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Article

Characterisation of Variants of Cyclic di-GMP Turnover Proteins Associated with Semi-Constitutive rdar Morphotype Expression in Commensal and Uropathogenic Escherichia coli Strains

by
Annika Cimdins-Ahne
1,
Ali-Oddin Naemi
2,
Fengyang Li
1,†,
Roger Simm
2,3 and
Ute Römling
1,*
1
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 171 77 Stockholm, Sweden
2
Institute of Oral Biology, University of Oslo, 0313 Oslo, Norway
3
Norwegian Veterinary Institute, 0106 Oslo, Norway
*
Author to whom correspondence should be addressed.
†
Current address: State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130015, China.
Microorganisms 2023, 11(8), 2048; https://doi.org/10.3390/microorganisms11082048
Submission received: 16 July 2023 / Revised: 1 August 2023 / Accepted: 7 August 2023 / Published: 9 August 2023
(This article belongs to the Special Issue The Latest Research on Microbial-Associated Biofilm)
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Abstract

:
Expression of rdar (red, dry, and rough) colony morphology-based biofilm formation in Escherichia coli is highly variable. To investigate the molecular mechanisms of semi-constitutive rdar morphotype formation, we compared their cyclic di-GMP turnover protein content and variability to the highly regulated, temperature-dependent morphotype of the historical and modern ST10 isolates E. coli MG1655 and Fec10, respectively. Subsequently, we assessed the effects of cyclic di-GMP turnover protein variants of the EAL phosphodiesterases YcgG and YjcC and the horizontally transferred diguanylate cyclase DgcX on biofilm formation and motility. The two YcgG variants with truncations of the N-terminal CSS signaling domain were oppositely effective in targeting downregulation of rdar biofilm formation compared to the full-length reference protein. Expression of the C-terminal truncated variants YjcCFec67 and YjcCTob1 showed highly diminished apparent phosphodiesterase activity compared to the reference YjcCMG1655. For YjcCFec101, substitution of the C-terminus led to an apparently inactive enzyme. Overexpression of the diguanylate cyclase DgcX contributed to upregulation of cellulose biosynthesis but not to elevated expression of the major biofilm regulator csgD in the “classical” rdar-expressing commensal strain E. coli Fec10. Thus, the c-di-GMP regulating network is highly complex with protein variants displaying substantially different apparent enzymatic activities.

1. Introduction

The genomic variability of organisms contributes to their adaptive potential. Microorganisms, particularly bacteria with their small genomes often in multiple copies, are masters of stress survival and adaptation. While a major focus has been on the effects of horizontally transferred accessory elements, which undoubtedly introduce novel features into bacterial genomes in quantum leaps, it has recently been realized that even single nucleotide polymorphisms (SNPs) can have a substantial effect on the cell’s physiology, metabolism, and virulence. These SNPs have been mainly explored upon being non-synonymous in open reading frames (ORFs). For example, non-synonymous mutations in a variety of ORFs have been shown to contribute to the enhanced virulence/reduced biofilm phenotype of the emerged African invasive ST313 clone of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium [1]. However, few studies have also explored SNPs in the more variable intergenic regions and in promoter regions. For example, the insertion/transversion of a single nucleotide in the promoter region of the major rdar biofilm regulator CsgD promotes temperature and stress sigma factor RpoS-independent expression of the downstream csgDEFG operon [2].
Biofilm formation, the capability of microorganisms to build up multicellular communities supported by extracellular matrix, is an intrinsic feature of almost any bacterial species. The multifunctional biofilm can thereby play diverse determinative roles in the interaction with the environment equally, as with a eukaryotic host. The exopolysaccharide cellulose, a C6 phosphoethanolamine modified β-1,4-glucan with a partial crystalline structure, and depolymerization-inert amyloid curli fimbriae are the extracellular matrix components, the biosynthesis of which is triggered by the csgD rdar biofilm activator [3,4,5,6]. The resulting spore-like biofilm structure promotes long-term survival in the environment equally, as it restricts acute virulence in mammalian hosts [7]. Although csgD expression is a major regulatory hub from the transcriptional to the post-translational level, a major determinant of csgD expression and cellulose biosynthesis is the complex signaling network of cyclic di-GMP, a ubiquitous bacterial second messenger [8]. Several of the 31 E. coli and 22 S. typhimurium proteins with a GGDEF diguanylate cyclase, an EAL phosphodiesterase domain, or an evolved EAL domain devoid of catalytic activity and cyclic di-GMP binding regulate the expression of this central rdar biofilm activator [9,10]. Notably, SNPs in cyclic di-GMP turnover proteins have been demonstrated to determine biofilm expression and virulence [11].
We recently observed a high diversity of expression patterns of rdar biofilm formation among commensal and uropathogenic isolates of E. coli [12,13,14]. This variability contrasts with the almost uniform appearance of rdar morphotype expression in S. typhimurium and related serovars of Salmonella enterica [15].
Additionally, in E. coli, mutations in the promoter region of csgD, the major biofilm activator gene, have been found to promote semi-constitutive (including temperature-independent) rdar biofilm formation [2,16]. However, not all E. coli strains with semi-constitutive rdar biofilm formation possess promoter mutations; thus, other mechanisms must cause upregulation of csgD expression. As others, we observed high variability in the biofilm regulating the cyclic di-GMP network genes of E. coli strains. Usually, the effects of gene variants are not immediately accessible unless amino acids in conserved signature motifs required for catalytic activity are directly addressed by non-synonymous nucleotide substitutions or frameshifts, leading to a premature stop in the open reading frame. However, we recently noted that amino acid (aa) substitutions that occur outside of signature motifs determinative for catalytic activity can be crucial for apparent enzymatic activity and consequently may contribute to differential expression of the rdar colony biofilm [17]. In this context, distinct, single aa substitutions in the FI-PAS-GGDEF-EAL domain protein YciR, associated with upregulated rdar biofilm formation, reduced the regulatory activity at ambient and body temperatures in the commensal E. coli Tob1, chosen as the test strain.
As multiple components might contribute to the emergence of semi-constitutive rdar morphotype formation, in this work, we aimed to further unravel the molecular basis of semi-constitutive rdar biofilm expression in commensal and uropathogenic E. coli strains by comparative genomic analyses. We compared their genome sequences with those of ST10 E. coli strains, the commensal strain E. coli K-12 MG1655, and the recently isolated human commensal strain E. coli Fec10, which express a highly regulated rdar biofilm exclusively at ambient temperature [18]. With a focus on investigating the correlation between sequence variations of proteins involved in the turnover of the second messenger c-di-GMP and semi-constitutive rdar biofilm formation [19], we addressed the effects of alterations on the cyclic di-GMP phosphodiesterases YcgG and YjcC and the horizontal transfer of the diguanylate cyclase DgcX on semi-constitutive rdar biofilm expression. Our results show that two YcgG variants with several aa substitutions and slightly different truncations of the N-terminal signaling domain downregulated the semi-constitutive rdar biofilm to different degrees compared to the reference YcgG of E. coli MG1655. Equally, YjcC variants with aa substitutions and truncation/substitution of the C-terminal aa sequence displayed highly downregulated or abolished apparent catalytic activity. On the other hand, as overexpression of the diguanylate cyclase DgcX contributed to upregulation of cellulose biosynthesis but did not elevate production of the rdar biofilm regulator CsgD, additional components might contribute to the semi-constitutive rdar morphotype and csgD expression at 37 °C in the commensal strain E. coli Fec101.

2. Materials and Methods

2.1. Strains and Growth Conditions

All commensal and uropathogenic E. coli strains analyzed in this work have been previously described and genome sequenced, and the cyclic di-GMP turnover network has been characterized [14]. E. coli strains expressing the rdar colony morphotype semi-constitutively were E. coli Tob1, Fec67, Fec101, B8638, B-11870, 80//6, and No. 12. Strains were propagated on LB agar or grown on LB without salt plates supplemented with 2% Congo red (CR) and Coomassie brilliant blue G stock solution (CR at 2 mg/mL, Brilliant Blue G at 1 mg/mL in 70% (v/v) ethanol) as described to visualize the rdar biofilm phenotype [19]. If required, 100 µg/mL ampicillin and 0.1% L-arabinose were added to the medium for plasmid propagation and gene expression, respectively. All strains used in the study are listed in Table S1.

2.2. Cloning Procedures and Site-Directed Mutagenesis

Genes of interest were amplified and ligated into the pBAD30 vector [20] via XbaI/HindIII restriction sites. Site-directed mutagenesis was performed with phusion® High-Fidelity DNA Polymerase and an overlap extension PCR approach using 17 cycles to introduce the mutations. A DpnI digest to remove methylated DNA ensured that only mutated plasmid was to be transformed. All constructs were verified by sequencing. All plasmids used in the study are listed in Table S2, and the primers are listed in Table S3.

2.3. Mutant Construction

Construction of the dgcX chromosomal deletion mutant was performed via λ red-mediated homologous recombination [21]. In brief, a kanamycin resistance cassette was amplified from the pKD4 template flanked by 40-bp homologous sequences at the beginning and end of the target gene. After electroporation into E. coli Fec101 carrying helper vector pKD46, the mutants were selected on 25 μg/mL and subsequently 50 μg/mL kanamycin, verified by PCR with primers flanking the replaced ORF and cured of pKD46 by incubation at 42 °C. Primers are listed in Table S3.

2.4. Analysis of rdar Colony Morphology and Type 1 Fimbriae Expression

Congo red LB without salt plates was used to visualize expression of the rdar morphotype. Dye binding to both cellulose and curli fibers stains the bacterial colony [2,9,12]. Strains were streaked for single colonies, or 5 μL of a suspension of OD600 = 5 was spotted onto the agar plates. If applicable, 100 µg/mL ampicillin and 0.1% L-arabinose were added. Development of the colony morphology and color was analyzed at 28 °C and 37 °C, respectively, and documented by taking photographs at distinct time points for up to 72 h.
To visualize cellulose expression, 5 μL of a suspension of OD600 = 5 were spotted onto LB without salt agar plates supplemented with 50 μg/mL Calcofluor white (Fluorescent Brightener 28, Sigma, St. Louis, MO, USA). Fluorescence was observed under UV light of a wavelength of 365 nm.
Type 1 fimbriae were assessed by the pellicle assay performed in standing culture in LB medium at 37 °C for 48 h. Pellicles were documented by taking photographs from above and as a side view.

2.5. Flagella-Based Motility

Strains were inoculated into LB motility plates containing 0.25% agar using a toothpick. If applicable, 100 µg/mL ampicillin and 0.1% L-arabinose were added. Agar plates were incubated at 28 °C and 37 °C for 6 h.

2.6. SDS PAGE and Western Blotting

To investigate CsgD production, the strains of interest were grown on LB without salt agar plates for 16–18 h. For the analysis of YcgG synthesis, strains were grown on LB without salt agar plates supplemented with 100 µg/mL ampicillin and 0.1% L-arabinose for 16 h. Five milligrams of cells were harvested and suspended in 1× SDS sample buffer, followed by boiling at 95 °C for 10 min. The samples were separated on a denaturing SDS PAGE (4% stacking and 12% (for detection of His-tagged YcgG) or 15% (for CsgD detection) resolving gel). Separated proteins in gels were visualized with a staining solution (0.1% Coomassie Brilliant Blue G-250, 2% (w/v) ortho-phosphoric acid, 10% (w/v) ammonium sulfate, (Sigma)) to allow for adjustment of equal protein content, or semidry western blotting was performed at 120 mA for 1 h to transfer the contained proteins onto a PVDF membrane (Immobilon P; Millipore, Burlington, MA, USA). CsgD protein was detected using a custom-made primary rabbit polyclonal anti-E. coli CsgD peptide antibody [22] and a goat anti-rabbit secondary antibody (Jackson Immuno Research) (both antibodies were diluted 1:3000). His-tagged proteins were detected using 1:1500 diluted mouse anti-Penta-His antibody (Qiagen, Hilden, Germany) and a 1:2000 diluted goat anti-mouse secondary antibody (Jackson Immuno Research). After treatment with Lumi-Light Western Blotting substrate (Roche, Basel, Switzerland), the resulting chemiluminescence was detected via an LAS-1000 detector (Fujifilm, Tokyo, Japan).

2.7. Bioinformatic Tools

The RAST-Server (Rapid Annotation using Subsystem Technology) was used for initial genome and proteome comparisons [23,24,25]. Prediction of functional protein domains was performed using SMART (accessed latest 8 June 2023, http://smart.embl-heidelberg.de) [26,27]. BLAST was used to retrieve homologous proteins [28]. The tool ClustalX 2.1 [29] or MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/; [30]), which is implemented in AliView (version 1.17.1, [31]), was used to perform multiple sequence alignment. The Sequence editor http://www.fr33.net/seqedit.php, the Expasy translate tool http://web.expasy.org/translate/, and GeneDoc were used to view and manage sequences. ESPript software, version 3.0, was used to visualize sequence alignments [32]. Structural models were either taken from UniProt (Alphafold models) [33] or constructed with Phyre2 [34]. Structural models were compared and visualized with Chimera software, version 1.14 [35]. Ident and Sim (www.bioinformatics.org/sms2/ident_sim.html; accessed latest 15 June 2023) were used to calculate the pairwise identity and similarity of aligned protein sequences.

2.8. Analyzed Protein Sequences

The YjcC sequences aligned in Figure 3 were (clockwise) YjcC_ECOL6, A0A0H2VD18_ECOL6 of E. coli CFT073; YjcC_ECO27, B7UPN0_ECO27 of EPEC strain E2348/69; YjcC_ECO8N, A0A0H3ESW7_ECO8N of AIEC strain NRG 857C; YjcC_ECO45, B7MJS8_ECO45 of ExPEC strain S88; YjcC_ECOK1, A0A0H2Z591_ECOK1 of APEC O1:K1; YjcC_ECO57, A0A0H3JK63_ECO57 of O157:H7; YjcC_ECOBD, A0A140NEW3_ECOBD of strain B BL21-DE3; YjcC_ECOLI, P32701 of E. coli K-12; YjcC_ECOHS, A0A7I6HB99_ECOHS of strain HS; YjcC_ECO24, A7ZUT3_ECO24 of ETEC strain E24377A; YjcC_ECO55, B7LB02_ECO55 of EAEC strain 55989; YjcC_SHISS, Q3YUS4_SHISS of Shigella sonnei Ss046; YjcC_CVM_N17EC1334, EAC1547616.1; YjcC_BLSE174, HCL8611405.1; YjcC_FSIS11705879, EFA9343023.1; YjcC_33, HBN0449758.1; and YjcC_EPECa14, EFZ42063.1.
The GGDEF domains investigated in Figure 4 were from the proteins (clockwise direction) Shigella sonnei EGF2695153.1 and EFZ2348867.1, E. coli WP_00058633.1, E. coli TW14182 WP_000293658.1, Salmonella kentucky EBV8389552.1, Escherichia albertii WP_105197891.1, Escherichia WP_105281683.1, Citrobacter sp. R56 WP_203358054.1, Escherichia fergusonii KWW06214.1, E. fergusonii WP_002432239.1, E. albertii KAF0954534.1, Escherichia WP_123058197.1, Escherichia WP_165382775.1, Klebsiella pneumoniae WP_134392503.1, E. fergusonii HAJ6531924.1, Enterobacter hormaechei subsp. xiangfangensis MBT1749060.1, and Enterobacter ludwigii WP_063214496.1.

3. Results

We identified two additional candidate genes encoding cyclic di-GMP turnover proteins associated with semi-constitutive rdar morphotype expression, the function of which is potentially altered/modulated by non-synonymous single nucleotide changes in combination with 5′ or 3′ alteration/truncation of the ORFs. These genes were ycgG coding for a redox-regulated cyclic di-GMP phosphodiesterase, previously shown to be involved in regulation of motility [36]; and yjcC encoding a phosphodiesterase (PDE), previously shown to mediate rdar morphotype regulation in E. coli [37,38]. In addition, dgcX coding for a horizontally transferred diguanylate cyclase (DGC) first identified in the 2001 EAEC outbreak strain [39] has been identified in the commensal strain Fec101.

3.1. The N-Terminal Sequence of Truncated YcgG Affects Protein Expression and rdar Morphotype Formation

In E. coli K-12 MG1655, the PDE YcgG consists of an N-terminal redox responsive CSS domain of around 200 aas [40,41] flanked by transmembrane domains and a C-terminal EAL domain. A full-length YcgG protein is present also in the modern equivalent of E. coli K-12. the commensal strain Fec10, in the commensal strain Fec101, and in the uropathogenic strain B-8638. E. coli Fec101 and B8628 both express a semi-constitutive rdar morphotype. As previously reported, YcgG from Fec10 and Fec101 possesses the aa substitution Y474N, while YcgG of B-8638 contains six additional aa substitutions [17]. In contrast, in the commensal strains Tob1 and Fec67 and the uropathogenic strains B-11870 (including the clonal variant 80//6) and No.12, YcgG has a truncated N-terminus, leaving basically only the EAL domain. Notably, the truncated nucleotide regions are not entirely identical, resulting in YcgG from E. coli Tob1 to possess a two aa longer and divergent 22 aa stretch at the N-terminus compared to the truncated YcgG variant of YcgGB-11870, where the N-terminus is congruent with the aa sequence of YcgGMG1655 at that position (Figure 1). Further, YcgGTob1 and YcgGB-11870 contain aa substitutions compared to YcgGMG1655 (Figure 1). The aa exchange A298V is unique to YcgGTob1; S306C, F358Y, E437A, L467F, and Y474N substitutions are present in YcgGTob1, and YcgGB-11870 and V504I are unique to YcgGB-11870 (Figure 1, [17]). Furthermore, the ORF of ycgGB-11870 has the rare start codon TTG in contrast to ycgGMG1655 and ycgGTob1, which have ATG as a start codon. However, changing TTG to the conventional ATG start codon has recently been demonstrated not to substantially alter protein expression in E. coli [42].
To investigate the consequences of the expression of the highly similar, yet distinct proteins with deletion of the CSS sensory domain, we analyzed the effect of expression of YcgGMG1655, YcgGTob1, and YcgGB-11870 on rdar morphotype formation and production of its major activator CsgD in the semi-constitutive rdar strain E. coli Tob1 (rdar28 °C/rdar37 °C) at 28 °C and 37 °C (Figure 2A). To restrict the assessment of functionality solely on the gene product, the coding regions were cloned under the control of the L-arabinose-inducible pBAD promoter in pBAD30 with an identical Shine–Dalgarno sequence. While full-length YcgGMG1655 slightly downregulated rdar morphotype formation and the protein level of the master rdar biofilm regulator CsgD, expression of truncated YcgGB-11870 unexpectedly led to a highly reduced rdar morphotype and low levels of CsgD production (Figure 2B). In contrast, expression of YcgGTob1 was less effective than YcgGMG1655 in downregulating the rdar colony morphotype and had no visible effect on CsgD synthesis. Notably, the effect of YcgG production on rdar morphotype formation was consistently observed at 28 °C and 37 °C. CsgD levels could only be reliably assessed at 28 °C. Although expression of soluble protein at 37 °C is at the detection limit of western blot, we judged that none of the constructs affected CsgD production at 37 °C (Figure 2B; [17]).
To investigate whether downregulation of rdar biofilm formation and its major activator is due to altered protein production levels or impaired enzymatic activity of YcgG upon truncation, we detected the production of YcgG via the C-terminal 6xHis-tag by western blotting. While production of YcgGMG1655 wild-type protein was detected at 28 °C and 37 °C, YcgGB-11870 was produced to a significantly greater extent. Notably, production of YcgGB-11870 was increased at 37 °C, suggesting that elevated temperature or decreased proteolysis stabilizes this YcgG variant. Concomitant with the high production of YcgGB-11870, a specific non-biofilm forming, smooth, and white (saw) morphotype emerged at 28 °C. In contrast, production of YcgGTob1 could not be observed by western blot analysis despite visible effects on rdar colony morphology (Figure 2C). In summary, the effect on rdar morphotype formation and CsgD expression of the YcgG variants strongly correlates with their protein production levels.

3.2. YjcC Proteins with C-Terminal and Individual Amino Acid Substitutions Show Diminished Apparent Phosphodiesterase Activity and Downregulation of the rdar Colony Morphotype

The phosphodiesterase YjcC is a major phosphodiesterase in S. typhimurium, determinative of temperature-dependent rdar colony morphotype expression [37]. In E. coli, MG1655 yjcC appears to play a more restricted role with alternative phosphodiesterases, being dominant with respect to affecting rdar biofilm colony morphology [38]. We noted variants of the phosphodiesterase YjcC in the semi-constitutive rdar morphotype-expressing strains E. coli Tob1, Fec101, and Fec67 (Figure S3). In strains Tob1 and Fec67, the C to T transition at position 1552 of the yjcC ORF created a stop codon leading to a 11 aa shorter protein (Figure S2). In strain Fec101, the deletion of an adenosine nucleotide at position 1433 led to a frameshift and the substitution of the last 49 C-terminal aa of YjcC with a 44 aa long unrelated sequence (Figure S3). In addition, YjcC proteins of strains Tob1 and Fec67 possess multiple, partly distinct, aa substitutions compared to reference YjcCMG1655, while YjcCFec101 displays two aa substitutions (P309L D403E) (Figure S3; [17]). To investigate the impact of the YjcC variants with the altered C-termini, we cloned the ORFs under the control of the same Shine–Dalgarno sequence and with the same extension of the nucleotide sequences 3′ of the stop codon (Table S1). Expression of these constructs and reference YjcCMG1655 in E. coli Tob1 showed production of YjcCMG1655 to substantially downregulate the rdar colony morphotype at 28 °C (Figure 3A). While the partially truncated protein variant YjcCTob1 and, to a minor extent, YjcCFec67 still downregulated the rdar morphotype at 28 °C, YjcCFec101 did not substantially alter the rdar colony morphology type compared to the vector control. Notably, YjcCMG1655 and YjcCFec67 expression downregulated the rdar morphotype also at 37 °C to a minor extent, while expression of YjcCTob1 displayed a substantially altered, although still downregulated, rdar colony morphology. We therefore must assume that the two aa substitutions that discriminate YjcCFec67 from YjcCTob1 are responsible for this different effect on the rdar colony morphotype.

3.3. YjcC Proteins with Similar C-Terminal and Amino Acid Substitutions Are Present in the Database

The YjcC phosphodiesterase proteins with C-terminal substitutions and truncations are not unique to our strain collection (Figure S2). YjcC proteins with the 11 aa shorter C-terminus and the same or similar aa substitutions, such as YjcCTob1/YjcCFec67, compared to YjcCMG1655 are found encoded by a number of isolates among the sequenced E. coli strains including the uropathogenic strain E. coli CFT073. Equally, at least eight human E. coli isolates, including a human commensal, pathogens such as strain EPECa14, animal, and wastewater strains, possess a YjcC with a C-terminal substituted aa sequence highly similar to YjcCFec101 with the second glutamate of the EVTE motif involved in divalent cation binding missing (Figure S2). Notably, distinct single nucleotide deletions and deletion of several nucleotides led to a similar C-terminal aa sequence substitution in YjcC in these epidemiologically unrelated strains. The phylogenetic tree clearly discriminates among these three distinct classes of YjcC protein variants (Figure 3B). Despite a divergent C-terminus, however, the structural model indicates a similar structure of the C-terminus of YjcCFec101 compared to YjcCMG1655 (Figure 3C).

3.4. The Novel Diguanylate Cyclase DgcX Is Critical for Biofilm Formation at Human Body Temperature

The DGC DgcX, not present in E. coli K-12 and Fec10, was first described as a highly expressed diguanylate cyclase in the E. coli O104:H4 2011 German outbreak strain, and it is considered to be a virulence determinant (Figure 4A; [39]). The genome of the commensal E. coli strain Fec101 showed only minor deviations in the c-di-GMP network and the biofilm components compared to the ST10 reference strains with respect to SNPs and gene rearrangements, but it contains the additional DGC DgcX, a MASE4-GGDEF domain protein [17,39,44]. DgcX was thus identified as a candidate diguanylate cyclase responsible for the observed upregulated rdar colony morphotype at 37 °C in the commensal strain Fec101.
Interestingly, the chromosomal location and gene context of dgcX vary among the strains harboring this gene. Database searches reveal that the O104:H4 outbreak strain and other EAEC strains harbor dgcX and the adjacent inserted prophage between ybhC (position complement 805998–807281 relative to the E. coli K-12 MG1655 genome) and ybhB (position complement 807433–807909). The nature of the prophage as such differs, however [39]. In the environmental strain E. coli SE11, dgcX, along with an adjacent prophage, is inserted between ydaW (position 1422701–1423200) and uspF (position complement 1435185–1435619) [39]. In Fec101, dgcX is present as part of a 9-kb insertion yet at another location between argW and dsdC (Figure S3A). In E. coli K-12 MG1655, the prophage CPS-53/KpLE1 [45] is inserted between argW (position 2466309–2466383 in K-12 MG1655) and dsdC (position complement 2476694–2477629).

3.5. Overexpression of DgcX Induces Cellulose Production at 37 °C in the Regulated rdar Colony Morphology Type Strain E. coli Fec10

DgcX harbors a GGDEF domain that is seemingly functional as a diguanylate cyclase, with an intact GGEEF catalytic motif and other conserved aa motifs required for catalytic activity, substrate binding, and metal ion coordination [46,47]. N-terminal of the GGDEF domain, DgcX contains a MASE4 domain [35] with six transmembrane regions (Figure 4A). The closest homologs to DgcX over the entire length of the protein in E. coli K-12 is the catalytically non-functional YeaI (CdgI) and the diguanylate cyclase YcdT (Figure 4B and Figure S3). Compared to other DGCs of E. coli, DgcX is highly expressed at both 28 °C and 37 °C [35].
To test the effect of DgcX against the Fec101 background, we constructed a dgcX deletion mutant (Figure S3B). Assessment of rdar morphotype expression, however, showed only a minor effect of the dgcX deletion on rdar biofilm formation, while the deletion mutant of the gene for the major biofilm activator csgD displayed a residual pink, dry, and rough (pdar) morphotype indicating cellulose but no curli fiber biosynthesis.
To investigate the effect of dgcX overexpression on rdar morphotype formation, dgcX was cloned with either an N- or C-terminal 6xHis-tag into the cloning vector pBAD30 under the control of an L-arabinose-controlled promoter. DgcX was subsequently expressed in strain E. coli Fec10, which features a highly regulated rdar morphotype at 28 °C and a saw morphotype at 37 °C, which facilitates assessment of the functionality of a diguanylate cyclase [12,18]. Overexpression of dgcX enhanced the rdar morphotype at 28 °C (Figure 4C, left panel) and resulted in a pdar morphotype and enhanced Calcofluor white binding at 37 °C (Figure 4C, right panel), suggesting csgD-independent cellulose production. Upregulation of cellulose expression is dependent on the catalytic activity, as DgcX variants with mutations of the GGEEF motif to GGEAF or GGAAF no longer induced upregulation of rdar morphotype expression, while modest upregulation in colony morphology was still observed in the GGAEF mutant. Remarkably, overexpression of DgcX did not alter production of CsgD in Fec10 at either 28 °C or 37 °C (Figure 5A). This outcome is in contrast to observations in the 2011 E. coli outbreak strain, in which DgcX elevated CsgD production, but cellulose was not synthesized due to a frame shift mutation in the gene encoding the c-di-GMP binding protein BcsE [39]. BcsE has recently been shown to be required for optimal cellulose production in E. coli and S. typhimurium [48]. Moreover, to clearly show the effect of DgcX at 28 °C, we expressed DgcX in the Fec10 ∆csgD strain. Expression of dgcX induced pdar morphotype expression also at 28 °C but to a lesser extent (Figure 5B). Thus, DgcX conditionally contributes to semi-constitutive rdar morphotype expression in Fec101 by upregulation of csgD-independent cellulose expression.
Notably, the effect of DgcX on cellulose expression is remarkably stronger for the N-terminally, rather than for the C-terminally, tagged construct with barely visible activity at 28 °C indicating that the tag either interferes with (at the C-terminus) or promotes (at the N-terminus) enzymatic activity and/or protein stability (Figure 4C and Figure 5B).

3.6. Effect of dgcX on Flagella-Based Motility in E. coli Fec101

Cyclic di-GMP signaling regulates the lifestyle transition from sessility (biofilm formation) to motility. We were therefore wondering whether dgcX affects motility. We have previously shown that Fec101 exhibits moderate motility [17]. The corresponding motility assay showed that E. coli Fec101 ΔdgcX has slightly higher motility than the Fec101 wild type at 28 °C and 37 °C (Figure S3B).
Consistent with the motility phenotype of the dgcX deletion mutant, overexpression of dgcX in Fec101 repressed motility at 28 °C and 37 °C. As expected with the predicted loss of the diguanylate cyclase activity, the GGAAF and GGEAF mutants did not repress motility, while the GGAEF mutant still displayed motility repression, suggesting, as in the case of regulation of rdar colony morphology, at least residual catalytic activity of the DgcX GGAEF variant (Figure 4D). Thus, in DgcX, unexpectedly, the second glutamate of the GGEEF motif is dominant in determining catalytic activity [46,47].

3.7. DgcX Is Restricted to E. coli Species

Previously, dgcX was found to be encoded by the genomes of the EAEC strains LB226692, 2011C-3493, HUSEC041, 55989, 2009EL-2071, 2009EL-2050, ETEC H10407, E24377A, and the commensal strain SE11 [39,44]. A protein BLAST search against the NCBI microbial genomes database revealed the presence of DgcX (>75% query cover and >50% identity of the aa sequence) in more than 1150 strains of the genus Escherichia, mostly E. coli, a few Shigella spp., and Escherichia albertii (Figure 4B and BLAST search performed in June 2023). Thus, the presence of dgcX, although obtained through horizontal gene transfer, is indeed restricted to E. coli. In conclusion, the gene product DgcX is highly conserved and present only in strains of E. coli and closely related species of the Escherichia genus, while the highly similar gene products YcdT and YeaI/CdgI can also be present in related genera (Figure 4B and Figure S3C,D). This outcome suggests recent diversifying radiation of the YcdT/DgcX/YeaI family specifically in E. coli with one of its members, DgcX, subject to extended mobilization. Identity/similarity analyses of the aligned sequences showed, however, that threshold identity/similarity values can be determined to categorize proteins of the YcdT/DgcX/YeaI subfamilies, in line with above-mentioned threshold values with the potential assignment of founders for novel protein subfamilies. (Figure S3C,D).

4. Discussion

Within a species, biofilm formation can be highly variable; however, the underlying molecular mechanisms in natural isolates remain to be determined. We have started to dissect the molecular basis of the temperature-independent rdar colony morphotype expression of three commensal and three uropathogenic E. coli strains compared to the commensal strain E. coli MG1655 and its recently isolated ‘modern’ ST10 counterpart E. coli Fec10. Both of these strains express a highly regulated rdar biofilm only at temperatures less than 30 °C [17,18]. These analyses subsequently revealed high variability in the enzymes that conduct turnover of the second messenger c-di-GMP associated with differential biofilm expression. In a subset of strains, distinct aa substitutions in the trigger diguanylate cyclase/phosphodiesterase YciR were experimentally coupled with reduced ability to downregulate csgD encoding the master regulator of rdar biofilm formation and, upon introduction of a nonsense mutation, even to upregulate csgD by a truncated FI-PAS-GGDEF protein acting as an apparent diguanylate cyclase [17]. However, additional alterations in alternative cyclic di-GMP turnover proteins might cumulatively contribute to semi-constitutive rdar morphotype expression in E. coli strains. In this work, we investigated the effect of genomic alterations occurring in ycgG and yjcC coding for redox-regulated cyclic di-GMP phosphodiesterases and the effect of the acquisition of dgcX, which codes for a horizontally transferred diguanylate cyclase in the commensal strain E. coli Fec101. DgcX was first identified in the 2011 EAEC outbreak strain [39]. Findings in this work could be extended to investigate the molecular basis of substantially different behaviors of protein variants equally as natural evolution toward potentially novel protein functionalities in their physiological and evolutionary contexts. For example, the specific feature of yjcC in regulating the temperature dependence of the rdar morphotype in S. typhimurium in contrast to E. coli representatives might be an acquired virulence trait specific for either Salmonella or E. coli, causing intestinal infection.
Although the EAL domain of the CSS-EAL protein YcgG is present in all investigated E. coli strains, the N-terminal part of the protein was truncated in a subset of isolates due to deletion of larger genomic sequences. Indeed, we have also observed a deletion of the CSS domain of YcgG in all strains of the multidrug-resistant pandemic E. coli ST131 clone (unpublished observations). However, the deletion of the N-terminal periplasmic CSS sensory domain left in all cases an ORF encoding an intact EAL phosphodiesterase domain. The deletions, however, also led to distinct protein variants that, upon overexpression, showed opposite performance in production levels and consequently downregulated the rdar morphotype relative to the ST10 E. coli wild-type protein. Thereby only the effect of the ycgGTob1 variant resulting in a lower protein level is in congruence with the expression of a semi-constitutive rdar morphotype. It is possible that the rare TTG start codon of ycgGTob1 causes significantly lower protein production. Alternatively, the novel unique 22-aa sequence at the truncated N-terminus and/or, less likely, the distinct nucleotide substitution A298V can affect protein folding, stability, or degradation, causing YcgGTob1 to be undetectable by standard western blot analyses, although its effect is still visible in the biological assay. However, it remains to be determined how ycgG expression has been altered upon introduction of the novel promoter in vivo in E. coli strain Tob1 (Figure S1D). Although expression of ycgG is almost undetectable in E. coli K-12 [36,50], deletion of a truncated ycgG (c1610), similar to ycgGB-11870, in the E. coli UPEC strain CFT073 increased the levels of the FimA subunit of type 1 fimbriae and enhanced adherence to bladder epithelial cells [51]. Thus, although we did not observe a decrease in type 1 fimbriae-dependent biofilm formation of E. coli Tob1 upon overexpression of the ycgG variants in laboratory assays (Figure S4), truncated ycgGB-11870 might decrease biofilm formation in other strains and/or under alternative conditions, such as in the host. In this context, ycgG in neonatal meningitis-causing E. coli (NMEC) and the uropathogenic isolate UTI89 is involved in the switch from iron acquisition to citrate fermentation via the citrate transporter CitT [52]. Whether the truncation of YcgG is causative for involvement in this metabolic activity has not been investigated.
In summary, the effect of ycgG variants is only partially congruent with enhanced rdar biofilm formation and might predominantly be involved in the regulation of alternative cyclic di-GMP-affected physiological and metabolic properties. YcgG belongs to a group of five redox sensing CSS-EAL domain proteins in E. coli [40,41]. Reducing conditions or deletion of the disulfide bond formation system components led to proteolytic cleavage of the YjcC and YlaB proteins, which resulted in reduced catalytic activity of the cytoplasmic EAL-only domain [41]. It is intriguing that the manifested genomic alterations observed here for ycgG in natural commensal and uropathogenic E. coli strains, alternative transcriptional start sites and additive proteolytic digest by the periplasmic proteases DegQ and DegP in the model strain E. coli K-12 under laboratory growth conditions led to a similar fragmentation pattern of a CSS-EAL protein [14,17,41,49,51,52,53].
In theory, at least three fundamentally different modes of regulation of semi-constitutive rdar biofilm expression by cyclic di-GMP turnover proteins can occur: reduced (apparent) phosphodiesterase activity, upregulated diguanylate cyclase activity of a chromosomally encoded diguanylate cyclase, and acquisition of a novel diguanylate cyclase (Figure 6). In the commensal strain E. coli Fec101, the situation might be more complex. The GGDEF-EAL domain protein variant YciR still substantially reduces rdar biofilm formation [17], and as shown in this work, the horizontally introduced diguanylate cyclase DgcX does not enhance production of the biofilm activator CsgD but contributes to rdar biofilm formation by upregulation of cellulose biosynthesis. Indeed, diguanylate cyclases transcriptionally independent of csgD have been shown to exclusively regulate cellulose biosynthesis in E. coli under alternative growth conditions [54]. The dedication of a diguanylate cyclase/phosphodiesterase to preferentially affect biofilm formation, a particular biofilm component, or motility is not uncommon.
In S. typhimurium, YjcC is the dominant phosphodiesterase-mediating temperature-dependent rdar morphotype [37]. In E. coli MG1655, yjcC has a less determinative role [38]. The investigated yjcC variants yjcCTob1, yjcCFec67, and yjcCFec101 showed subsequently decreased activity. Although YjcCFec101 seems to be catalytically inactive (as it lacks a glutamate required for divalent cation binding), it remains to be shown whether lack of this phosphodiesterase activity would be sufficient to promote CsgD production in Fec101 at 37 °C. On the other hand, the substitution of the last 49 C-terminal aa with a 44 aa-long unrelated sequence in YjcCFec101 might have led to a protein with a novel functionality that is not immediately visible by substantial alterations in the rdar colony morphology biofilm type. However, the substantially different appearance of the colony morphology of E. coli Tob1 upon overexpression of yjcCFec101 at 28 °C indicates expression and a physiological role of yjcCFec101.

5. Conclusions

In conclusion, characterization of the effects of variants of cyclic di-GMP specific phosphodiesterases on the downregulation of rdar colony morphotype expression and regulation of motility provided indications for their contributions to the semi-constitutive rdar morphotype, which is seen at high frequency in E. coli strains compared to the closely related species S. typhimurium.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms11082048/s1, Figure S1: Nucleotide sequence of the ycgG region from E. coli K-12 MG1655, commensal Tob1, and uropathogenic B-11870; Figure S2: Multiple alignment of YjcC of E. coli strains MG1655, Tob1, Fec67, and Fec101; Figure S3: Flanking region of the DGC encoding gene dgcX in Fec101; Figure S4: Pellicle assay indicative for the expression of type 1 fimbriae. Table S1: Strains constructed or used in this study; Table S2: Plasmids constructed or used in this study; Table S3: Oligonucleotides used in this study. References [55,56] are cited in the supplementary materials as reference [6] and [7], respectively.

Author Contributions

Conceptualization, A.C.-A. and U.R.; methodology, A.C.-A., A.-O.N., F.L., R.S. and U.R.; formal analysis, A.C.-A., A.-O.N., F.L., R.S. and U.R.; investigation, A.C.-A., A.-O.N., F.L., R.S. and U.R.; resources, R.S. and U.R.; writing—original draft preparation, A.C.-A. and U.R.; writing—review and editing, A.C.-A., F.L., R.S. and U.R.; supervision, U.R. and R.S.; project administration, U.R.; funding acquisition, A.C.-A. and U.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research has been supported by the German Research Foundation [Deutsche Forschungsgemeinschaft; CI 239/1-1 and CI 239/2-1, A.C.-A.] and by the Swedish Research Council for Natural Sciences and Engineering [621-2013-4809, U.R.].

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Sokaribo, A.S.; Balezantis, L.R.; MacKenzie, K.D.; Wang, Y.; Palmer, M.B.; Chung, B.; Herman, N.J.; McCarthy, M.C.; Chen, J.M.; White, A.P. A SNP in the cache 1 signaling domain of diguanylate cyclase STM1987 leads to increased in vivo fitness of invasive Salmonella strains. Infect. Immun. 2021, 89, e00810-20. [Google Scholar] [CrossRef] [PubMed]
  2. Römling, U.; Sierralta, W.D.; Eriksson, K.; Normark, S. Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter. Mol. Microbiol. 1998, 28, 249–264. [Google Scholar] [CrossRef] [PubMed]
  3. Thongsomboon, W.; Serra, D.O.; Possling, A.; Hadjineophytou, C.; Hengge, R.; Cegelski, L. Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose. Science 2018, 359, 334–338. [Google Scholar]
  4. Zogaj, X.; Nimtz, M.; Rohde, M.; Bokranz, W.; Römling, U. The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix. Mol. Microbiol. 2001, 39, 1452–1463. [Google Scholar] [CrossRef]
  5. Chapman, M.R.; Robinson, L.S.; Pinkner, J.S.; Roth, R.; Heuser, J.; Hammar, M.; Normark, S.; Hultgren, S.J. Role of Escherichia coli curli operons in directing amyloid fiber formation. Science 2002, 295, 851–855. [Google Scholar] [CrossRef] [Green Version]
  6. Hammar, M.; Arnqvist, A.; Bian, Z.; Olsén, A.; Normark, S. Expression of two csg operons is required for production of fibronectin- and congo red-binding curli polymers in Escherichia coli K-12. Mol. Microbiol. 1995, 18, 661–670. [Google Scholar] [CrossRef]
  7. Lamprokostopoulou, A.; Römling, U. Yin and Yang of biofilm formation and cyclic di-GMP signaling of the gastrointestinal pathogen Salmonella enterica Serovar Typhimurium. J. Innate Immun. 2022, 14, 275–292. [Google Scholar] [CrossRef]
  8. Römling, U.; Galperin, M.Y.; Gomelsky, M. Cyclic di-GMP: The first 25 years of a universal bacterial second messenger. Microbiol. Mol. Biol. Rev. 2013, 77, 1–52. [Google Scholar] [PubMed] [Green Version]
  9. Ahmad, I.; Cimdins, A.; Beske, T.; Römling, U. Detailed analysis of c-di-GMP mediated regulation of csgD expression in Salmonella typhimurium. BMC Microbiol. 2017, 17, 27. [Google Scholar] [CrossRef] [Green Version]
  10. Sarenko, O.; Klauck, G.; Wilke, F.M.; Pfiffer, V.; Richter, A.M.; Herbst, S.; Kaever, V.; Hengge, R. More than enzymes that make or break cyclic di-GMP-local signaling in the interactome of GGDEF/EAL domain proteins of Escherichia coli. mBio 2017, 8, e01639-17. [Google Scholar]
  11. Römling, U.; Cao, L.-Y.; Bai, F. Evolution of cyclic di-GMP signaling on a short and long term time scale. Microbiology 2023, 169, 001354. [Google Scholar] [CrossRef] [PubMed]
  12. Bokranz, W.; Wang, X.; Tschäpe, H.; Römling, U. Expression of cellulose and curli fimbriae by Escherichia coli isolated from the gastrointestinal tract. J. Med. Microbiol. 2005, 54, 1171–1182. [Google Scholar] [CrossRef]
  13. Kai-Larsen, Y.; Lüthje, P.; Chromek, M.; Peters, V.; Wang, X.; Holm, A.; Kadas, L.; Hedlund, K.O.; Johansson, J.; Chapman, M.R.; et al. Uropathogenic Escherichia coli modulates immune responses and its curli fimbriae interact with the antimicrobial peptide LL-37. PLoS Pathog. 2010, 6, e1001010. [Google Scholar]
  14. Cimdins, A.; Lüthje, P.; Li, F.; Ahmad, I.; Brauner, A.; Römling, U. Draft genome sequences of semiconstitutive red, dry, and rough biofilm-forming commensal and uropathogenic Escherichia coli isolates. Genome Announc. 2017, 5, e02492-18. [Google Scholar] [CrossRef] [Green Version]
  15. Römling, U.; Bokranz, W.; Rabsch, W.; Zogaj, X.; Nimtz, M.; Tschäpe, H. Occurrence and regulation of the multicellular morphotype in Salmonella serovars important in human disease. Int. J. Med. Microbiol. 2003, 293, 273–285. [Google Scholar] [CrossRef]
  16. Uhlich, G.A.; Keen, J.E.; Elder, R.O. Mutations in the csgD promoter associated with variations in curli expression in certain strains of Escherichia coli O157:H7. Appl. Environ. Microbiol. 2001, 67, 2367–2370. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Cimdins, A.; Simm, R.; Li, F.; Lüthje, P.; Thorell, K.; Sjöling, A.; Brauner, A.; Römling, U. Alterations of c-di-GMP turnover proteins modulate semi-constitutive rdar biofilm formation in commensal and uropathogenic Escherichia coli. Microbiologyopen 2017, 6, e00508. [Google Scholar] [CrossRef] [PubMed]
  18. Kamal, S.M.; Cimdins-Ahne, A.; Lee, C.; Li, F.; Martin-Rodriguez, A.J.; Seferbekova, Z.; Afasizhev, R.; Wami, H.T.; Katikaridis, P.; Meins, L.; et al. A recently isolated human commensal Escherichia coli ST10 clone member mediates enhanced thermotolerance and tetrathionate respiration on a P1 phage-derived IncY plasmid. Mol. Microbiol. 2021, 115, 255–271. [Google Scholar] [PubMed]
  19. Römling, U. Genetic and phenotypic analysis of multicellular behavior in Salmonella typhimurium. Methods Enzymol. 2001, 336, 48–59. [Google Scholar] [PubMed]
  20. Guzman, L.M.; Belin, D.; Carson, M.J.; Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 1995, 177, 4121–4130. [Google Scholar] [PubMed] [Green Version]
  21. Datsenko, K.A.; Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 2000, 97, 6640–6645. [Google Scholar] [CrossRef] [PubMed]
  22. Monteiro, C.; Saxena, I.; Wang, X.; Kader, A.; Bokranz, W.; Simm, R.; Nobles, D.; Chromek, M.; Brauner, A.; Brown, R.M., Jr.; et al. Characterization of cellulose production in Escherichia coli Nissle 1917 and its biological consequences. Environ. Microbiol. 2009, 11, 1105–1116. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Aziz, R.K.; Bartels, D.; Best, A.A.; DeJongh, M.; Disz, T.; Edwards, R.A.; Formsma, K.; Gerdes, S.; Glass, E.M.; Kubal, M.; et al. The RAST Server: Rapid annotations using subsystems technology. BMC Genom. 2008, 9, 75. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Brettin, T.; Davis, J.J.; Disz, T.; Edwards, R.A.; Gerdes, S.; Olsen, G.J.; Olson, R.; Overbeek, R.; Parrello, B.; Pusch, G.D.; et al. RASTtk: A modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci. Rep. 2015, 5, 8365. [Google Scholar] [CrossRef] [Green Version]
  25. Overbeek, R.; Olson, R.; Pusch, G.D.; Olsen, G.J.; Davis, J.J.; Disz, T.; Edwards, R.A.; Gerdes, S.; Parrello, B.; Shukla, M.; et al. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res. 2014, 42, D206–D214. [Google Scholar] [CrossRef]
  26. Letunic, I.; Doerks, T.; Bork, P. SMART: Recent updates, new developments and status in 2015. Nucleic Acids Res. 2015, 43, D257–D260. [Google Scholar] [CrossRef] [Green Version]
  27. Schultz, J.; Milpetz, F.; Bork, P.; Ponting, C.P. SMART, a simple modular architecture research tool: Identification of signaling domains. Proc. Natl. Acad. Sci. USA 1998, 95, 5857–5864. [Google Scholar] [CrossRef]
  28. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar] [CrossRef]
  29. Higgins, D.G.; Sharp, P.M. CLUSTAL: A package for performing multiple sequence alignment on a microcomputer. Gene 1988, 73, 237–244. [Google Scholar] [CrossRef]
  30. Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792–1797. [Google Scholar]
  31. Larsson, A. AliView: A fast and lightweight alignment viewer and editor for large datasets. Bioinformatics 2014, 30, 3276–3278. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Robert, X.; Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014, 42, W320–W324. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Apweiler, R.; Bairoch, A.; Wu, C.H.; Barker, W.C.; Boeckmann, B.; Ferro, S.; Gasteiger, E.; Huang, H.; Lopez, R.; Magrane, M.; et al. UniProt: The Universal Protein knowledgebase. Nucleic Acids Res. 2004, 32, D115–D119. [Google Scholar] [CrossRef] [PubMed]
  34. Kelley, L.A.; Mezulis, S.; Yates, C.M.; Wass, M.N.; Sternberg, M.J. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 2015, 10, 845–858. [Google Scholar] [CrossRef] [Green Version]
  35. Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605–1612. [Google Scholar] [CrossRef] [Green Version]
  36. Reinders, A.; Hee, C.S.; Ozaki, S.; Mazur, A.; Boehm, A.; Schirmer, T.; Jenal, U. Expression and genetic activation of cyclic di-GMP-specific phosphodiesterases in Escherichia coli. J. Bacteriol. 2016, 198, 448–462. [Google Scholar] [CrossRef] [Green Version]
  37. Simm, R.; Lusch, A.; Kader, A.; Andersson, M.; Römling, U. Role of EAL-containing proteins in multicellular behavior of Salmonella enterica serovar Typhimurium. J. Bacteriol. 2007, 189, 3613–3623. [Google Scholar] [CrossRef] [Green Version]
  38. Sommerfeldt, N.; Possling, A.; Becker, G.; Pesavento, C.; Tschowri, N.; Hengge, R. Gene expression patterns and differential input into curli fimbriae regulation of all GGDEF/EAL domain proteins in Escherichia coli. Microbiology 2009, 155, 1318–1331. [Google Scholar] [CrossRef] [Green Version]
  39. Richter, A.M.; Povolotsky, T.L.; Wieler, L.H.; Hengge, R. Cyclic-di-GMP signalling and biofilm-related properties of the Shiga toxin-producing 2011 German outbreak Escherichia coli O104:H4. EMBO Mol. Med. 2014, 6, 1622–1637. [Google Scholar] [CrossRef]
  40. Römling, U. Characterization of the rdar morphotype, a multicellular behaviour in Enterobacteriaceae. Cell. Mol. Life Sci. 2005, 62, 1234–1246. [Google Scholar] [CrossRef]
  41. Herbst, S.; Lorkowski, M.; Sarenko, O.; Nguyen, T.K.L.; Jaenicke, T.; Hengge, R. Transmembrane redox control and proteolysis of PdeC, a novel type of c-di-GMP phosphodiesterase. EMBO J. 2018, 37, e97825. [Google Scholar] [CrossRef]
  42. Himeno, H.; Masaki, H.; Kawai, T.; Ohta, T.; Kumagai, I.; Miura, K.; Watanabe, K. Unusual genetic codes and a novel gene structure for tRNA(AGYSer) in starfish mitochondrial DNA. Gene 1987, 56, 219–230. [Google Scholar] [PubMed]
  43. Römling, U.; Rohde, M.; Olsen, A.; Normark, S.; Reinköster, J. AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways. Mol. Microbiol. 2000, 36, 10–23. [Google Scholar] [CrossRef] [PubMed]
  44. Povolotsky, T.L.; Hengge, R. Genome-based comparison of cyclic di-GMP signaling in pathogenic and commensal Escherichia coli strains. J. Bacteriol. 2016, 198, 111–126. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Rudd, K.E. Novel intergenic repeats of Escherichia coli K-12. Res. Microbiol. 1999, 150, 653–664. [Google Scholar] [CrossRef] [PubMed]
  46. Schirmer, T. C-di-GMP Synthesis: Structural Aspects of Evolution, Catalysis and Regulation. J. Mol. Biol. 2016, 428, 3683–3701. [Google Scholar] [CrossRef] [Green Version]
  47. Römling, U.; Liang, Z.X.; Dow, J.M. Progress in understanding the molecular basis underlying functional diversification of cyclic dinucleotide turnover proteins. J. Bacteriol. 2017, 199, e00790-16. [Google Scholar] [CrossRef] [Green Version]
  48. Fang, X.; Ahmad, I.; Blanka, A.; Schottkowski, M.; Cimdins, A.; Galperin, M.Y.; Römling, U.; Gomelsky, M. GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria. Mol. Microbiol. 2014, 93, 439–452. [Google Scholar] [CrossRef] [Green Version]
  49. Hufnagel, D.A.; Evans, M.L.; Greene, S.E.; Pinkner, J.S.; Hultgren, S.J.; Chapman, M.R. The Catabolite Repressor Protein-Cyclic AMP Complex Regulates csgD and Biofilm Formation in Uropathogenic Escherichia coli. J. Bacteriol. 2016, 198, 3329–3334. [Google Scholar] [CrossRef] [Green Version]
  50. Weber, H.; Pesavento, C.; Possling, A.; Tischendorf, G.; Hengge, R. Cyclic-di-GMP-mediated signalling within the sigma network of Escherichia coli. Mol. Microbiol. 2006, 62, 1014–1034. [Google Scholar] [CrossRef]
  51. Spurbeck, R.R.; Tarrien, R.J.; Mobley, H.L. Enzymatically active and inactive phosphodiesterases and diguanylate cyclases are involved in regulation of motility or sessility in Escherichia coli CFT073. mBio 2012, 3, e00307-12. [Google Scholar] [CrossRef] [Green Version]
  52. Zlatkov, N.; Uhlin, B.E. Absence of Global Stress Regulation in Escherichia coli promotes pathoadaptation and novel c-di-GMP-dependent metabolic capability. Sci. Rep. 2019, 9, 2600. [Google Scholar] [CrossRef] [Green Version]
  53. Thomason, M.K.; Bischler, T.; Eisenbart, S.K.; Förstner, K.U.; Zhang, A.; Herbig, A.; Nieselt, K.; Sharma, C.M.; Storz, G. Global transcriptional start site mapping using differential RNA sequencing reveals novel antisense RNAs in Escherichia coli. J. Bacteriol. 2015, 197, 18–28. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Bernal-Bayard, J.; Gomez-Valero, L.; Wessel, A.; Khanna, V.; Bouchier, C.; Ghigo, J.M. Short genome report of cellulose-producing commensal Escherichia coli 1094. Stand. Genom. Sci. 2018, 13, 13. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Rochon, M.; Römling, U. Flagellin in combination with curli fimbriae elicits an immune response in the gastrointestinal epithelial cell line HT-29. Microbes Infect. 2006, 8, 2027–2033. [Google Scholar] [CrossRef] [PubMed]
  56. Lee, C.; Franke, K.B.; Kamal, S.M.; Kim, H.; Lünsdorf, H.; Jäger, J.; Nimtz, M.; Trček, J.; Jänsch, L.; Bukau, B.; et al. Stand-alone ClpG disaggregase confers superior heat tolerance to bacteria. Proc. Natl. Acad. Sci. USA 2018, 115, E273–E282. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Microorganisms 11 02048 g001
Figure 1. YcgG is truncated in commensal and uropathogenic E. coli strains. (A) Domain organization of YcgG from E. coli K-12 (MG1655 and Fec10), commensal Tob1, and uropathogenic B-11870 as depicted by SMART server (accessed latest 8 June 2023, http://smart.embl-heidelberg.de/). Tob1 and B-11870 lack the N-terminal CSS signaling domain (indicated in green) compared to K-12. The N-terminal region of truncated YcgG is unique to E. coli Tob1 (black line). (B) Alignment of aa sequences of YcgG from E. coli K-12, Tob1, and B-11870. YcgG proteins from Tob1 and B-11870 are truncated at the N-terminus compared to the K-12 reference protein. The alignment was created with MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/). Amino acid substitutions are indicated in red letters. Star * indicates conservation of amino acid, : indicates conservative substitution; blank indicates non-conservative substitution; . indicates semi-conservative substitution.
Figure 1. YcgG is truncated in commensal and uropathogenic E. coli strains. (A) Domain organization of YcgG from E. coli K-12 (MG1655 and Fec10), commensal Tob1, and uropathogenic B-11870 as depicted by SMART server (accessed latest 8 June 2023, http://smart.embl-heidelberg.de/). Tob1 and B-11870 lack the N-terminal CSS signaling domain (indicated in green) compared to K-12. The N-terminal region of truncated YcgG is unique to E. coli Tob1 (black line). (B) Alignment of aa sequences of YcgG from E. coli K-12, Tob1, and B-11870. YcgG proteins from Tob1 and B-11870 are truncated at the N-terminus compared to the K-12 reference protein. The alignment was created with MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/). Amino acid substitutions are indicated in red letters. Star * indicates conservation of amino acid, : indicates conservative substitution; blank indicates non-conservative substitution; . indicates semi-conservative substitution.
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Figure 2. Differential activity of YcgG from E. coli Tob1, B-11870, and MG1655 ST10 strains. (A) Biofilm formation on LB without salt agar plates supplemented with Congo red or Calcofluor white after 48 h of incubation at 28 °C or 37 °C. (B) Western blot detection of CsgD from cells grown on LB agar plates for 16 h at 28 °C or 37 °C. CsgD was detected with an anti-CsgD antibody as described previously [43,44]. (C) Western blot detection of 6xHis-tagged YcgG from cells grown on LB agar plates without salt for 16 h at 28 °C or 37 °C. Recombinant YcgG was detected with an anti-Penta-His antibody (Qiagen). 1 = Tob1 (pBAD30), 2 = Tob1 (pYcgGMG1655-6xHis), 3 = Tob1 (pYcgGTob1-6xHis), 4 = Tob1 (pYcgGB-11870-6xHis). C1 = Tob1 ∆csgD incubated at 28 °C for 16 h, C2 = Tob1 ∆csgD incubated at 37 °C for 16 h. All plates supplemented with 100 µg/mL ampicillin and 0.1% L-arabinose. pc = ClpG-6xHis of Pseudomonas aeruginosa SG17M. Blue and red stars indicate the signals detected for YcgGMG1655 and YcgGB-11870, respectively.
Figure 2. Differential activity of YcgG from E. coli Tob1, B-11870, and MG1655 ST10 strains. (A) Biofilm formation on LB without salt agar plates supplemented with Congo red or Calcofluor white after 48 h of incubation at 28 °C or 37 °C. (B) Western blot detection of CsgD from cells grown on LB agar plates for 16 h at 28 °C or 37 °C. CsgD was detected with an anti-CsgD antibody as described previously [43,44]. (C) Western blot detection of 6xHis-tagged YcgG from cells grown on LB agar plates without salt for 16 h at 28 °C or 37 °C. Recombinant YcgG was detected with an anti-Penta-His antibody (Qiagen). 1 = Tob1 (pBAD30), 2 = Tob1 (pYcgGMG1655-6xHis), 3 = Tob1 (pYcgGTob1-6xHis), 4 = Tob1 (pYcgGB-11870-6xHis). C1 = Tob1 ∆csgD incubated at 28 °C for 16 h, C2 = Tob1 ∆csgD incubated at 37 °C for 16 h. All plates supplemented with 100 µg/mL ampicillin and 0.1% L-arabinose. pc = ClpG-6xHis of Pseudomonas aeruginosa SG17M. Blue and red stars indicate the signals detected for YcgGMG1655 and YcgGB-11870, respectively.
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Figure 3. Characterization of YjcC variants of semi-constitutive E. coli isolates compared to YjcC of E. coli K-12 MG1655. (A) Variants of yjcC from E. coli Tob1, Fec67, Fec101, and K-12 MG1655 were cloned into pBAD30 using an identical Shine–Dalgarno sequences and an extended region downstream of the stop codon. The constructs were expressed in E. coli Tob1. VC = vector control pBAD30. LB without salt plates supplemented with 100 µg/mL ampicillin, 0.1% arabinose, and Congo red incubated at 28 °C (left panel) and 37 °C (right panel) for 48 h and 24 h, respectively. (B) Phylogenetic tree of YjcC variants of investigated E. coli strains MG1655, Tob1, Fec67, and Fec101 [17] and additional variants retrieved by Blast search from NCBI and UniProt databases (Section 2 [28,33]). Alignment was conducted with ClustalX 2.1, and phylogenetic relationships were assessed with the maximum-likelihood approach using all sites and 1000 bootstrap replications. YjcC (by strain name) subgroups are indicated by different branch colors. (C) Overlaid Phyre2 structural models of the EAL domains of YjcCMG1655 (red) and YjcCFec101 (blue). Diverging C-terminal sequences are in yellow (YjcCMG1655) and dark blue (YjcCFec101). Amino acids involved in catalysis, substrate, and divalent ion binding and loop six stabilization are indicated with their side chains. Functional aas with yellow side chains not present in YjcCFec101 are involved in divalent cation binding.
Figure 3. Characterization of YjcC variants of semi-constitutive E. coli isolates compared to YjcC of E. coli K-12 MG1655. (A) Variants of yjcC from E. coli Tob1, Fec67, Fec101, and K-12 MG1655 were cloned into pBAD30 using an identical Shine–Dalgarno sequences and an extended region downstream of the stop codon. The constructs were expressed in E. coli Tob1. VC = vector control pBAD30. LB without salt plates supplemented with 100 µg/mL ampicillin, 0.1% arabinose, and Congo red incubated at 28 °C (left panel) and 37 °C (right panel) for 48 h and 24 h, respectively. (B) Phylogenetic tree of YjcC variants of investigated E. coli strains MG1655, Tob1, Fec67, and Fec101 [17] and additional variants retrieved by Blast search from NCBI and UniProt databases (Section 2 [28,33]). Alignment was conducted with ClustalX 2.1, and phylogenetic relationships were assessed with the maximum-likelihood approach using all sites and 1000 bootstrap replications. YjcC (by strain name) subgroups are indicated by different branch colors. (C) Overlaid Phyre2 structural models of the EAL domains of YjcCMG1655 (red) and YjcCFec101 (blue). Diverging C-terminal sequences are in yellow (YjcCMG1655) and dark blue (YjcCFec101). Amino acids involved in catalysis, substrate, and divalent ion binding and loop six stabilization are indicated with their side chains. Functional aas with yellow side chains not present in YjcCFec101 are involved in divalent cation binding.
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Figure 4. DgcX is an additional diguanylate cyclase horizontally transferred into E. coli Fec101. (A) Domain organization of DgcX by the SMART server. (B) Phylogenetic tree of the GGDEF domains of GGDEF and GGDEF-EAL domain proteins of E. coli ST10 [17] compared to the GGDEF domains of the newly introduced DgcX and selected proteins with high aa identity belonging to the MASE4-GGDEF family. In E. coli, the MASE4-GGDEF family includes the functional diguanylate cyclases DgcX and YcdT (DgcT) and the catalytically inactive protein YeaI (CdgI) as described in the text. (C) Colony morphology of E. coli Fec10 (rdar28 °C/saw37 °C) expressing DgcX or catalytic mutants, grown for 24 h on Congo-red (upper panel) or Calcofluor white (lower panel) agar plates at 28 °C and 37 °C. 1 = Fec10 VC, 2 = pDgcXN, 3 = pDgcXC, 4 = pDgcXE360A, 5 = pDgcXE359A, 6 = pDgcXE359A, E360A. VC = pBAD30. (D) Swimming motility of E. coli Fec10 (rdar28 °C/saw37 °C) expressing DgcX or catalytic mutants, grown for 6 h on LB swimming plates at 28 °C and 37 °C. 1. pBAD30, 2. pDgcXN, 3. p:DgcXC, 4. p gcXE359A,E360A, 5. pDgcXE360A, 6. pDgcXE359A. (C,D): Plates supplemented with 100 µg/mL ampicillin and 0.1% arabinose. pDgcX = dgcX cloned in pBAD30.
Figure 4. DgcX is an additional diguanylate cyclase horizontally transferred into E. coli Fec101. (A) Domain organization of DgcX by the SMART server. (B) Phylogenetic tree of the GGDEF domains of GGDEF and GGDEF-EAL domain proteins of E. coli ST10 [17] compared to the GGDEF domains of the newly introduced DgcX and selected proteins with high aa identity belonging to the MASE4-GGDEF family. In E. coli, the MASE4-GGDEF family includes the functional diguanylate cyclases DgcX and YcdT (DgcT) and the catalytically inactive protein YeaI (CdgI) as described in the text. (C) Colony morphology of E. coli Fec10 (rdar28 °C/saw37 °C) expressing DgcX or catalytic mutants, grown for 24 h on Congo-red (upper panel) or Calcofluor white (lower panel) agar plates at 28 °C and 37 °C. 1 = Fec10 VC, 2 = pDgcXN, 3 = pDgcXC, 4 = pDgcXE360A, 5 = pDgcXE359A, 6 = pDgcXE359A, E360A. VC = pBAD30. (D) Swimming motility of E. coli Fec10 (rdar28 °C/saw37 °C) expressing DgcX or catalytic mutants, grown for 6 h on LB swimming plates at 28 °C and 37 °C. 1. pBAD30, 2. pDgcXN, 3. p:DgcXC, 4. p gcXE359A,E360A, 5. pDgcXE360A, 6. pDgcXE359A. (C,D): Plates supplemented with 100 µg/mL ampicillin and 0.1% arabinose. pDgcX = dgcX cloned in pBAD30.
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Figure 5. DgcX stimulates cellulose biosynthesis but not csgD expression. (A) Western blot detection of CsgD in E. coli Fec10 harboring plasmids expressing wild-type and catalytic mutants of DgcX. N = negative control E. coli Tob2 (Tob1 ΔcsgD; pBAD30), P = positive control E. coli Tob1 (pBAD30). (B) rdar morphotype of E. coli Fec10 ∆csgD (saw28 °C/saw37 °C) harboring plasmids expressing wild-type and catalytic mutants of DgcX were grown on Congo red or Calcofluor white plates for 24 h at 28 °C and 37 °C. (A,B): 1 = Fec10 VC, 2 = pDgcXN, 3 = pDgcXC, 4 = pDgcXE360A, 5 = pDgcXE359A, 6 = pDgcXE359A, E360A. pDgcX = dgcX cloned in pBAD30. VC = pBAD30. Plates were supplemented with 100 µg/mL ampicillin and 0.1% arabinose. (C) Model regulation of cellulose biosynthesis by different genetic factors. CsgD activates curli expression directly and provides a positive feedback loop on its own expression in E. coli [49]. Cellulose biosynthesis is indirectly activated by csgD via expression of the DGC AdrA. However, expression of the DGC DgcX, equally as the DGC YdeQ, can stimulate cellulose expression independently of csgD at both 28 °C and 37 °C.
Figure 5. DgcX stimulates cellulose biosynthesis but not csgD expression. (A) Western blot detection of CsgD in E. coli Fec10 harboring plasmids expressing wild-type and catalytic mutants of DgcX. N = negative control E. coli Tob2 (Tob1 ΔcsgD; pBAD30), P = positive control E. coli Tob1 (pBAD30). (B) rdar morphotype of E. coli Fec10 ∆csgD (saw28 °C/saw37 °C) harboring plasmids expressing wild-type and catalytic mutants of DgcX were grown on Congo red or Calcofluor white plates for 24 h at 28 °C and 37 °C. (A,B): 1 = Fec10 VC, 2 = pDgcXN, 3 = pDgcXC, 4 = pDgcXE360A, 5 = pDgcXE359A, 6 = pDgcXE359A, E360A. pDgcX = dgcX cloned in pBAD30. VC = pBAD30. Plates were supplemented with 100 µg/mL ampicillin and 0.1% arabinose. (C) Model regulation of cellulose biosynthesis by different genetic factors. CsgD activates curli expression directly and provides a positive feedback loop on its own expression in E. coli [49]. Cellulose biosynthesis is indirectly activated by csgD via expression of the DGC AdrA. However, expression of the DGC DgcX, equally as the DGC YdeQ, can stimulate cellulose expression independently of csgD at both 28 °C and 37 °C.
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Figure 6. Different modes of regulation of rdar biofilm expression by genetic and environmental factors. Modes of regulating rdar morphotype formation can be at the transcriptional level by mutations in the csgD promoter, leading to altered expression at 28 °C, 37 °C, and 42 °C (I) [2,16], by different genetic factors including global transcriptional regulators and small RNAs targeting the csgD promoter and beyond (II); by alterations of cyclic di-GMP turnover protein activity and presence, such as altered catalytic activity, including phosphodiesterase and diguanylate cyclase activity; or by deletion and acquisition of novel diguanylate cyclases (III) [10,38,39]. Cyclic di-GMP is a major determinative factor for overriding most other regulatory determinants. CsgD subsequently activates curli expression directly. Cellulose biosynthesis can be regulated independently of csgD-mediated rdar biofilm expression (IV) [4,8,54].
Figure 6. Different modes of regulation of rdar biofilm expression by genetic and environmental factors. Modes of regulating rdar morphotype formation can be at the transcriptional level by mutations in the csgD promoter, leading to altered expression at 28 °C, 37 °C, and 42 °C (I) [2,16], by different genetic factors including global transcriptional regulators and small RNAs targeting the csgD promoter and beyond (II); by alterations of cyclic di-GMP turnover protein activity and presence, such as altered catalytic activity, including phosphodiesterase and diguanylate cyclase activity; or by deletion and acquisition of novel diguanylate cyclases (III) [10,38,39]. Cyclic di-GMP is a major determinative factor for overriding most other regulatory determinants. CsgD subsequently activates curli expression directly. Cellulose biosynthesis can be regulated independently of csgD-mediated rdar biofilm expression (IV) [4,8,54].
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Cimdins-Ahne, A.; Naemi, A.-O.; Li, F.; Simm, R.; Römling, U. Characterisation of Variants of Cyclic di-GMP Turnover Proteins Associated with Semi-Constitutive rdar Morphotype Expression in Commensal and Uropathogenic Escherichia coli Strains. Microorganisms 2023, 11, 2048. https://doi.org/10.3390/microorganisms11082048

AMA Style

Cimdins-Ahne A, Naemi A-O, Li F, Simm R, Römling U. Characterisation of Variants of Cyclic di-GMP Turnover Proteins Associated with Semi-Constitutive rdar Morphotype Expression in Commensal and Uropathogenic Escherichia coli Strains. Microorganisms. 2023; 11(8):2048. https://doi.org/10.3390/microorganisms11082048

Chicago/Turabian Style

Cimdins-Ahne, Annika, Ali-Oddin Naemi, Fengyang Li, Roger Simm, and Ute Römling. 2023. "Characterisation of Variants of Cyclic di-GMP Turnover Proteins Associated with Semi-Constitutive rdar Morphotype Expression in Commensal and Uropathogenic Escherichia coli Strains" Microorganisms 11, no. 8: 2048. https://doi.org/10.3390/microorganisms11082048

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