Enzymatically Active and Inactive Phosphodiesterases and Diguanylate Cyclases Are Involved in Regulation of Motility or Sessility in Escherichia coli CFT073

ABSTRACT Intracellular concentration of cyclic diguanylate monophosphate (c-di-GMP), a second messenger molecule, is regulated in bacteria by diguanylate cyclases (DGCs) (synthesizing c-di-GMP) and phosphodiesterases (PDEs) (degrading c-di-GMP). c-di-GMP concentration ([c-di-GMP]) affects motility and sessility in a reciprocal fashion; high [c-di-GMP] typically inhibits motility and promotes sessility. A c-di-GMP sensor domain, PilZ, also regulates motility and sessility. Uropathogenic Escherichia coli regulates these processes during infection; motility is necessary for ascending the urinary tract, while sessility is essential for colonization of anatomical sites. Here, we constructed and screened 32 mutants containing deletions of genes encoding each PDE (n = 11), DGC (n = 13), PilZ (n = 2), and both PDE and DGC (n = 6) domains for defects in motility, biofilm formation, and adherence for the prototypical pyelonephritis isolate E. coli CFT073. Three of 32 mutations affected motility, all of which were in genes encoding enzymatically inactive PDEs. Four PDEs, eight DGCs, four PDE/DGCs, and one PilZ regulated biofilm formation in a medium-specific manner. Adherence to bladder epithelial cells was regulated by [c-di-GMP]. Four PDEs, one DGC, and three PDE/DGCs repress adherence and four DGCs and one PDE/DGC stimulate adherence. Thus, specific effectors of [c-di-GMP] and catalytically inactive DGCs and PDEs regulate adherence and motility in uropathogenic E. coli.

Here, we conducted comprehensive screens to identify effects on motility, biofilm formation, adherence, curli synthesis, and cellulose production by construction of individual deletions of each DGC, PDE, and PilZ domain-encoding gene in uropathogenic E. coli CFT073. The effect of chemically complementing the deletion mutants with demonstrable phenotypes was assessed by expression of exogenous PDE or DGC in each mutant background. Thus, the phenotypes conferred by both enzymatically active and inactive proteins were determined.
Both ⌬yhdA and ⌬ycgF were tested for their effect on motility in the Fim L-ON background to determine if the motility phenotypes of these mutants were affected by type 1 fimbrial expression. As observed previously, the Fim L-ON construct has reduced motility, with an average diameter of 10.0 mm, compared to the wild-type parental strain (average diameter of 42.4 mm). Both mutants further decreased motility in the Fim L-ON background  Since deletion of ydiV (average diameter of 18.5 mm) partially restores motility in E. coli CFT073 Fim L-ON, mutation of yhdA and ycgF was assessed in a ⌬ydiV Fim L-ON background to determine if either the low-or high-motility phenotype was dominant. Deletion of yhdA significantly decreased motility in the ⌬ydiV Fim L-ON background (3.7 mm; P ϭ 0.0013) but compared to ⌬yhdA Fim L-ON, ⌬yhdA ⌬ydiV Fim L-ON was significantly more motile (P ϭ 0.034), suggesting that ⌬yhdA and ⌬ydiV likely act through different regulatory pathways and thus have an additive effect. When placed in the ⌬ydiV Fim L-ON background, ⌬ycgF no longer affected motility; thus, the ydiV deletion is dominant over ycgF.
Motility phenotypes correlate with flagellin production. Since deletion of yhdA, ycgF, and ydiV affected the swimming motility of E. coli CFT073, we estimated the production of flagellin by Western blotting to determine whether the effects on motility were due to a change in the amount of flagellin synthesized. Consistent with their reduced-motility phenotypes, both the ⌬yhdA and ⌬ycgF mutants yielded reduced levels of flagellin protein compared to the wild-type strain (Fig. 1C), while the ⌬ydiV mutant demonstrated increased FliC expression (as previously shown [13]), commensurate with its increased motility.
Exogenous phosphodiesterase or diguanylate cyclase activity and motility in mutants. While c-di-GMP affects motility in many bacterial species (5,6), in E. coli CFT073 all three deletions affecting motility were of degenerate PDEs. Therefore, we endeavored to determine whether E. coli CFT073 produces this cyclic dinucleotide. However, under all conditions tested (culture in LB and in minimal medium, at both exponential and stationary growth phases), the level of c-di-GMP was below the 7.8 nM limit of detection by liquid chromatography-tandem mass spectrometry. We next induced expression of WspR, a known active diguanylate cyclase from Pseudomonas aeruginosa, from an IPTG (isopropyl-␤-d-thiogalactopyranoside)-inducible promoter (14) to determine if increasing [c-di-GMP] in E. coli CFT073 affects motility. Likewise, a known active phosphodiesterase from Vibrio cholerae, VC1086, under the control of an IPTG-inducible promoter (15) was utilized to determine if decreasing [c-di-GMP] affects motility. However, induction of neither WspR nor VC1086 in E. coli CFT073 significantly affected motility. Therefore, either [c-di-GMP] does not regulate motility in E. coli CFT073 or [c-di-GMP] is so tightly regulated in E. coli CFT073 that the addition of an exogenous PDE or DGC does not sufficiently alter [c-di-GMP] to produce a visible change in swimming diameter.
Biofilm formation by PDE, DGC, and PilZ mutants of E. coli CFT073. Biofilm formation, a sessile phenotype regulated by c-di-GMP, is increased as levels of c-di-GMP increase in some bacteria (3). Therefore, all 32 mutants were tested using the crystal violet assay to determine the contribution of individual PDE, DGC, and PilZ domain-encoding genes to biofilm formation when bacteria were cultured statically for 24 h at 37°C in LB without salt or M9 minimal salts medium with either 0.2% glucose or 0.4% glycerol as carbon source. In LB medium without salt, wild-type E. coli CFT073 forms a robust biofilm ring on the side of the well at the air-liquid interface. When corrected for the optical density at 600 nm (OD 600 ) to account for any growth differences in the mutants and normalized to wild-type, the ⌬ycdT, ⌬yeaI, and ⌬yfiN mutants (DGCs), the ⌬c1246, ⌬yoaD, and ⌬rtn mutants (PDEs), and the ⌬yhjK, ⌬yfgF, and ⌬yfeA mutants (PDE/DGCs) produced amounts of biofilm significantly different from that of the wildtype strain (P Ͻ 0.044) ( Fig. 2A; Table 2). Deletion of yhjK or c1246 increased biofilm biomass, whereas ⌬ycdT, ⌬yeaI, ⌬yfiN, ⌬yoaD, ⌬rtn, ⌬yfgF, and ⌬yfeA reduced biofilm formation.
In M9 minimal salts medium, with either glucose or glycerol as the carbon source, the ⌬ydiV mutant produced significantly more biofilm (P ϭ 0.0048 and P ϭ 0.0073, respectively) while the ⌬yhdA mutant formed significantly less biofilm (P ϭ 0.0078, and P ϭ 0.0398, respectively) than the wild-type strain ( Fig. 2B and C; Table 2). None of the other deletions affected biofilm formation when glucose served as the carbon source. However, with glycerol as the sole carbon source, the ⌬c1918, ⌬yneF, ⌬yeaP, ⌬yedQ, and ⌬yeaI mutants (DGCs) and the ⌬bscA (PilZ) mutant also significantly decreased biofilm formation compared to the wild-type strain (P Ͻ 0.03) ( Fig. 2C and Table 2).
Complementation of biofilm defects by exogenous phosphodiesterase or diguanylate cyclase activity. To determine if the biofilm defects observed were due to a change in [c-di-GMP] because of loss of either PDE or DGC activity, the mutants were complemented with either WspR (IPTG-inducible DGC) or VC1086 (IPTG-inducible PDE). An enzymatically inactive mutant of the PDE (VC1086*) was included as a control. In M9 minimal salts with glucose, induction of VC1086 decreased biofilm formation (52% Ϯ 6% of that of VC1086*; P Ͻ 0.0001) (see In M9 minimal medium with glycerol, induction of VC1086 unexpectedly increased biofilm formation in wild-type E. coli CFT073 (144% Ϯ 21% of that of VC1086*); therefore, no conclusions could be drawn about PDE activity under this condition. However, induction of WspR increased biofilm formation in wild-type E. coli CFT073 (160% Ϯ 13% of uninduced wild-type; P ϭ 0.0066) (see Fig. S1C in the supplemental material) as expected since an increase in [c-di-GMP] generally increases biofilm formation. ⌬yhdA, ⌬yeaP, ⌬yneF, and ⌬yedQ were all complemented by induction of WspR (see Fig. S1C), suggesting that the biofilm defects in these mutants are due to a loss of DGC function. However, ⌬yeaI and ⌬c1918 could not be complemented by expression of WspR (see Fig. S1C), suggesting that YeaI and C1918 do not actively produce c-di-GMP in this medium and therefore that they affect biofilm formation by another mechanism.
Wild-type E. coli CFT073 biofilm formation increases in nosalt LB medium when WspR is induced (121% Ϯ 6% of that of the uninduced wild type; P ϭ 0.0130) (see Fig. S2A), demonstrating that DGC activity promotes biofilm development and that biofilm formation decreases when VC1086 is induced (67% Ϯ 7% of that of VC1086*; P ϭ 0.0016) (see Fig. S2B), due to PDE activity. Three mutants with single DGC domains, the ⌬yfiN, ⌬yeaI, and ⌬ycdT mutants, were complemented by induction of WspR (see Fig.  S2A), suggesting that these are active DGCs in no-salt LB medium. All three mutants with single PDE domains, the ⌬yoaD, ⌬c1246, and ⌬rtn mutants, were complemented by induction of VC1086encoded PDE (see Fig. S2B). However, ⌬rtn was also complemented by VC1086*; therefore, no conclusion could be drawn for Rtn, as it may be PDE activity or another regulatory function underlying the phenotype. The cause of the biofilm defects in the ⌬yfgF, ⌬yfeA, and ⌬yhjK (PDE and DGC) mutants were also undetermined, as induction of both VC1086 and the WspR-encoded DGC was able to complement these mutations; therefore, no conclusion could be drawn as to which domain is enzymatically active under this condition (see Fig. S2A and B).
Curli and cellulose biosynthesis. In environmental E. coli biofilms, the extracellular matrix may be comprised of cellulose and curli, which are both regulated by [c-di-GMP] in E. coli K-12 and Salmonella strains (16,17). Using E. coli strain UTI89 as a positive control for curli production (18), all 32 mutants, along with wildtype E. coli CFT073, were tested for curli biosynthesis during culture on YESCA agar containing Congo red. Plates were incubated for 24 h at 30°or 37°C. Curli production was observed as red growth due to binding of Congo red by the curli amyloid structure (19). Wild-type E. coli CFT073 remained white under all conditions, while E. coli UTI89 turned red at 30°C, demonstrating that this strain produced curli (Fig. 2D). Two mutants in E. coli CFT073, ⌬yciR and ⌬rtn, produced red colonies at 30°C suggesting that YciR and Rtn suppress curli synthesis ( Fig.  2D; Table 2). Furthermore, after incubation for 96 h at 30°C, E. coli UTI89 produced the typical red, dry, and rough (rdar) phenotype, whereas wild-type E. coli CFT073 remained white, mucoid, and smooth. The E. coli CFT073 ⌬yciR mutant, however, produced a red, mucoid, and rough phenotype, whereas the ⌬rtn mutant produced the rdar phenotype (Fig. 2D). Thus, it appears that Rtn represses curli synthesis, while YciR may be involved in repression of PGA or cellulose synthesis.
Cellulose production was determined for all 32 mutants and   2E; Table 2) and the ⌬yhdA mutant ( Fig. 2F; Table 2) fluoresced at 30°C and at 37°C, respectively. These data suggest that E. coli CFT073, unlike UTI89 or K-12 strains, has developed additional regulatory elements that suppress cellulose and curli biosynthesis, resulting in a different extracellular matrix during biofilm formation. Phosphodiesterases and diguanylate cyclases regulate E. coli CFT073 adherence to bladder epithelial cells. Adherence to epithelial cells is a sessile phenotype that is vitally important during urinary tract infection. If E. coli cannot adhere to the bladder or kidney epithelium, the flow of urine may expel the invading pathogen. In general, c-di-GMP increases the synthesis of adhesins utilized for binding to epithelial cell surfaces (10,20). We tested all 32 mutants for adherence to the immortalized bladder epithelial cell line, UM-UC-3. Thirteen mutants displayed quantitative adherence to bladder cells that was significantly different (P Ͻ 0.05) from wild-type E. coli CFT073 (Fig. 3A Exogenous phosphodiesterase or diguanylate cyclase activity and adherence of mutants. To determine if the effect of the mutations on adherence to epithelial cells was due to the modulation of [c-di-GMP], we complemented each mutant with either exogenous PDE (VC1086) or DGC (WspR). Wild-type E. coli CFT073 adherence increased when WspR was induced (122% Ϯ 4% of the adherence of the uninduced strain; P ϭ 0.0146) (see Fig.  S3A in the supplemental material), demonstrating that DGC activity promotes adherence to epithelial cells, and decreased when VC1086 was induced (82% Ϯ 4% of the adherence of the VC1086* strain; P ϭ 0.0193) (see Fig. S3B), due to PDE activity. The active PDE VC1086 complemented ⌬ydiV, ⌬yhjH, ⌬c1610, ⌬yfgF, and ⌬yhjK, but ⌬yhdA and ⌬yegE were unaffected (see Fig. S3B). This suggests that YhdA and YegE do not act as PDEs, whereas YdiV, YhjH, C1610, YfgF, and YhjK affect c-di-GMP levels. Interestingly, ⌬ydiV, which increased motility, biofilm formation in minimal media, and adherence to bladder epithelial cells, was complemented only by VC1086 in adherence assays. This suggests that YdiV may indirectly affect the concentration of c-di-GMP, possibly by repression of an active PDE. The inactive PDE VC1086* did not affect adherence in wild-type E. coli CFT073 or the PDE mutants, with the exception of ⌬rtn; therefore, no conclusion could be made as to whether [c-di-GMP] contributes to the adherence phenotype of ⌬rtn.
Type 1 fimbria expression is increased in the ⌬yhjH and ⌬c1610 mutants but reduced in the ⌬ydeH mutant. Western blots against FimA, the main subunit of type 1 fimbriae, were conducted to determine if expression of these fimbriae is affected in mutants with adherence phenotypes. Three of the 13 mutants revealed levels of FimA different from that of wild-type E. coli CFT073, with the ⌬yhjH and ⌬c1610 strains expressing more and the ⌬ydeH strain expressing less FimA than the wild type (Fig. 3B).
To determine if the adherence effects of ⌬ydeH, ⌬yhjH, and ⌬c1610 were due to the aberrant expression of type 1 fimbriae, these mutants were constructed in the Fim L-ON background, and adherence of each mutant to UM-UC-3 bladder epithelial cells was compared to that of the Fim L-ON parental strain. In Fim L-ON, the ⌬ydeH (122% Ϯ 23%, P ϭ 0.1524), ⌬yhjH (111% Ϯ 29%, P ϭ 0.5185), and ⌬c1610 (99% Ϯ 17%, P ϭ 0.9557) strains were not significantly different in adherence from the Fim L-ON parental strain, suggesting that all three mutations affect adherence through type 1 fimbriae.

DISCUSSION
The regulatory network of UPEC involving c-di-GMP is complex, partly due to the large number of DGCs and PDEs encoded in the genome. Although the role of DGCs and PDEs in regulating the switch between motile and sessile lifestyles is being elucidated, one must ask how all of these enzymes affect the concentration of one molecule yet have individualized effects within a single bacterium. That several of the proteins annotated as PDEs or DGCs are degenerate, and may not be enzymatically active, but still are involved in regulation of motility and sessility adds another complication to interpretation. To address these issues, we conducted a comprehensive screen of single-deletion mutants of each PDE-, DGC-, PDE/DGC-, and PilZ-encoding gene in E. coli CFT073 to determine which regulates motility, adherence, biofilm formation, curli production, and cellulose synthesis. Furthermore, complementation of these mutants with an exogenous PDE and/or DGC demonstrated that some of these proteins indeed act by affecting [c-di-GMP], while others act by a [c-di-GMP]independent mechanism. Finally, strain differences between E. coli K-12 and uropathogenic E. coli CFT073 are apparent in the c-di-GMP regulatory network, suggesting that c-di-GMP signaling is regulated differently in pathogens than environmental or commensal bacteria.
From a screening of all 32 deletions of PDE, DGC, PDE/DGC, and PilZ domainencoding genes for effects on motility in E. coli CFT073, three were implicated in regulation of motility, all of which encode degenerate PDE domains. YhdA and YcgF promote motility, whereas YdiV acts as a repressor of motility. These proteins affect motility through a mechanism independent of effects on [c-di-GMP], as expression of an active PDE could not complement the observed effects. All three degenerate PDEs affect the expression of FliC, the main subunit comprising flagella. Epistatic analysis demonstrates that YcgF and YdiV affect motility in a shared regulatory pathway, with the increased motility phenotype of ⌬ydiV masking the decreased motility of ⌬ycgF. On the other hand, ⌬yhdA had an additive effect on motility with ⌬ydiV, demonstrating that these mutations affect motility via different regulatory pathways. Interestingly, no functional DGCs or PDEs appear to be involved in regulation of motility, which is very different from E. coli K-12, in which deletion of the DGC-encoding genes ycdT, yegE, ydeH, yeaI, yeaJ, yneF, yfiN, and yedQ increased swimming motility (21,23,24,26) and deletion of yhjH and ycgR reduced motility (29,30). Thus, E. coli CFT073 diverged from E. coli K-12 to regulate motility in such a way that single deletions of active PDEs or DGCs do not affect motility.
The focus of current research is on active PDEs and DGCs and the effect of fluctuations in [c-di-GMP] mediated by these enzymes; however, enzymatically inactive degenerate domains also clearly mediate phenotypic changes. For instance, in P. fluorescens, LapD, an enzymatically inactive PDE/DGC, binds c-di-GMP with the degenerate PDE domain and mediates signal transduction to cause the sequestration of a protease, LapG, thus preventing cleavage of an adhesin LapA (10,31). Furthermore, YdiV, the degenerate PDE protein that inhibited motility in our study, also inhibits motility in Salmonella enterica serovar Typhimurium and E. coli K-12 by binding to FlhD, causing a decrease in fliA transcription (32)(33)(34). However, there are strain differences between E. coli K-12 and CFT073. In E. coli K-12, deletion of ydiV does not affect motility, since this strain does not naturally express ydiV (32), whereas E. coli CFT073 ⌬ydiV is significantly more motile than the wild type (reference 13 and this study). In E. coli K-12, ydiV expression is inhibited posttranscriptionally (15,32), which was postulated to be due to a secondary structure formed in the 5= untranslated region occluding the translational start site, since this structure does not occur in Salmonella, where ydiV is fully expressed (32). By alignment of the intergenic region between ydiV and the closest gene upstream in E. coli K-12 and CFT073, we found there is a 1-bp difference between the two strains that would improve the predicted secondary structure, not abolish it. Therefore, the absence of YdiV in E. coli K-12 cannot solely be due to the secondary structure in the transcript. High [c-di-GMP] induces sessile phenotypes, such as adherence to epithelial cells or biofilm formation on abiotic surfaces in bacteria (35). In E. coli CFT073, 13 deletions affected adherence to the bladder epithelial cells, indicating that these proteins may play a role in regulation of adherence factors important for colonization of the human bladder. Of the eight mutations that increased adherence to bladder epithelial cells, five (⌬ydiV, ⌬yhjH, ⌬c1610, ⌬yfgF, and ⌬yhjK) were complemented by expression of a functional PDE, demonstrating that YhjH, C1610, YfgF, and YhjK inhibit adherence by directly decreasing [c-di-GMP] and that YdiV indirectly decreases [c-di-GMP] (Fig. 4). The five mutations that decreased adherence to epithelial cells, ⌬ydeH, ⌬yeaJ, ⌬yneF, ⌬yfiN, and ⌬yegE, were complemented by expression of an active DGC, suggesting that these enzymes promote adherence by increasing [c-di-GMP]. This is consistent with increased [c-di-GMP] in the bacterial cell inducing and decreased [c-di-GMP] repressing the sessile lifestyle. Furthermore, ⌬ydeH, ⌬c1610, and ⌬yhjH affect adherence to bladder cells through regulation of type 1 fimbriae (Fig. 4). In the Crohn's disease-associated adherent-invasive E. coli strain LF82, YhjH activates expression of type 1 fimbriae through the degradation of c-di-GMP (20). The mechanism(s) by which the ten other mutations affect adherence has yet to be elucidated.
Both degenerate and functional PDEs and DGCs affect biofilm formation in a medium-specific manner, as the analysis of the effect of the mutations on biofilm formation does not perfectly conform to dogma for all conditions tested. For example, in LB without salt, YcdT, YeaI, and YfiN (active DGCs) promote and C1246 and YhjK (active PDEs) inhibit biofilm formation, conforming to the hypothesis that increased [c-di-GMP] induces biofilm formation (Fig. 4). However, YoaD and YfgF (active PDEs) promote biofilm formation, contrary to this hypothesis. Carbon source had an effect in M9 minimal medium, where biofilm formation is regulated by YdiV and YhdA through a [c-di-GMP]-independent mechanism in glucose ( Fig. 4). However, in glycerol, both [c-di-GMP]-independent regulation (YdiV inhibited and YhdA, C1918, and YeaI promoted biofilm formation) and a [c-di-GMP]-dependent mechanism occur (YneF, YedQ, and YeaP [active DGC] and BscA [PilZ] promoted biofilm formation) (Fig. 4). These findings are in direct contrast with results from E. coli K-12 strain BW25113, where YfiN, YedQ, and YeaI inhibit biofilm formation (26). Thus, these proteins have evolved to regulate the same phenotype by disparate mechanisms in UPEC and commensal E. coli.
In this work, we demonstrated that active PDEs and DGCs regulate adherence to host cells and biofilm formation but do not regulate motility. Instead, motility is regulated in a [c-di-GMP]independent manner by three enzymatically inactive PDEs. The roles of enzymatically active and inactive PDEs and DGCs in the transition between sessile and motile lifestyles have diverged in E. coli CFT073 from E. coli K-12. Future studies will determine the molecular mechanism(s) by which these active and inactive enzymes modulate motility and sessility of UPEC.

MATERIALS AND METHODS
Construction and complementation of mutants for PDE, DGC, PDE/ DGC, and PilZ domain-encoding genes. Deletion mutants were constructed in E. coli CFT073 using the lambda red recombinase system as described by Datsenko and Wanner (36). Primers containing sequences homologous to the 5= and 3= ends of the target sequence were designed and used to amplify the resistance cassette from either template plasmid pKD3 or pKD4 (encoding chloramphenicol or kanamycin resistance, respectively). Lambda red-mediated recombination was used to replace tween experiments, data are expressed as OD 570 /OD 600 relative to the wildtype strain.