AdnA/FleQ is a master regulator for both flagellum-based motility and surface attachment in Pseudomonas spp. We have identified 103 genes in the AdnA regulon of P. fluorescens Pf0-1 by using a whole-genome microarray (see Table S1 in the supplemental material). About half of these genes are predicted orthologs of genes known to be involved in some aspect of motility. Of the remaining genes, 39 do not have orthologs in P. aeruginosa PAO1. The majority of these 39 genes are predicted to specify hypothetical proteins that do not have defined functional domains (see Table S1 in the supplemental material). By comparing the regulons of AdnA and FleQ in their respective organisms, one notes that while both are involved in regulation of motility genes, they are also involved in the regulation of a vastly different set of genes.
By investigating four AdnA-controlled genes not previously associated with motility in Pf0-1 (Pfl01_1508, Pfl01_1516, Pfl01_1517, and Pfl01_1572), we have identified new features of flagellum-dependent motility. This study and others (7
) have added to the list of genes that are involved in some aspect of flagellar synthesis and/or motility, highlighting the complexity of flagellar motility in Gram-negative bacteria. The ability of Pseudomonas
to swarm or swim can also affect its ability to form biofilms (40
; current study). This finding is not necessarily due to the known genes related to flagellar synthesis or transport (42
Two of the genes, Pfl01_1508 and Pfl01_1517, identified have protein sequence identity with glycosyl transferases. Orthologs of these protein products, found in P. syringae
and P. aeruginosa
, affect virulence, motility, and biofilm formation in P. syringae
) and virulence in P. aeruginosa
). P. syringae
possesses two flagellar glycosyl transferases, FgtA1 and FgtA2; deletion of either gene affects swarming motility in this strain (61
). Deletion of fgtA1
has the same effect on swimming motility in viscous media (60
) and decreased adhesion to polystyrene compared to the wild-type strain (61
). In P. aeruginosa
, FgtA is not involved in motility (2
) and its role in biofilm formation has not been reported. A phylogenetic tree of Pseudomonas
glycosyl transferases identified in GenBank was constructed (see Fig. S1 in the supplemental material). We have named Pfl01_1508 and Pfl01_1517 fgtA1
based on their protein sequence identity to FgtA1 (57%, Psyrps6_010100004493) and FgtA2 (50%, Psyrps6_010100004488) from P. syringae
642. Many other studies have been performed using different genera and have indicated that flagellar glycosylation is involved in the production and assembly of flagella, not just motility and virulence (reviewed in reference 32
The Pfl01_1508 (fgtA1
) deletion mutant is different from P. syringae
carrying its ortholog, in that it has motility defects on semisolid medium (0.3% agar). fgtA1
mutants also show biofilm defects, which may result from an inability to interact with borosilicate glass (hydrophilic) or PVC (hydrophobic) because of the altered properties of flagella that are not glycosylated appropriately. In P. syringae
6605, an fgtA1
mutant has defects in binding to another hydrophobic surface, polystyrene (61
In contrast to the mutants lacking the second putative glycosyl transferase, the Pfl01_1517 (fgtA2
) mutant produces wild-type biofilm on PVC and has increased biofilm formation on borosilicate glass when grown at 30°C (). A deletion of either fgtA1
in Pf0-1 results in the loss of swimming motility, unlike deletion of their counterparts in P. syringae
, where only a mutant with disruption of fgtA1
has that phenotype in viscous medium, 0.6% agar or higher (60
). Motility on agar surfaces with agar concentrations of 0.6% or higher is usually associated with swarming motility (22
). Further investigation is required to determine whether indeed these two proteins have glycosyl transferase functions and which residues of FliC they glycosylate.
Pfl01_1516 was annotated as a cephalosporin hydroxylase but clearly has an important role in motility and biofilm formation and was therefore named flhH. Deletion of flhH resulted in greatly reduced motility ( and ), even though the strain still produced flagella that are visually indistinguishable from those of Pf0-1 (data not shown). Biofilm formation on both borosilicate glass and PVC is also impaired in this mutant ().
Colonies of the Pfl01_1573 deletion mutant have a fried egg phenotype on motility plates (A). TEM showed that this deletion mutant produced flagella which were detached from the P. fluorescens cells (data not shown). While the loss of some flagella may be due to sample preparation-facilitated detachment, the complete lack of attached flagella was not seen with wild-type Pf0-1 or any other mutants prepared under similar conditions. Since the Pfl01_1573 mutant was still motile (albeit motility was reduced), the function is not in attachment of the flagella per se but in the tightness of the flagellar attachment. Clearly, flagella are made and are functional (A and data not shown); however, the inability to maintain attachment leads to reduced motility.
By complementing each of the motility mutants with its respective gene, the wild-type Pf0-1 colony phenotype was restored (B). However, the motility of each of these strains exhibited on soft agar was only 35 to 90% of wild-type levels (). The one exception is the Pfl01_1573 deletion mutant, which required a wild-type Pfl01_1572 gene, as described below. Biofilm formation and chemotaxis were also restored in these mutants as well (). The restoration of chemotaxis in the mutants ranged from 53 to 92% of that of the wild type.
In order to complement the motility defect of the Pfl01_1573 deletion mutant, a wild-type copy of the Pfl01_1572 gene was needed. During replacement of the wild-type Pfl01_1573 gene by a gentamicin-resistance cassette, both Pfl01_1572 and Pfl01_1573 could be affected since they are part of the same transcript (A). RT-PCR results showed that the Pfl01_1572 transcript was present in the Pfl01_1573 deletion mutant (B). The presence of the transcript verifies that the Pfl01_1572 mRNA transcript is stable (B); however, translation of the Pfl01_1572 protein may still be the limiting factor.
To help determine if the phenotype that we see in the Pfl01_1573 deletion mutant is linked to a disruption of translation of Pfl01_1572 or a deletion of Pfl01_1573, we complemented the strain with wild-type Pfl01_1572. Motility was restored in the Pfl01_1573 deletion mutant (to 55% of that of wild type using pMQ71B) bearing Pfl01_1572 (A). To further investigate these findings, a Pfl01_1572 deletion mutant was created. Removal of Pfl01_1572 completely abolished motility, unlike what was seen in the Pfl01_1573 deletion mutant ( and A). Biofilm experiments were also carried out using the Pfl01_1572 deletion mutant. The results from this assay showed similar trends in both the Pfl01_1572 and Pfl01_1573 deletion mutants (). Chemotaxis results showed a drastic reduction in chemotaxis in the Pfl01_1573 mutant and a lack of chemotaxis in the Pfl01_1572 mutant ( and data not shown). We performed RT-PCR on the Pfl01_1572 deletion mutant in order to determine if Pfl01_1573 was transcribed downstream of the gentamicin-resistance cassette (B). The results showed that Pfl01_1573 is transcribed in this mutant.
The complementation results indicate that Pfl01_1572 is responsible for the motility defects seen in the Pfl01_1573 mutant. Pfl01_1572 specifies a putative CheW protein, and the ortholog in P. aeruginosa
PAO1 was shown to be involved in chemotaxis (25
). Kato et al. (25
) also noted no motility defects in the cheW
mutant on soft agar, but the effects on biofilm formation were not examined. To our knowledge, this is the first report of a CheW mutant affecting biofilm formation and completely abolishing swimming motility.
Both Pfl01_1572 and Pfl01_1573 deletion mutants exhibited reduced biofilm development compared to Pf0-1 over a 48-h period on both borosilicate glass (A) and PVC (B). Flagella are important for attachment by a number of organisms (reviewed in reference 66
). The TEMs show a Pfl01_1573 mutant with detached flagella, which, in conjunction with other results, stems from the lack of a functional Pfl01_1572.
Recently, a protein, FlgT, which was identified to be an anchoring protein for flagella, was found in Vibrio cholerae
mutations result in detached flagella. FlgT was found to be localized in the periplasm, which could indicate an interaction with either the P or L rings, which are part of the flagellar basal body (34
). The mutant with the deletion of Pfl01_1573 in Pf0-1 had a similar phenotype, with the detachment of flagella, but no protein sequence identity between FlgT and Pfl01_1572 or Pfl01_1573 was seen (data not shown). This phenotype may also be related to the loss of a functional Pfl01_1572 in the Pfl01_1573 mutant.
To date our laboratories have identified only four genes important for colonization/survival in soil either under natural field conditions, adnA
), or in soil competition experiments in a laboratory setting, cosA
) and both ppk
). So far, the only gene to increase soil colonization of wild-type Pf0-1 has been cosA
). Under the current laboratory conditions, the adnA
mutant does not exhibit the same soil colonization defects (53
) seen under natural field conditions (33
). These differences in results show that there is a greater level of complexity in the natural field environment that cannot be duplicated in the laboratory. Thus, our results using the current soil methods (see Fig. S2 in the supplemental material) may not completely reflect the entire story that is happening in the natural soil environment. Further experiments are needed in order to determine each of these gene's roles under natural conditions.
The identification of novel genes involved in both motility and biofilm formation and the differences seen between organisms in the same genus and even species indicate that we do not fully understand motility in the Pseudomonas genus. Further investigation in other Pseudomonas species may help to better define the functional basis for these differences.