encoding outer membrane receptors involved in iron perception or uptake. virulence factors pseudomonads produce a wide-range of secondary metabolites with potential benefit or detriment to plants and microbes. many are synthesized by non-ribosomal peptide synthases or polyketide synthases. we found evidence for several nrps-encoding genes. because of their modular architecture, most nrps-encoding genes of wh were fragmented and found on small contigs that failed to assemble or reorder. therefore, it was not possible to determine the structure of the repeats or infer functions based on homology. we were, however, able to identify several other candidate toxins and virulence factors. *table derived from; candidates were identified using blastp. there are wh proteins with homology to candidate tat-secreted proteins of p. syringae pv tomato dc; ^no orthologs were detected in genomes of other p. fluorescens. we identified several secretion systems in wh unique to host-associated bacteria and/or necessary for full virulence of pathogenic bacteria. wh appears to encode a complete and functional type iii secretion system. we named its genes according to the nomenclature first proposed for sbw. there is strong homology and synteny between the t3ss-encoding regions of wh and p. syringae, raising the possibility of a recent acquisition of the t3ss-encoding locus by wh similar to kd. phylogenetic analyses of rscn, however, placed wh with the group of biocontrol isolates of p. fluorescens. additionally, kb of sequences on either side of the t3ss-encoding region of wh were highly syntenic to regions flanking the t3ss-encoding region of sbw with the exception of the type iii effector gene, rope. together, these data argue against a recent acquisition of the t3ss-encoding region by wh there were some differences between t3ss-encoding regions of wh and p. syringae. the rspr, rspz, and rspv genes of wh were not present and we failed to detect any homology between the rspf/hrpf, rspa/hrpa, and rspg/hrpg genes. data, however suggest these differences likely have little to no effect on t3ss function. hrpr and hrps are highly similar and are functionally redundant. in some erwinia strains, hrps by itself is demonstrably sufficient for t3ss function. deletion mutants of hrpz are still functional and hrpv functions as a negative regulator of the t3ss. hrpf and hrpa are homologous to each other and are structural components of the t3ss. they are the most polymorphic proteins encoded within the t3ss-cluster and the absence of significant homology between rspf and rspa to their counterparts of p. syringae was therefore not surprising. our automated annotation approach failed to identify rspg but upon visual inspection, we noted a small cds that encodes a potential product of amino acids. blast searches failed to detect homology to hrpg, but given its position in the t3ss-encoding region and similarity in size to the translated product of hrpg, we have annotated it as rspg. in total, these data support the notion that wh encodes a complete and functional t3ss, although, its role in the lifestyle of wh remains unknown. candidate type iii effectors of wh6 we used a homology-based approach to search for type iii effector genes in the genome of wh our database of type iii effectors included those from t3ss-using phytopathogens and some mammal pathogens. we only identified one translated sequence with homology to pipb from salmonella, and another with homology to hopi from p. syringae. however, neither appeared to be strong candidates for a type iii effector. we identified a homolog of pipb in the genome of pf- which does not encode the t3ss. hopi encodes a j domain and its homolog in wh was annotated as the molecular chaperone, dnaj. these results suggest that if wh does encode type iii effectors, they are very divergent in sequence. sbw in contrast, had at least five genes with homology to known type iii effectors, of which two were expressed to sufficiently high levels and delivered by a heterologous t3ss-encoding bacterium. computational approaches have been successfully used to identify candidate type iii effectors from p. syringae, based in part on identifying a cis-regulatory element upstream of their genes and also some genes of the t3ss. this so-called hrp-box is recognized by hrpl, an extracytoplasmic function σ factor encoded within the t3ss-encoding region of p. syringae. we therefore used a hidden markov model trained using known hrpl-regulated genes of p. syringae pv tomato dc to mine the genome of wh for hrp-boxes. we found hrp-boxes in the genome of wh but only were within bp of a cds. two were located upstream of rspf and rscr in the t3ss-encoding region, with bit scores of and, respectively. we also identified a hrp-box upstream of rspj but it had a lower bit score of. fifteen of the cdss downstream of candidate hrp-boxes had annotated functions not typically associated with type iii effectors and we did not list them as possible candidates. the remaining eight cdss downstream of hrp-boxes were annotated as hypothetical proteins and the five with the highest bit scores for their corresponding hrp-boxes were not present in the genomes of pf- and pf0-1; all but pfwh6_ were unique to wh. further investigation of their first amino-terminal residues indicated that three have characteristics suggestive of t3ss-dependent secretion. only coding sequences with no predicted homology or identifiable orthologs in pf- or pf0- are shown. *pfwh6_# unless otherwise noted; ^n-terminal translocation signal scores were assigned based on "a"; >10% serine in first amino acids, "b"; absence of aliphatic amino acids in position or and/or "c"; absence of negatively-charged amino acids in the first amino acids. our two computational approaches yielded very few candidate type iii effectors. one possible explanation is that because rspl and hrpl have only 50% identity, they recognize slightly different cis-regulatory sequences and our hmm was not adequately trained for the cis-regulatory sequence recognized by rspl. this is an unlikely scenario. three sequences with strong similarity to the hrp-box of p. syringae were found in the t3ss-encoding regions for wh and sbw. additionally, it has been observed that all hrpl-dependent phytopathogenic bacteria share an identical motif in the hrp-box despite having as little as 52% similarity. furthermore, in σ factors, dna binding specificity is conferred by the helix-turn-helix domain. domain of the wh rspl is highly similar to the corresponding domain of hrpl. an alternative explanation is that wh encodes very few type iii effectors with little homology to those that have been identified. this is not unheard of. p. aeruginosa for example, has only three type iii effectors. type vi secretion systems the type vi secretion system is another secretion apparatus that is common to host-associated bacteria. computational approaches suggest the t6ss may also be in p. fluorescens. we found evidence for two complete and functional t6sss in wh we have named these two systems t6ss- and t6ss-. it is not uncommon for organisms to possess multiple t6sss that are of different lineages and acquired independently. additionally, in other strains that have been characterized, different t6sss appear to be independently regulated, suggesting each t6ss may have functions specific to different aspects of the lifestyle of the bacterium. whether this is also the case with wh awaits further characterization. t6ss- belongs to the group a lineage and shares homology and synteny to hsi-i of p. aeruginosa pao. we therefore named the corresponding genes in wh according to the nomenclature established for hsi-i. synteny extended beyond the t6ss-encoding region and included the tagqrst genes bordering ppka. we did not, however, find evidence for tagj in wh. t6ss- is a group b secretion system. less is understood about the group b secretion systems but t6ss- showed strong homology and synteny to a corresponding t6ss encoded in the genome of the phytopathogen p. syringae pv tomato dc. there are few proteins that are demonstrable type vi effectors. three homologs of vgrg and hcp have been shown to be secreted by the t6ss but both likely have functions for the t6ss itself. we found four vgrg genes, of which only one was associated with t6ss- the other three genes were found elsewhere in the genome. whether products from these latter three are secreted proteins of the t6ss or merely homologous in sequence is unknown. both t6sss of wh had a homolog of hcp. recently, three additional proteins from p. aeruginosa pao were shown to be secreted by the t6ss, but their orthologs were not found in wh.  p. fluorescens is a genetically and physiologically diverse species found in many habitats. we sequenced the genome of the isolate wh because it produces germination-arrest factor, an herbicide that specifically arrests seed germination of graminaceous species. comparisons of the wh genome to genomes of sbw pf- and pf0- helped better define this species, with wh and sbw forming one lineage. comparative studies revealing substantial similarity in gene inventory and synteny supported its placement and the argument of at least two major lineages of p. fluorescens. with the genome sequence, we were able to deduce potential functions for two genes necessary for gaf activity. one encoded a candidate anti-sigma factor. our previous results suggest that prtr is an activator and suggests it has a role in regulating expression of genes necessary for gaf. the second gene encoded a candidate aminotransferase, which tentatively supports our previous speculation that gaf is a small peptide or amino acid analog. further studies are required to confirm their functions. a less labor-intensive and saturating screen will also be necessary for a fuller understanding of the pathway controlling gaf expression and biosynthesis. the genome sequence will certainly facilitate such future endeavors. we also identified a number of mechanisms that potentially affect plant health and some typically associated with host-associated bacteria. one of the more extensively characterized mechanisms is the type iii secretion system. wh appears to encode the necessary repertoire of genes for a complete and functional t3ss. we also identified two t6sss in wh further studies are necessary to identify the role these secretion systems and their effectors play in the lifestyle of wh 
the improved high-quality draft genome sequence of wh contributes towards resolving the p. fluorescens species, providing additional impetus for establishing two separate lineages in p. fluorescens. p. fluorescens isolate wh produces germination-arrest factor, a predicted small peptide or amino acid analog with herbicidal activity that specifically inhibits germination of seeds of graminaceous species. from this assembly of megabases, we predicted genes, of which were core to p. fluorescens and were unique to wh comparative genomic studies of wh revealed high similarity in synteny and orthology of genes with p. fluorescens sbw a phylogenomic study also placed wh in the same lineage as sbw in a previous non-saturating mutagenesis screen we identified two genes necessary for gaf activity in wh mapping of their flanking sequences revealed genes that encode a candidate anti-sigma factor and an aminotransferase. pseudomonas fluorescens is a genetically and physiologically diverse species of bacteria present in many habitats and in association with plants. despite the high levels of orthology and synteny to sbw wh still had a substantial number of unique genes and represents another source for the discovery of genes with implications in affecting plant growth and health. we used a hybrid next-generation sequencing approach to develop a high-quality draft genome sequence for p. fluorescens wh we employed automated, manual, and experimental methods to further improve the draft genome sequence. finally, wh is the first isolate of p. fluorescens reported to encode a complete t3ss. this species of bacteria produces a large array of secondary metabolites with potential as natural products. two genes are demonstrably necessary for gaf and further characterization of their proteins is important for developing natural products as control measure against grassy weeds.
 pseudomonas fluorescens is a genetically and physiologically diverse species of bacteria present in many habitats and in association with plants. this species of bacteria produces a large array of secondary metabolites with potential as natural products. p. fluorescens isolate wh produces germination-arrest factor, a predicted small peptide or amino acid analog with herbicidal activity that specifically inhibits germination of seeds of graminaceous species.  we used a hybrid next-generation sequencing approach to develop a high-quality draft genome sequence for p. fluorescens wh we employed automated, manual, and experimental methods to further improve the draft genome sequence. from this assembly of megabases, we predicted genes, of which were core to p. fluorescens and were unique to wh comparative genomic studies of wh revealed high similarity in synteny and orthology of genes with p. fluorescens sbw a phylogenomic study also placed wh in the same lineage as sbw in a previous non-saturating mutagenesis screen we identified two genes necessary for gaf activity in wh mapping of their flanking sequences revealed genes that encode a candidate anti-sigma factor and an aminotransferase. finally, we discovered several candidate virulence and host-association mechanisms, one of which appears to be a complete type iii secretion system.  the improved high-quality draft genome sequence of wh contributes towards resolving the p. fluorescens species, providing additional impetus for establishing two separate lineages in p. fluorescens. despite the high levels of orthology and synteny to sbw wh still had a substantial number of unique genes and represents another source for the discovery of genes with implications in affecting plant growth and health. two genes are demonstrably necessary for gaf and further characterization of their proteins is important for developing natural products as control measure against grassy weeds. finally, wh is the first isolate of p. fluorescens reported to encode a complete t3ss. this gives us the opportunity to explore the role of what has traditionally been thought of as a virulence mechanism for non-pathogenic interactions with plants. 