Metabolic Activation of CsgD in the Regulation of Salmonella Biofilms

Among human food-borne pathogens, gastroenteritis-causing Salmonella strains have the most real-world impact. Like all pathogens, their success relies on efficient transmission. Biofilm formation, a specialized physiology characterized by multicellular aggregation and persistence, is proposed to play an important role in the Salmonella transmission cycle. In this manuscript, we used luciferase reporters to examine the expression of csgD, which encodes the master biofilm regulator. We observed that the CsgD-regulated biofilm system responds differently to regulatory inputs once it is activated. Notably, the CsgD system became unresponsive to repression by Cpx and H-NS in high osmolarity conditions and less responsive to the addition of amino acids. Temperature-mediated regulation of csgD on agar was altered by intracellular levels of RpoS and cyclic-di-GMP. In contrast, the addition of glucose repressed CsgD biofilms seemingly independent of other signals. Understanding the fine-tuned regulation of csgD can help us to piece together how regulation occurs in natural environments, knowing that all Salmonella strains face strong selection pressures both within and outside their hosts. Ultimately, we can use this information to better control Salmonella and develop strategies to break the transmission cycle.


Introduction
Salmonella enterica strains that cause gastroenteritis and typhoid fever were recently ranked first and second in terms of global disease impact (i.e., disability adjusted life years) among 22 of the most common food borne pathogens [1]. S. enterica strains are distributed within >2000 serovars, with yearly estimates of approximately 94 million cases of gastroenteritis [2] and 21 million cases of typhoid fever worldwide [3]. The serovars associated with typhoid fever (i.e., Typhi, Paratyphi and few others [4]) consist of human-restricted strains and are collectively referred to as typhoidal The regulation of Salmonella biofilms is also strongly influenced by the intracellular levels of the second messenger, cyclic-di-GMP (c-di-GMP). It is synthesized from two guanosine 5 -triphosphate molecules by diguanylate cyclases (DGCs) and degraded by specific phosphodiesterases (PDEs). In general, high levels of c-di-GMP are associated with biofilm formation, sessility and persistence, and low levels of c-di-GMP are associated with motility and virulence [43]. The change in c-di-GMP levels in S. enterica is controlled by the enzymatic activity of 17 different DGCs and PDEs. For biofilms, the cellulose synthase enzyme, BcsA, is allosterically activated by c-di-GMP that is produced by AdrA, a DGC that is transcriptionally activated by CsgD. Expression of CsgD itself is influenced by c-di-GMP synthesis and breakdown by a network of DGC and PDE enzymes [44]. The importance of c-di-GMP regulation is underscored by the observation that S. enterica isolates that are defective in the production of DGCs are both avirulent and unable to form biofilms [45].
In this manuscript, we analyzed the regulation of csgD transcription and the activity of CsgD through activation of curli biosynthesis (csgBAC) and cellulose production (adrA). We examined the response to different environmental signals (i.e., temperature, osmolarity, nutrients) and discovered that there is a hierarchy of regulation. These environmental signals were selected because their effects on csgD expression before induction have been well established and we hypothesize that these conditions would be encountered during food processing and in both host and non-host environments. We established that the CsgD system responds differently or not at all to known regulatory inputs once it has been activated. This is similar to some dedicated, point of no return processes, such as sporulation in Bacillus subtilis [46]; however, we show that the CsgD system can be reversed by other signals, such as glucose. The implications for the Salmonella lifecycle are discussed.

Bacterial Strains, Media, and Growth Conditions
The bacterial strains used in this study are listed in Table 1. For standard growth, strains were inoculated from frozen stocks onto LB agar (lysogeny broth, 1% NaCl, 1.5% agar) supplemented with appropriate antibiotic (50 µg mL −1 kanamycin (Kan), or 5 µg mL −1 tetracycline (Tet)) and grown overnight at 37 • C. Isolated colonies were used to inoculate 5 mL LB broth and the culture was incubated for 18 h at 37 • C with agitation at 200 RPM.  [48] For analysis of colony morphology and gene expression, 4 µl of overnight culture was spotted on 1% tryptone agar supplemented with 0.2% freshly made glucose, 25 mM salt or 100 mM salt (agar supplemented with glucose were used within 24 h). Plates were incubated at 28 • C or 37 • C for two days. Visible and luminescence images were captured with a spectrum CT in vivo imaging system (PerkinElmer, Waltham, MA, USA).

Generation of S. typhimurium 14028 Mutant Strains
Lambda red recombination [49] was used to generate ∆cpxR and ∆iraP S. Typhimurium mutant strains. Primers containing 50-nuclelotide sequences on either side of cpxR or iraP (Table 2) were used to amplify the cat gene from pKD3 using Phusion high-fidelity DNA polymerase (New England Bio-Labs, Ipswich, MA, USA). The PCR products were solution purified and electroporated into S. Typhimurium 14028 cells containing pKD46. Mutants were first selected by growth at 37 • C on LB agar supplemented with 10 µg ml −1 chloramphenicol (Cam) before streaking onto LB agar containing 34 µg ml −1 Cam. PCR primers upstream and downstream of cpxR or iraP ( Table 2) were used to amplify sequence from the genome of mutant S. typhimurium 14028 strains and verify loss of the corresponding open reading frames. The ∆cpxR or ∆iraP mutations were moved into a clean S. typhimurium strain background with P22 phage [50]. The cat gene was resolved from the chromosome using pCP20 [49].

Generation of Bacterial Luciferase Reporters and Other Plasmid Vectors
Luciferase fusion reporter plasmids containing the promoters of csgDEFG, csgBAC and adrA have been previously described [12]. The cpxP reporter plasmid was generated to monitor the levels of CpxA/CpxR activation within the cell. The intergenic region containing the cpxR and cpxP promoter sequences was PCR amplified from S. Typhimurium 14028 using primers cpxR1 and cpxR2 (Table 2) and Phusion high-fidelity DNA polymerase (New England BioLabs, Ipswich, MA, USA). The resulting PCR product was purified, sequentially digested with XhoI and BamHI, and ligated (in the cpxP direction) using T4 DNA ligase (New England BioLabs, Ipswich, MA, USA) into pU220 digested with XhoI and BamHI. The stm1987 luciferase reporter plasmid was generated similarly using primers STM14_2408for1 and STM14_2408rev2 (Table 2), with cloning into pCS26. PCR screening with primers pZE05 and pZE06 was used to verify the successful fusion of promoter regions to luxCDABE.
For plasmid-based overexpression of cyclic-di-GMP related enzymes, fragments containing stm1987 and yhjH genes with their native promoters were PCR amplified from S. typhimurium 14028 gDNA using Phusion high-fidelity DNA polymerase and appropriate primers (Table 2). Resulting PCR products were purified, digested with EcoRI and AatII, and ligated using T4 DNA ligase into EcoRI/AatII-digested pBR322. The pACYC-rpoS plasmid vector has previously been described [48]. Reporter plasmids and overexpression plasmids were co-transformed into S. typhimurium strains by electroporation and selected by growth at 37 • C on LB agar supplemented with 50 µg mL −1 Kan (pCS26) and 10 µg mL −1 Tet (pBR322 or pACYC).

Luciferase Reporter Assays
96-well bioluminescence assays were performed with S. Typhimurium luciferase reporter strains. Overnight cultures were diluted 1 in 600 into individual wells of black, clear bottom 96-well plates (9520 Costar; Corning Life Sciences, Tewksbury, MA, USA) containing 150 µL of 1% tryptone broth supplemented with 50 µg mL −1 of kanamycin (Kan). When noted, media was supplemented before growth with NaCl (25,150 mM), sucrose (50,150 mM), CuCl 2 (1 mM), casamino acids (12%) or individual amino acids (15 mM) to the final concentrations as indicated. For the addition of media supplements during growth, cells were inoculated into 135 µL of media and grown for 18 h at 28 • C before supplements were added as 15 µL aliquots to the appropriate wells. This included glucose ranging from 25-150 mM. To minimize evaporation of the media during the assays, cultures were overlaid with 50 µL of mineral oil per well. Cultures were assayed for absorbance (600 nm, 0.1 s) and luminescence (1s; in counts per second (CPS)) every 30 min during growth at 28 • C with agitation in a Victor X3 multilabel plate reader (Perkin-Elmer, Waltham, MA, USA).

Osmolarity Has No Effect Once csgD Transcription Is Activated
Osmolarity is a key regulatory factor for Salmonella biofilm formation in vitro [29,51]. In the presence of high concentrations of NaCl, csgD transcription is abolished [52]. In E. coli, this repression is mediated through the CpxA/R two-component system [37]. We performed transcription experiments with S. enterica serovar Typhimurium ATCC 14028 (i.e., S. typhimurium 14028). Consistent with E. coli, expression of csgD was highest in low osmolarity media (i.e., no salt) and reduced sequentially in media supplemented with increasing concentrations of NaCl ( Figure 1A). Reduced csgD expression in media supplemented with 75 mM or more salt correlated with basal expression of csgBAC (curli biosynthesis) and adrA (cellulose biosynthesis) ( Figure 1B,C). To gauge the activity of the CpxA/R system, and its potential role in repression, we monitored expression of cpxP, a known regulatory target of CpxR [53]. Expression of cpxP was highest in media supplemented with 150 mM NaCl ( Figure 1D), which was inversely correlated with csgD expression levels. This was consistent with CpxR-mediated repression of csgD transcription.
Regulation of csgD expression via the CpxA/R system is thought to be a dynamic process involving surface-sensing and feedback during curli production [36,54]. Therefore, we performed experiments where salt was added to growing cultures after 18 h of growth, rather than being premixed into the media before growth. At 18 h of growth, csgD expression level is rapidly increasing and csgBAC and adrA expression are just beginning to increase [26]. Under these conditions, csgD expression did not change when increasing concentrations of salt were added during growth; the expression curves were nearly superimposable regardless of the amount of salt added ( Figure 1E). Expression of csgBAC was also not inhibited by the addition of salt and was actually increased at high salt concentrations ( Figure 1F). For adrA, mild repression was observed, but expression was well above background levels, even in the presence of 150 mM salt ( Figure 1G). cpxP expression, on the other hand, was similar to the premixed experiments, with highest expression in the 150 mM salt media and lowest expression in non-supplemented media ( Figure 1H). These results indicated that the Cpx system was activated by

Repressive Effect of CpxR on csgD Transcription Is Alleviated During Growth
To examine the effects of Cpx-mediated repression of csgD transcription in more detail, we monitored gene expression in a cpxR mutant background. The Cpx system can be activated by high concentrations of metals and a variety of other signals, with each thought to represent a form of periplasmic stress [54,55]. Growth of S. Typhimurium 14028 in media supplemented with 1 mM copper chloride resulted in activation of the Cpx system, as measured by an increase in cpxP

Repressive Effect of CpxR on csgD Transcription Is Alleviated During Growth
To examine the effects of Cpx-mediated repression of csgD transcription in more detail, we monitored gene expression in a ∆cpxR mutant background. The Cpx system can be activated by high concentrations of metals and a variety of other signals, with each thought to represent a form of periplasmic stress [54,55]. Growth of S. typhimurium 14028 in media supplemented with 1 mM copper chloride resulted in activation of the Cpx system, as measured by an increase in cpxP expression ( Figure 2A, + inducer). As expected, cpxP expression was off in the ∆cpxR strain background ( Figure 2A, red line). In the presence of copper chloride, csgD expression reached high levels, similarly to when in the presence of non-supplemented media ( Figure 2B). There was also a slight increase in the ∆cpxR strain, which was consistent with CpxR being a repressor of csgD transcription. This effect was more pronounced for csgBAC, as expression was approximately four times higher in the ∆cpxR strain ( Figure 2C). We performed the same experiment with the addition of copper chloride after 18 h of growth. The Cpx system was activated normally, as shown by elevated cpxP expression levels in the presence of the inducer ( Figure 2D). However, expression of csgDEFG and csgBAC was unchanged in the ∆cpxR mutant strain, showing no evidence of CpxR-mediated repression ( Figure 2E,F). This indicated that once the csgD network was activated, the system was unresponsive to CpxR.  (Figure 2A, + inducer). As expected, cpxP expression was off in the cpxR strain background ( Figure 2A, red line). In the presence of copper chloride, csgD expression reached high levels, similarly to when in the presence of non-supplemented media ( Figure 2B). There was also a slight increase in the cpxR strain, which was consistent with CpxR being a repressor of csgD transcription. This effect was more pronounced for csgBAC, as expression was approximately four times higher in the cpxR strain ( Figure 2C). We performed the same experiment with the addition of copper chloride after 18 h of growth. The Cpx system was activated normally, as shown by elevated cpxP expression levels in the presence of the inducer ( Figure 2D). However, expression of csgDEFG and csgBAC was unchanged in the cpxR mutant strain, showing no evidence of CpxR-mediated repression (Figure 2 E,F). This indicated that once the csgD network was activated, the system was unresponsive to CpxR.  We also measured biofilm gene expression after the addition of sucrose ( Figure 3). In E. coli, sucrose has been shown to repress csgD transcription, due to the activity of H-NS [36]. Sucrose is also a cleaner measure of osmolarity because unlike salt, it does not result in a change of ionic strength. In general, the csgDEFG, csgBAC and adrA expression profiles were consistent with what was measured in response to salt addition. When sucrose was added to the media before growth, significant repression was observed for all three promoters ( Figure 3A-C). However, when sucrose was added to growing cultures at 18 h, there was no repression measured ( Figure 3E-G). The addition of sucrose had minimal  Figure 3D,H), and, therefore, did not appear to engage the CpxR/A system, similar to what was observed in E. coli [36]. These results indicated that the S. typhimurium csgD biofilm network is not influenced by changes in osmolarity after it has been activated. Moreover, this appears to be a general effect that is not restricted to repression by the Cpx system. We also measured biofilm gene expression after the addition of sucrose ( Figure 3). In E. coli, sucrose has been shown to repress csgD transcription, due to the activity of H-NS [36]. Sucrose is also a cleaner measure of osmolarity because unlike salt, it does not result in a change of ionic strength. In general, the csgDEFG, csgBAC and adrA expression profiles were consistent with what was measured in response to salt addition. When sucrose was added to the media before growth, significant repression was observed for all three promoters (Figure 3 A,B,C). However, when sucrose was added to growing cultures at 18 h, there was no repression measured (Figure 3 E,F, G). The addition of sucrose had minimal effect on cpxP expression (Figure 3 D, H), and, therefore, did not appear to engage the CpxR/A system, similar to what was observed in E. coli [36]. These results indicated that the S. Typhimurium csgD biofilm network is not influenced by changes in osmolarity after it has been activated. Moreover, this appears to be a general effect that is not restricted to repression by the Cpx system.

Temperature and Glucose Repress csgD Expression
The idea that the biofilm system can become unresponsive to known regulatory inputs once it is activated fits with one of the hallmarks of a bistable gene expression system, in that a proportion of cells can remain activated even when the inducer is absent [56]. There are other bacterial physiologies, such as sporulation, where the cellular differentiation process is irreversible [46]. The csgD biofilm network has been shown to have bistable expression [40,42]. We wondered if the response we had observed with osmolarity was representative of a non-reversible system.
In most Salmonella and E. coli strains, csgD expression and biofilm formation is activated at temperatures below 30 • C and repressed at higher temperatures [52]. There are strains that produce biofilms at higher temperatures (i.e., 37 • C), but these typically possess single nucleotide polymorphisms in the csgD promoter region that allows for disregulated expression [50,52,57,58]. We tested whether increased temperature could shut off activated biofilm gene expression by first growing cells at 28 • C for 18 h and then shifting the temperature to 30, 32, 35, or 37 • C. At 30 • C or 32 • C, there was a measurable drop in csgDEFG, csgBAC and adrA expression, but it was still above background levels ( Figure 4A-C). However, a temperature shift above 32 • C reduced gene expression to baseline levels ( Figure 4A-C). This showed that high temperature was able to override the activation of csgD and biofilm related genes.

Effect of Casamino Acids on Biofilm Formation
Expression of csgD is known to be activated once cells reach a critical density and nutrients start to run out [30]. Since 1% tryptone is primarily an amino acid-based media [60], we speculated that the addition of amino acids would reduce or delay expression of csgD and other biofilm genes. Casamino acids (CAA) are a complex mixture of amino acids and small peptides that are used for nutritional investigations of bacterial growth. The addition of CAA to the medium prior to S. Typhimurium growth reduced csgDEFG expression approximately 15-fold in the presence of 0.5, 1.0 or 2.0% CAA ( Figure 5A). csgBAC and adrA expression dropped to near baseline levels when CAA was added at the beginning of growth ( Figure 5B,C). The addition of CAA to growing S. Typhimurium 14028 cultures also reduced the expression of all three promoters, but in a more dosedependent manner. Expression of csgDEFG was reduced to ~75%, 50% and 25% of initial levels after the addition of 0.5% CAA, 1.0% CAA and 2% CAA, respectively ( Figure 5D). Expression of csgBAC was reduced after the addition of 0.5% or 1.0% CAA, but the promoter was still considered active, Glucose is another powerful repressor of csgD expression and biofilm formation in vitro [26,59]. For S. typhimurium, the addition of glucose to growing cultures at 18 h rapidly abolished csgDEFG ( Figure 4D), csgBAC ( Figure 4E) and adrA ( Figure 4F) expression, even at the lowest added concentration of 25 mM. We tested lower concentrations of glucose and found that in each case, csgD transcription was immediately repressed but was restored at later timepoints, presumably when all glucose was metabolized. This showed that glucose was a powerful repressive signal. Together, these experiments showed that activation of the S. Typhimurium csgD biofilm network is a reversible process and suggested the existence of a regulatory hierarchy.

Effect of Casamino Acids on Biofilm Formation
Expression of csgD is known to be activated once cells reach a critical density and nutrients start to run out [30]. Since 1% tryptone is primarily an amino acid-based media [60], we speculated that the addition of amino acids would reduce or delay expression of csgD and other biofilm genes. Casamino acids (CAA) are a complex mixture of amino acids and small peptides that are used for nutritional investigations of bacterial growth. The addition of CAA to the medium prior to S. Typhimurium growth reduced csgDEFG expression approximately 15-fold in the presence of 0.5, 1.0 or 2.0% CAA ( Figure 5A). csgBAC and adrA expression dropped to near baseline levels when CAA was added at the beginning of growth ( Figure 5B,C). The addition of CAA to growing S. typhimurium 14028 cultures also reduced the expression of all three promoters, but in a more dose-dependent manner. Expression of csgDEFG was reduced to~75%, 50% and 25% of initial levels after the addition of 0.5% CAA, 1.0% CAA and 2% CAA, respectively ( Figure 5D). Expression of csgBAC was reduced after the addition of 0.5% or 1.0% CAA, but the promoter was still considered active, whereas expression returned to baseline after the addition of 2.0% CAA ( Figure 5E). adrA expression returned to near baseline levels, even with the addition of 0.5% CAA ( Figure 5F). These experiments demonstrated that there is a metabolic feedback into csgD expression and that the system responds differently once it has been activated. whereas expression returned to baseline after the addition of 2.0% CAA ( Figure 5E). adrA expression returned to near baseline levels, even with the addition of 0.5% CAA ( Figure 5F). These experiments demonstrated that there is a metabolic feedback into csgD expression and that the system responds differently once it has been activated.

Differing Effects of Individual Amino Acids on csgD Gene Expression
We wanted to test how individual amino acids contributed to the repression of biofilm gene expression caused by CAA. We measured csgBAC expression i.e., curli production) as a proxy for biofilm formation and as readout for CsgD activity. Only Asn, Pro and Arg had a direct repressive

Differing Effects of Individual Amino Acids on csgD Gene Expression
We wanted to test how individual amino acids contributed to the repression of biofilm gene expression caused by CAA. We measured csgBAC expression i.e., curli production) as a proxy for biofilm formation and as readout for CsgD activity. Only Asn, Pro and Arg had a direct repressive effect on csgBAC expression when added individually ( Figure 6A; blue bars). The expression curves were lower for the entirety of growth ( Figure 6B). Six amino acids had no significant effect ( Figure 6A, grey bars; examples in 6C) and seven amino acids caused an increase in expression ( Figure 6A, pink bars). The addition of Gly and Thr yielded an approximately three-fold boost to csgBAC expression ( Figure 6D), which was unexpected. These results indicated that the repression caused by CAA must have been due to the cumulative effect of multiple amino acids.
When individual amino acids were added to S. Typhimurium cultures after 18 h of growth, the effects on csgBAC expression were not predictable based on their previous groupings ( Figure 6E; see color distribution). No amino acids caused a decrease in expression, and some that were repressive when added before growth (i.e., Arg, Pro), now caused a significant boost in expression ( Figure 6E,F). Eight amino acids had no signficant difference from the water control ( Figure 6G, Lys, Ser). Val, Ala, Gln and Thr led to increased csgBAC expression when added before or during growth, suggesting that these amino acids have a positive effect on curli fimbriae synthesis. Glycine, on the other hand, had no significant effect when added during growth ( Figure 6H). Overall, we could not explain the differing effects of individual amino acids when added during growth. However, the results were consistent with our previous observation that the S. typhimurium biofilm network responds differently to regulatory inputs after the csgD network has been activated.

Regulation of Rdar Morphotype on Agar-Containing Media
In the bistable expression of CsgD, the proportion of cells in the "ON" state is thought to be maintained by a feed-forward loop consisting of RpoS, the stationary phase sigma factor that controls csgD transcription, IraP, a protein that stabilizes RpoS, and CsgD itself [35,42]. In addition, csgD expression and CsgD activity can be influenced by the bacterial secondary messenger, cyclic-di-GMP (c-di-GMP) [44]. We wanted to investigate how these additional regulatory components influenced metabolic control of the S. typhimurium biofilm regulatory network. Strains were grown at 28 • C or 37 • C on 1% tryptone agar, with different components added to the media. To modulate intracellular c-di-GMP levels, strains were transformed with plasmids over-expressing stm1987, encoding a DGC enzyme that generates c-di-GMP, or yhjH, encoding a PDE enzyme that breaks down c-di-GMP. To analyze the proposed feed-forward loop, we utilized a plasmid over-expressing RpoS and measured gene expression in ∆rpoS and ∆iraP strains. Each strain was transformed with a luciferase reporter plasmid so that we could visualize csgBAC expression.  The vector-only S. Typhimurium 14028 control strain displayed robust light production at 28 • C, with faint csgBAC signals also observed in the presence of 25mM salt (Figure 7, vector). Over-expression of rpoS appeared to elevate csgBAC expression under most conditions, including in the presence of salt and at 37 • C (Figure 7, rpoS). The importance of RpoS was emphasized in that the ∆rpoS strain had no visible csgBAC expression under all tested conditions, unless it was co-transformed with pACYC/rpoS (Figure 7; 28 • C ∆rpoS). A strong stimulatory effect was also caused by over-expression of stm1987, which allowed for robust csgBAC expression and biofilm colony morphology under most conditions (Figure 7, stm1987). The strain transformed with pBR322/stm1987 was the only one to have detectable csgBAC expression at 37 • C in the presence of 25 mM salt (Figure 7). This indicated that elevated levels of c-di-GMP may be enough to overcome temperature-based repression of csgBAC. Emphasizing the importance of c-di-GMP, the expression of yhjH was sufficient to abolish csgBAC expression at 28 • C (Figure 7, yhjH), as well as in all other tested conditions. In contrast, deletion of iraP appeared to have little effect on csgBAC expression, with only a mild reduction observed at 28 • C (Figure 7, iraP). Finally, the presence of glucose in the media abolished csgBAC expression in all strain and plasmid combinations (Figure 7, 0.2% Glc). This experiment indicated that increased levels of RpoS and c-di-GMP could partially overcome some csgBAC repression, and that glucose was perhaps the most powerful metabolic signal feeding into the S. typhimurium csgD regulatory network.
37°C on 1% tryptone agar, with different components added to the media. To modulate intracellular c-di-GMP levels, strains were transformed with plasmids over-expressing stm1987, encoding a DGC enzyme that generates c-di-GMP, or yhjH, encoding a PDE enzyme that breaks down c-di-GMP. To analyze the proposed feed-forward loop, we utilized a plasmid over-expressing RpoS and measured gene expression in ΔrpoS and ΔiraP strains. Each strain was transformed with a luciferase reporter plasmid so that we could visualize csgBAC expression.
The vector-only S. Typhimurium 14028 control strain displayed robust light production at 28°C, with faint csgBAC signals also observed in the presence of 25mM salt (Figure 7, vector). Overexpression of rpoS appeared to elevate csgBAC expression under most conditions, including in the presence of salt and at 37°C (Figure 7, rpoS). The importance of RpoS was emphasized in that the rpoS strain had no visible csgBAC expression under all tested conditions (data not shown), unless it was co-transformed with pACYC/rpoS (Figure 7; 28°C rpoS). A strong stimulatory effect was also caused by over-expression of stm1987, which allowed for robust csgBAC expression and biofilm colony morphology under most conditions (Figure 7, stm1987). The strain transformed with pBR322/stm1987 was the only one to have detectable csgBAC expression at 37°C in the presence of 25 mM salt (Figure 7). This indicated that elevated levels of c-di-GMP may be enough to overcome temperature-based repression of csgBAC. Emphasizing the importance of c-di-GMP, the expression of yhjH was sufficient to abolish csgBAC expression at 28°C (Figure 7, yhjH), as well as in all other tested conditions (data not shown). In contrast, deletion of iraP appeared to have little effect on csgBAC expression, with only a mild reduction observed at 28°C ( Figure  iraP). Finally, the presence of glucose in the media abolished csgBAC expression in all strain and plasmid combinations (Figure 7, 0.2% Glc). This experiment indicated that increased levels of RpoS and c-di-GMP could partially overcome some csgBAC repression, and that glucose was perhaps the most powerful metabolic signal feeding into the S. Typhimurium csgD regulatory network. Figure 7. Visualization of S. Typhimurium curli expression in response to changing growth conditions. S. Typhimurium 14028 wild-type, rpoS or iraP reporter strains containing a csgBAC promoter-luciferase fusion were transformed with pBR322 (vector), pACYC/rpos (rpoS), pBR322/stm1987 (stm1987) or pBR322/yhjH (yhjH) plasmids. Cells were inoculated onto T agar or T agar supplemented with 0.2% glucose, 25 mM or 100 mM NaCl and grown at 28°C or 37ºC. Colony morphology (left column) and luminescence (right column) was recorded after 48 h growth. Control Figure 7. Visualization of S. typhimurium curli expression in response to changing growth conditions. S. typhimurium 14028 wild-type, ∆rpoS or ∆iraP reporter strains containing a csgBAC promoter-luciferase fusion were transformed with pBR322 (vector), pACYC/rpos (rpoS), pBR322/stm1987 (stm1987) or pBR322/yhjH (yhjH) plasmids. Cells were inoculated onto T agar or T agar supplemented with 0.2% glucose, 25 mM or 100 mM NaCl and grown at 28 • C or 37ºC. Colony morphology (left column) and luminescence (right column) was recorded after 48 h growth. Control strains containing pACYC were also tested, but the csgBAC expression profiles were similar to strains transformed with pBR322; therefore, only the pBR322 pictures are shown.

Discussion
Biofilm formation is subject to tight and complex regulation through transcription factor CsgD. In S. Typhimurium, the intergenic region between divergent csgDEFG and csgBAC operons is among the longest non-coding region with 582 bp, which allows for a highly sophisticated signaling network. CsgD expression is regulated at the transcriptional, post transcriptional, translational and post translation level, in response to a variety of external and internal signals [8]. In this study we show that once activated, the CsgD biofilm network responds differently to metabolic inputs.
The ability of S. enterica strains to form biofilms is thought to be critical for the success of Salmonella as pathogens, particularly for gastroenteritis-causing strains [14]. With bistability of CsgD synthesis resulting in distinct cell types-multicellular aggregates associated with persistence (CsgD-ON), and single cells associated with virulence (CsgD-OFF) [42]-there is a need to have a flexible and dynamic response. We speculated that this phenotypic heterogeneity was a form of bet-hedging. A bet-hedging strategy ensures that at least one group of cells will be more adapted for a specific set of conditions that is encountered [61]. For some bacterial processes, such as sporulation, the advantage of the sporulating cell is obvious; however, for the non-sporulating cells, the advantage lies in being capable of more rapid growth when an influx of new nutrients occurs [62]. For Salmonella, there is a lot of energy devoted to generating the polymers associated with biofilm aggregates [26,63]; in the virulent, single cell group, synthesis of the type three secretion apparatus also requires a significant outlay of energy [64]. This type of population split makes the most sense in response to the unpredictability of transmission [65] or perhaps for modulating host-pathogen interactions, as observed for Vibrio cholerae [66]. We analyzed regulation before csgD activation, which has been tested before in S. typhimurium and E. coli and generally had the expected results, and compared this to regulation after csgD activation, which to our knowledge has not been tested before. We observed that csgD transcription and activation of downstream biofilm components was no longer repressed by increased osmolarity, and that the response to nutrient addition was also different, either as individual amino acids or a set of pooled amino acids. In contrast, the addition of glucose and temperatures above 32 • C rapidly repressed csgD, csgB and adrA expression even after induction. We approached these experiments from the point of view of biofilm formation as a developmental process [67,68], and our results show that CsgD biofilm formation is reversible, but can also be viewed as irreversible, depending on the signal. Our results, therefore, suggest the existence of a regulatory hierarchy among external signals that regulate biofilm formation.
For osmolarity, it has been well established that the optimal conditions for csgD expression and rdar biofilm formation in vitro include low osmolarity [51,52,69]. Key transcription factors have been identified (i.e., OmpR, CpxR, H-NS, MlrA and others) and binding within the csgD promoter region has been characterized [29,36,37,70,71]. Yet, there are still some intriguing aspects; for example, S. enterica biofilm cells produce high levels of osmoprotectants even when growing in low osmolarity conditions [26]. To explain the accumulation of osmoprotectants, we hypothesized that there could be high osmolarity microenvironments created within biofilms due to nutrient and ion trapping by the extracellular matrix [25,72]. The presence of hyperosmolar environments was recently observed with E. coli biofilms [73]. Our experiments show that once CsgD has activated downstream target genes (i.e., csgBAC (curli) and adrA (cellulose), transcription of all units becomes unresponsive to increases in osmolarity. This was specific to the CpxR/A two-component system in high salt conditions and by activating CpxR in ways that are not expected to significantly a change in osmolarity (i.e., metal stress) [55]. There has been some recent controversy about the role of CpxR in surface sensing or adhesion [74], but it is a well-established regulator of csgD [75]. The osmolarity effect was also general, as similar gene expression patterns were observed after the addition of sucrose, which was shown to repress csgD transcription in E. coli by acting through H-NS [36]. In our experiments, the CpxR/A system was not activated by the addition of sucrose, therefore, we assume that the same H-NS-mediated signaling occurs in Salmonella. To explain the results with csgD, it is possible that the presence of osmoprotectants produced early on during biofilm formation could mute the signaling effects associated with high external osmolarity [76]. Although we have shown that several osmoprotectant-associated genes are produced in time with csgBAC and adrA [26], we do not know the detailed time course for the appearance of the molecules themselves. The biological relevance for a lack of response to increased osmolarity is not clear, however, a recent paper described a real-world scenario where such a characteristic could be favored. Grinberg et al. 2019 [77] demonstrated that bacterial aggregates have enhanced survival on the surfaces of leaves in microdroplets that are not visible to the naked eye. As liquid evaporates from the leaf surfaces, solutes become concentrated and the microdroplets become hyperosmolar solutions. One could envision S. enterica biofilm aggregates surviving well in this scenario due to their stress-resistance adaptations and the altered csgD regulatory program identified here. We hypothesize that these microdroplets represent an environment where biofilms, and presumably biofilm-forming strains, would be favored over individual cells that do not aggregate together or strains that do not form biofilms.
Nutrient limitation was one of the first activating signals identified for csgD transcription [30,51]. In 1% tryptone or lysogeny broth, which are predominantly comprised of amino acids [60], csgD transcription occurs when cell density increases and cells start to run out of nutrients [8,63]. While this was initially attributed to phosphate and nitrogen depletion [30], we tested if supplementation with additional amino acids would delay or prevent activation of csgD transcription. When amino acids were added together (i.e., casamino acids), the transcription of csgD and downstream biofilm genes was delayed for almost the entire 70-h growth period, well after high cell densities were reached. When CAA were added during growth, csgD expression was shifted down in a dose-dependent manner. This showed that after induction, csgD expression was still responsive to negative regulation by CAA. The dose response could represent a subpopulation of S. typhimurium cells that retain metabolic flexibility [78] and are able to shift their metabolism away from biofilm formation. Based on the results with CAA, we predicted that individual amino acids might also have a repressive effect on biofilm formation. We measured the expression of the curli biosynthesis operon (i.e., csgBAC), a direct target of CsgD. Only Asn, Pro and Arg reduced csgB expression when added before growth, while Ile, Val, Gln, Met, Ala, Thr and Gly all increased expression. This indicated that the repression observed with CAA was the cumulative effect of the individual amino acids, as recently observed [79]. When added during growth, Leu, Arg, His, Val, Pro, Ala, Gln and Thr increased csgB expression, and no single amino acid decreased expression. This again showed that the CsgD biofilm network responds differently once it is activated. The production of sugars from gluconeogenesis is important for biofilm formation, as S. Typhimurium strains with mutations of pckA and ppsA are unable to form biofilms [26]. PckA and PpsA are important gluconeogenic enzymes required for the synthesis of phosphoenolpyruvate (PEP). Pck catalyzes the conversion of oxaloacetate to PEP [80], while Pps catalyzes the conversion of pyruvate to PEP. Ala, Gly and Thr are gluconeogenic amino acids that enter the gluconeogenic pathway through pyruvate [81]. In support of this, Ala and Thr increased csgB expression when introduced before and during growth. Gly also increased csgB expression when added before and during growth, but the change was not statistically significant. CsgD was shown to directly stimulate Gly biosynthesis during E. coli biofilm formation [82], presumably to ensure there is enough Gly supply to produce large quantities of the major curli subunit, CsgA (i.e., 16% Gly residues). Increased csgB expression in the presence of Ala, Gly and Thr is consistent with their conversion to pyruvate contributing to gluconeogenesis. For the aromatic amino acids, due to solubility and concentration problems, we only tested Phe, which had no significant effect on csgB expression. This was unfortunate since S. enterica strains defective in aromatic amino acid biosynthesis are unable to form biofilms [83], and tryptophan has been shown to have an important role in S. Typhimurium biofilms [84]. Tryptophan was also not present in CAA, as it is destroyed during the acid hydrolysis process [85]. More research is needed to understand the impact of individual amino acids on csgD expression.
Glucose was the most powerful external signal tested in our experiments. Under all growth conditions, the presence of exogenous glucose completely repressed the transcription of csgD, csgB and adrA. Expression of csgD was repressed in the presence of glucose even when rpoS was over-expressed from a plasmid or when levels of c-di-GMP were enhanced due to STM1987 activity. In the initial paper on carbon source foraging [86], the presence of glucose had a streamlining effect on the metabolism of E. coli when compared with growth on lower-quality carbon sources. This study was a genome-wide illustration of carbon catabolite repression [87], where growth on optimal carbon sources occurs first and genes for the metabolism of other carbon sources are repressed, usually acting through cyclic AMP (cAMP) and cAMP receptor proteins (CRP). Glucose had a repressive effect on biofilm formation in both S. Typhimurium and E. coli [12,59,79,88], however how the regulation is mediated is reported to be the opposite. High levels of cAMP repress csgD transcription in S. Typhimurium [79], but stimulate csgD transcription in E. coli [88]. It was also initially reported in S. Typhimurium that cAMP/CRP had no effect on csgD transcription [30]. It is hard to believe that the conserved divergent csgDEFG and csgBAC operons [89], biofilm networks and large intergenic region are capable of having opposite regulation in S. Typhimurium and E. coli. However, as pointed out by Hufnagel et al. [88], E. coli and S. Typhimurium have different evolutionary histories, hence could have differing regulatory responses to glucose. Another important aspect of cAMP/CRP regulation and glucose metabolism pertains to the quality of nitrogen source available [90], making this complex regulatory network in need of further study. It should be noted that the repressive effect of glucose did not change according to whether csgD transcription was activated or not, which was in contrast to the other nutritional signals that we tested.
The effects of temperature and c-di-GMP on csgD transcription were also evaluated. Temperature was one of the first conditions identified to regulate biofilm formation [50,52]. Activation at temperatures below 30 • C is known to represent RpoS-dependent transcription of csgD. S. enterica strains with csgD promoter mutations can alleviate temperature-based repression by shifting transcription to be RpoD-dependent [51,52]. This may be a way for natural rpoS mutant strains to retain the ability to form biofilms, as there are always a few isolates within natural collections that display temperature-independent biofilm formation [12,21,50]. Temperature was able to shut off the biofilm network even after csgD was activated, proving that it is also a strong regulatory signal. S. typhimurium biofilm colonies were only formed at 37 • C if c-di-GMP levels were enhanced by stm1987 overexpression, with partial restoration if rpoS was overexpressed. Although these conditions are somewhat artificial, the c-di-GMP regulatory principles could be an important observation. We recently discovered that curli can be synthesized by S. typhimurium during murine infections, with csgD transcription activated at 37 • C in vivo [91]. It is also of note that iron limitation [52] and exposure to bile [92] can alleviate temperature-based repression of csgD transcription. Finally, expression of the c-di-GMP-degrading enzyme, YhjH (or STM3611), was enough to repress csgD expression in all tested conditions, which is similar to previous observations [93,94].

Conclusions
We have started to dissect the external signal hierarchy that regulates csgD transcription and CsgD-mediated biofilm formation in S. enterica. Most significantly, we identified differences in the regulatory responses based on whether or not csgD was activated before being exposed to a signal. These findings are summarized in Figure 8A,B. We hypothesize that the differences upon activation are related to the bistable expression of CsgD [40,42], similar to dedicated processes in other bacterial species. Even seemingly well-understood processes, such as diauxie-the switching of E. coli growth between two carbon sources-is subject to heterogeneity, as one sub-population of cells ceases growth once glucose has been exhausted, while the other subpopulation begins to grow on the second carbon source without delay [78]. The diauxic behavior was originally interpreted as the whole population of cells stopping growth during a transition period before starting growth on the second carbon source [95]. We hypothesize that many of the csgD regulatory elements that we have examined here are consistent between S. Typhimurium and E. coli [41], with some notable differences. With respect to phenotypic heterogeneity, we may only fully understand biofilm regulation once we are able to examine the fate of individual cells [27].  and csgC) with the intergenic region highlighted by transcription factor binding sites that have been experimentally verified in Salmonella (CpxR-black bars; H-NS-grey box; OmpR-hatched boxes). Phosphorylated OmpR binds the proximal, high affinity site under conditions of low osmolarity, which activates csgD transcription, and binds the distal, low affinity sites under conditions of high osmolarity, which represses csgD transcription [38]. The different regulatory elements that we have tested are shown: glucose; amino acids; growth temperature; and osmolarity, with sodium chloride, which is known to act via the CpxR/A system [37], and sucrose, which is known to act via H-NS [36]. The adrA gene encodes a diguanylate cyclase, which produces cyclic-di-GMP and allosterically activates cellulose production. (A) Glucose (> 25 mM), amino acids (> 0.5% casamino acids), temperature (> 32°C), salt and sucrose (> 25 mM) caused a reduction in csgD transcription and blocked transcription of csgBAC and adrA, preventing curli and cellulose biosynthesis. The effect of reduced c-di-GMP was tested by overexpression of the YhjH phosphodiesterase. The addition of individual amino acids was variable, with three leading to reduced csgD transcription (Asn, Pro, Arg), and seven leading to increased csgD transcription (Ile, Val, Gln, Met, Ala, Thr, Gly). (B) When the same regulatory components were tested after 18 h of growth, the effects were different. We assume that by this time point, the CsgD-IraP-RpoS feedforward loop [35] is activated, although deletion of iraP in our experiments had little effect. The addition of salt and sucrose had no effect on csgD transcription, and casamino acids were not as repressive. The effect of increased c-di-GMP was tested by overexpression of the diguanylate cyclase STM1987, which was able to relieve temperature-based repression of csgD transcription. The response to individual amino acids was again variable, however, none caused a reduction in csgD transcription and eight were stimulatory (Leu, Arg, His, Val, Pro, Ala, Gln, Thr). The question mark signifies that we do not fully understand the regulatory effects of individual amino acids. The divergent csg operons are shown (without csgFG and csgC) with the intergenic region highlighted by transcription factor binding sites that have been experimentally verified in Salmonella (CpxR-black bars; H-NS-grey box; OmpR-hatched boxes). Phosphorylated OmpR binds the proximal, high affinity site under conditions of low osmolarity, which activates csgD transcription, and binds the distal, low affinity sites under conditions of high osmolarity, which represses csgD transcription [38]. The different regulatory elements that we have tested are shown: glucose; amino acids; growth temperature; and osmolarity, with sodium chloride, which is known to act via the CpxR/A system [37], and sucrose, which is known to act via H-NS [36]. The adrA gene encodes a diguanylate cyclase, which produces cyclic-di-GMP and allosterically activates cellulose production. (A) Glucose (>25 mM), amino acids (>0.5% casamino acids), temperature (>32 • C), salt and sucrose (> 25 mM) caused a reduction in csgD transcription and blocked transcription of csgBAC and adrA, preventing curli and cellulose biosynthesis. The effect of reduced c-di-GMP was tested by overexpression of the YhjH phosphodiesterase. The addition of individual amino acids was variable, with three leading to reduced csgD transcription (Asn, Pro, Arg), and seven leading to increased csgD transcription (Ile, Val, Gln, Met, Ala, Thr, Gly). (B) When the same regulatory components were tested after 18 h of growth, the effects were different. We assume that by this time point, the CsgD-IraP-RpoS feed-forward loop [35] is activated, although deletion of iraP in our experiments had little effect. The addition of salt and sucrose had no effect on csgD transcription, and casamino acids were not as repressive. The effect of increased c-di-GMP was tested by overexpression of the diguanylate cyclase STM1987, which was able to relieve temperature-based repression of csgD transcription. The response to individual amino acids was again variable, however, none caused a reduction in csgD transcription and eight were stimulatory (Leu, Arg, His, Val, Pro, Ala, Gln, Thr). The question mark signifies that we do not fully understand the regulatory effects of individual amino acids.