Transcriptional Control of Dual Transporters Involved in α-Ketoglutarate Utilization Reveals Their Distinct Roles in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) are the primary causative agents of urinary tract infections. Some UPEC isolates are able to infect renal proximal tubule cells, and can potentially cause pyelonephritis. We have previously shown that to fulfill their physiological roles renal proximal tubule cells accumulate high concentrations of α-ketoglutarate (KG) and that gene cluster c5032-c5039 contribute to anaerobic utilization of KG by UPEC str. CFT073, thereby promoting its in vivo fitness. Given the importance of utilizing KG for UPEC, this study is designed to investigate the roles of two transporters KgtP and C5038 in KG utilization, their transcriptional regulation, and their contributions to UPEC fitness in vivo. Our phylogenetic analyses support that kgtP is a widely conserved locus in commensal and pathogenic E. coli, while UPEC-associated c5038 was acquired through horizontal gene transfer. Global anaerobic transcriptional regulators Fumarate and nitrate reduction (FNR) and ArcA induced c5038 expression in anaerobiosis, and C5038 played a major role in anaerobic growth on KG. KgtP was required for aerobic growth on KG, and its expression was repressed by FNR and ArcA under anaerobic conditions. Analyses of FNR and ArcA binding sites and results of EMS assays suggest that FNR and ArcA likely inhibit kgtP expression through binding to the -35 region of kgtP promoter and occluding the occupancy of RNA polymerases. Gene c5038 can be specifically induced by KG, whereas the expression of kgtP does not respond to KG, yet can be stimulated during growth on glycerol. In addition, c5038 and kgtP expression were further shown to be controlled by different alternative sigma factors RpoN and RpoS, respectively. Furthermore, dual-strain competition assays in a murine model showed that c5038 mutant but not kgtP mutant was outcompeted by the wild-type strain during the colonization of murine bladders and kidneys, highlighting the importance of C5038 under in vivo conditions. Therefore, different transcriptional regulation led to distinct roles played by C5038 and KgtP in KG utilization and fitness in vivo. This study thus potentially expanded our understanding of UPEC pathobiology.

Introduction

Urinary tract infection (UTI) is one of the most common bacterial infections in humans, and represents a significant clinical issue worldwide (Foxman, 2010). The primary causative agent of UTIs is uropathogenic Escherichia coli (UPEC), which is responsible for ∼80–90% of community-acquired UTIs (Russo and Johnson, 2003; Spurbeck and Mobley, 2013). Despite that most previous studies of UPEC pathogenesis have been focused on traditional virulence factors, increasing attention is being paid to UPEC’s metabolic adaptive mechanisms, which impact colonization and survival in host’s urinary tract (Alteri and Mobley, 2015; Conover et al., 2016). UPECs have evolved multiple systems and strategies to snatch needed iron, combatting extremely low iron availability within host (Opal et al., 1990; Russo et al., 2001, 2002; Torres et al., 2001). Possession of a complete dsdCXA locus responsible for the detoxification of D-serine allows UPEC to use D-serine as the sole carbon and nitrogen source in urine (Roesch et al., 2003). In addition, most UPEC strains carry a genomic island involved in utilizing α-ketoglutarate (KG) under anaerobic conditions, which significantly increased UPEC’s fitness in a mouse model for UTI (Cai et al., 2013). KG is an abundant metabolite in the UPEC’s infection site – renal proximal tubule cells (Mobley et al., 1990; Chassin et al., 2008; Melican et al., 2008; Pichon et al., 2009), with an intracellular concentration of up to 400 μM (Boyd and Goldstein, 1979; Martin et al., 1989; Pritchard, 1995). KG lies at the intersection between the carbon and nitrogen metabolic pathways, and has long been recognized to coordinate carbon and nitrogen metabolism (Kim and Gadd, 2008).

A BLAST search revealed that the great majority of UPEC strains encode at least two transporters possibly associated with utilization of KG (Cai et al., 2013). A genomic island-encoded gene c5038 was predicted to encode a putative dicarboxylate transporter with 13 transmembrane alpha-helices, showing 49% similarity to citrate/succinate antiporter CitT (Pos et al., 1998) at the amino acid level. Its expression is activated by two-component regulatory system (TCS) KguSR in response to KG under anaerobic conditions (Cai et al., 2013). Fumarate and nitrate reduction (FNR) positively regulates c5038 expression by directly affecting the expression of KguSR (KG utilization sensor and regulator) (Barbieri et al., 2014). The other gene, kgtP (KG permease), encodes a KG:H+ symporter in E. coli K-12 strain (Seol and Shatkin, 1991, 1992). Alkaline phosphatase fusion study showed that KgtP contains 12 transmembrane segments (Seol and Shatkin, 1993). Genome-wide analysis of ArcA binding sites and its modulon in E. coli K-12 revealed that kgtP is negatively affected by ArcA (aerobic respiratory control), which binds to the upstream region of kgtP in vivo under anaerobic conditions (Liu and De Wulf, 2004; Park et al., 2013). Despite the knowledge gained of KgtP in K-12 strain and C5038, the individual roles and transcriptional regulation of C5038 and KgtP in UPEC during KG utilization remain unknown.

In this study, we describe KgtP and C5038 contribute differentially to growth on KG under aerobic and anaerobic conditions. Further analyses of their expression levels in various growth conditions and of roles played by regulators ArcA, FNR, RpoN, RpoS, and CRP (catabolite receptor protein) can account for the findings of different contributions to growth on KG and to colonization of murine urinary tracts. Overall, our results suggest that C5038 is a specialized, anaerobic KG importer, but KgtP is a generalized, aerobic KG importer. This report, therefore, should substantially improve our understanding of UPEC physiology.

Materials and Methods

Ethics Statement

All animal experimental procedures were conducted according to the guidelines of Experimental Animal Management Measures of Jiangsu Province and were approved by the Laboratory Animal Monitoring Committee of Jiangsu Province (China).

Bacterial Strains and Culture Conditions

Strains and plasmids used in this study are listed in Supplementary Table S1. Aerobic growth was achieved by shaking in air at 160 rpm, and anaerobic growth by incubating in a BugBox chamber (Ruskinn, UK) filled with a gas mixture (N2, 85%; CO2, 5%; H2, 10%). For genetic manipulations, all E. coli strains were grown routinely in lysogenic broth (LB) medium (OXOID). For growth and gene expression studies, bacteria were generally grown aerobically or anaerobically in M9 minimal salts with certain carbon sources indicated, supplemented with 2 mM MgSO4, 0.1 mM CaCl2, and 1 μg ml-1 vitamin B1 (Cai et al., 2013). Glucose (0.4% m/v) or glycerol (0.4% v/v) (Sinopharm, China) was added as energy substrates, as indicated. When used, electron acceptor trimethylamine N-oxide (TMAO) and dicarboxylates were present at 40 mM. Selective antibiotics and IPTG (Isopropyl β-D-1-thiogalactopyranoside) were added when necessary at the following concentrations: ampicillin (Amp), 100 μg ml-1; kanamycin (Kan), 50 μg ml-1;chloramphenicol (Chl), 25 μg ml-1; IPTG, 1 mM. All reagents were purchased from Sigma-Aldrich unless otherwise noted.

For growth studies, fresh colonies of wild-type (WT) CFT073 and its derivative mutant strains were picked and inoculated in 3 ml LB medium. After OD600 of bacterial culture reached about 1.0, bacteria were washed once with phosphate-buffered saline (PBS) and normalized, followed by being subcultured 1:100 into M9 medium. OD600 value at different time points was measured and recorded using spectrophotometer (Eppendorf, basic model).

Recombinant DNA Techniques

Polymerase chain reaction (PCR), DNA ligation, electroporation and DNA gel electrophoresis were performed according to Sambrook and Russell (2001) unless otherwise indicated. All oligonucleotide primers were purchased from BGI Technology Solutions Co., Ltd. (BGI, Guangzhou, China) and are listed in Supplementary Table S2. All restriction and DNA-modifying enzymes were purchased from New England Biolabs and were used based on the suppliers’ recommendations. Recombinant plasmids, PCR products, and restriction fragments were purified using TaKaRa PCR purification kit or gel extraction kit (TaKaRa, a Clontech company) as recommended by the supplier. DNA sequencing was performed at Shanghai Sunny Biotech Co., Ltd. DNA and amino acid sequence analyses were performed using Clone Manager software (Scientific & Educational Software, Morrisville, NC, USA) and the search engine[1] was used to find homologous sequences. ClustalW program was used to perform multiple sequences alignments. Phylogenetic trees were made using MEGA software (Tamura et al., 2011; Hall, 2013).

Deletion mutants were constructed using the lambda red recombinase system described by Datsenko and Wanner (2000). For complementation studies, the coding sequences of genes plus their promoter regions (400 bp upstream of start codons) were amplified from the CFT073 genome and independently cloned into pGEN-MCS (Lane et al., 2007) using EcoRI and SalI restriction sites. Plasmid pCJ112 for plasmid-borne LacZ fusion studies was constructed by replacing the R6K origin in pVIK112 [this plasmid was created for making chromosomal transcriptional fusions of lacZ reporter gene (Kalogeraki and Winans, 1997)] with p15A origin from pBAD30 (Guzman et al., 1995) plasmid using EcoRI and BamHI restriction sites (See Supplementary Figure S1 for detail). The resulting plasmid was tested to show the functionality and an undetectable basal level without inserting any promoters upstream of promoterless lacZ. Pbla constitutively expressed promoter was cloned from pGEN-MCS plasmid by amplifying 400 bp upstream of bla gene as previous described (Sperandio et al., 1999). All constructs were verified by Sanger Sequencing (Shanghai Sunny Biotech Co., Ltd).

β-Galactosidase Assays

Overnight LB cultures of CFT073 and its derivative strains containing the gene of interest-lacZ fusions were washed with PBS once and then were diluted 1:100 in LB or M9 medium with the carbon sources indicated. Empty vector pCJ112 and pCJ112 containing Pbla-lacZ were used under all conditions as controls. Cultures were grown at 37°C to log phase or stationary phase. Samples were diluted 1:1 in Z buffer and assayed for β-galactosidase activity using ortho-Nitrophenyl-β-galactoside (ONPG) as a substrate as described previously (Miller, 1972).

5’-RACE (Rapid Amplification of cDNA Ends) PCR to Identify Transcriptional Start Site (TSS)

The transcriptional start site (TSS) site of c5038 was identified by using ExactSTART 5′- and 3′ -RACE Kit (Epicentre) with some modifications. Briefly, bacteria were grown in M9 media supplemented with glycerol and KG to log phase. RNA was stabilized by RNAprotect Bacterial Reagent (QIAGEN) and extracted using an RNeasy Mini Kit (QIAGEN) with a 1-h in-tube DNase digestion (QIAGEN) to remove possible DNA contamination according to the manufacturer’s instructions. Complete removal of DNA contamination was confirmed by using reverse transcription PCR. Three biological replicates of each sample were prepared. The concentration of RNA was determined using a Spectrophotometer (Eppendorf, basic model). The Steps A and B were omitted as they were designed for treating Eukaryotic RNA. About 5 μg of RNA were used for the Step C in the manual: 5’-RACE Acceptor Oligo Ligation step. Control reactions were included according to the manual. The primers which yielded reliable PCR products were listed in Supplementary Table S2.

Electrophoretic Mobility Shift Assays

To study the binding of proteins of interest to DNA probes, electrophoretic mobility shift assays (EMSAs) were performed using the commercialized EMSA kit (Invitrogen, Carlsbad, CA, USA) (Shen et al., 2011; Cai et al., 2013). pET28a prokaryotic expression system (Novagen) was used to over-produce recombinant proteins. His6-ArcA (Jiang et al., 2015), His6-(FNR-FNR)2 (Shan et al., 2012) and His6-RpoN fusion proteins were individually purified to homogeneity using Ni-NTA Spin Columns (QIAGEN) and dialyzed against the binding buffer. Protein concentrations were measured using BCA protein assay kit (Pierce). DNA probes (230 bp upstream of the start codon of kgtP or c5038) were PCR amplified using specific primers and gel-purified. The negative control probe was amplified from the coding region of Chloramphenicol resistance gene-cat. The concentrations of DNA fragments were determined by Biospectrometer (Eppendorf). EMSAs were performed by adding increasing amounts of purified fusion proteins (0240 nM) to DNA probes (10 nM) in binding buffer [10 mM Tris (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 50 mM KCl, 50 mM MgCl2, 1 μg ml-1 bovine serum albumin (NEB)) and incubating for 30 min at room temperature (His6-ArcA and His6-RpoN), or 20 min at 37°C (His6-(FNR-FNR)2]. Reaction mixtures were then mixed with the loading dye (bromophenol blue, 0.25%; xylene cyanol FF, 0.25%; Ficoll 400, 15%), followed by electrophoresis on a 6% polyacrylamide gel in 0.5 × TBE buffer (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA, pH 8.0) at 200V for 45 min in an ice-bath. The gel was stained in 0.5 × TBE buffer containing 1 × SYBR Gold nucleic acid staining solution (Life technology, USA) for 15 min. Gels were then visualized and photographed using GelDoc-it Imager (UVP).

Experimental UTIs

Mouse infection studies were performed according to the methods of Johnson et al. (2005). Female Balb/C mice (about 6 to 7 weeks of age) were anesthetized by ketamine/xylazine/Midazolam and inoculated via transurethral catheterization with a 50 μl (∼109 CFU) challenge inoculum per mouse. Overnight statically grown LB cultures of CFT073 and the mutant strains were pelleted and resuspended in sterile PBS, mixed in equal number, and adjusted to make challenge inocula. To determine the initial CFU/ml, dilutions of each inoculum were plated onto LB plates with and without chloramphenicol (kgtP::ChlR mutant) or kanamycin (c5038::KanR mutant). After 48 h, the mice were sacrificed and the bladder and kidneys were aseptically removed and homogenized in tubes containing PBS using a Bioprep-24 bullet homogenizer (Allsheng, Hangzhou in China). Dilutions (10 μl in each droplet) of the homogenized tissue were then spotted onto quadruplicate LB plates with and without antibiotics to determine the bacterial load. The numbers of colonies on selective plates were subtracted from those on LB plates to obtain the number of WT bacteria. A group of 10 or 6 (for complementation studies) mice for each dual-strain challenge were used to determine alterations in fitness, and assays were performed at least twice. Competitive index is defined as the ratio of mutant to the WT isolated divided by the ratio of mutant to the WT in the inoculums. the single-gene deletion mutants

Statistical Analyses

Statistical tests were performed using GraphPad Prism (version 5.0 for Windows, San Diego, CA, USA). For animal experiments data, Wilcoxon signed rank test was used to determine significance of competitive indices with a hypothetical median of 1 (numberWT = numbermutant). For other data, unpaired Student t-test was used to estimate differences between samples. The threshold for statistical significance was a P-value < 0.05.

Results

C5038 and KgtP Played Differential Roles in KG Utilization under Different Oxygen Tension Conditions

Gene c5038 is encoded on a metabolic island implicated in the anaerobic utilization of KG in UPEC (Cai et al., 2013). Bioinformatic analysis revealed that C5038 belongs to the Divalent Anion:Na+ Symporter (DASS) Family (Saier et al., 2006). Characterized DASS members in bacteria include succinate transporter VcINDY, citrate transporter CitT, L-tartrate/succinate transporter TtdT, C4-dicarboxylates uptake system DccT, YbhI, and SdcS (Markovich, 2012). C5038 on average shares 29% homology with these proteins. Sequence alignment of these DASS members (Supplementary Figure S2A) revealed that C5038 contains a SAT (serine–alanine–threonine) signature, which is similar to the relatively conserved SNT (serine–asparagine–threonine) motif involved in substrates-binding (Mancusso et al., 2012). The kgtP locus in K-12 is 96.4% identical to kgtP in UPEC str. CFT073 at DNA level, with 47 differences between them. At the amino acid level, only three differences exist between them, all of which are non-charged amino acids. KgtP shares no homology with C5038 at both DNA and amino acid levels. The promoter regions of kgtP in K-12 and CFT073 have an 88% sequence identity to each other (Supplementary Figure S2B).

To determine the roles of C5038 and KgtP in growth of CFT073 in various media, we compared the growth of the WT, the single-gene deletion mutants (Δc5038 and ΔkgtP, respectively), and the double-gene deletion mutant (Δc5038ΔkgtP) in different media under aerobic conditions. We observed no significant difference in growth kinetics among these strains in LB or M9 medium supplemented with glucose (Gluc) or glycerol (Glyc) as the sole carbon source (data not shown).

When cultured in M9 using KG as the sole carbon source under aerobic conditions, the Δc5038 mutant showed similar growth kinetics as WT, indicating that c5038 was not important for growth under these conditions (Figure ). In contrast, deletion of kgtP abolished CFT073’s growth (P < 0.0001) and kgtP mutant displayed no detectable growth within 40 h. To rule out the possible masking effect of kgtP on c5038’s role, we compared the growth of two mutants, ΔkgtP mutant and Δc5038ΔkgtP mutant. The results showed no significant difference in growth between ΔkgtP mutant and Δc5038ΔkgtP mutant (Figure ), further supporting that c5038 played a negligible role in KG utilization in aerobiosis. Additionally, the introduction of a complementation plasmid (pkgtP) carrying kgtP controlled by its native promoter restored the growth of kgtP mutant (Figure ). These results suggest that kgtP is required whereas c5038 is dispensable for growth on KG under aerobic conditions.

Contributions of and anaerobic (C,D) conditions. Various strains were grown in M9 medium containing KG as the sole carbon and energy source. Trimethylamine N-oxide (TMAO) was added as electron acceptor under anaerobic conditions. Optical density at different time points was determined. Growth curves represent the average measurement at each time point from triplicate experiments. Student t-test was used for statistical analysis. e. v., empty vector; pc5038, complementation plasmid for c5038; pkgtP, complementation plasmid for kgtP.

Under anaerobic conditions, deletion of kgtP did not affect CFT073’s growth, whereas loss of c5038 dramatically reduced its growth in M9 with KG as the sole carbon source, as compared to WT (Figure ). Transformation of the c5038 mutant with a complementation plasmid (pc5038) carrying c5038 controlled by its native promoter significantly improved c5038 mutant’s growth (Figure ). To rule out the possible masking effect of c5038 on kgtP’s role under these conditions, we compared the growth of Δc5038 mutant and Δc5038ΔkgtP mutant. We observed a difference in growth between Δc5038 mutant and Δc5038ΔkgtP mutant at 60 h time point and thereafter (P < 0.05) (Figure ). These data indicate that under anaerobic conditions, c5038 played a major role and kgtP a minor role in growth on KG. Of note, in both aerobiosis and anaerobiosis, the complementation strains apparently had much shorter lag phases and higher growth rates than the WT strain carrying the empty vector. This was likely due to the fact that the complementation plasmid vector is a multi-copy plasmid (∼15 copies per cell), which can cause overexpression of kgtP or c5038.

Anaerobiosis Induced c5038 but Repressed kgtP Expression

Given that kgtP and c5038 contributed differentially to growth on KG under aerobic and anaerobic conditions, we sought to unravel their transcriptional levels under these conditions. The TSS of c5038 was obtained using 5′-RACE PCR (Figure ). The –10 and –35 sites of kgtP promoter were predicted by Virtual Footprint program (Munch et al., 2005) and Bprom program (Solovyev and Salamov, 2011), which presented consistent output with high reliability. The putative ribosome binding sites were also indicated. The predicted TSS of kgtP in CFT073 was highly similar to that in K-12 identified by 5′-RACE and deep sequencing (Cho et al., 2009). The promoter regions of kgtP and c5038 were then cloned to drive promoterless lacZ gene on a plasmid, respectively, and the resulting plasmid constructs were individually introduced into LMP10 strain (CFT073 ΔlacZYA). In parallel, a control plasmid carrying constitutively expressed promoter Pbla-lacZ fusion was also transformed into LMP10 strain. β-galactosidase activities were measured to indicate expression levels. As shown in Figure , c5038 expression was not detectable in aerobiosis, but was induced in anaerobiosis. The expression of kgtP was moderate under aerobic conditions, but was greatly repressed under anaerobic conditions, with a 40-fold reduction. It is also evident that anaerobic expression level of c5038 was significantly higher than aerobic expression level of kgtP (about 10-fold difference). In the control strain, Pbla-lacZ expression did not exhibit difference between under aerobic and anaerobic conditions. These results clearly showed that under anaerobic conditions, c5038 was highly expressed whereas kgtP was severely repressed, thereby at least partially explaining the phenotypic difference in growth.

Promoter region of kgtP. (B) Promoter region of c5038. Italic, start codon; italic and underlined, putative ribosome binding site; bold, transcription start site; bold and underlined, RpoN binding site/σ54 motif (–12 and –24 regions); italic and bold, inverted repeats for KguR binding; shaded, –10 and –35 regions; Underlined (solid), perfect ArcA binding site; underlined (dashed), imperfect ArcA binding site; boxed, FNR binding site. (C) kgtP and c5038 expression under aerobic and anaerobic conditions. Bacteria were grown aerobically or anaerobically in M9 medium containing KG as the sole carbon source. TMAO was used as an electron acceptor for anaerobic tests. Expression of c5038-lacZ or kgtP-lacZ was indicated by β-galactosidase activity. Pbla-lacZ constitutive expression was used as a control. Bars represent means ± SEM from three independent experiments performed in triplicate.

FNR and ArcA Induced c5038 But Repressed kgtP Expression

Fumarate and nitrate reduction and ArcA are two master regulators mediating bacterial adaptation to environmental oxygen availability (Constantinidou et al., 2006; Park et al., 2013; Jiang et al., 2015). To probe the regulatory roles of arcA and fnr in expression of c5038 and kgtP, we examined the effects of single deletion and double deletion of fnr and arcA on expression levels of c5038 and kgtP under aerobic and anaerobic conditions. Under aerobic conditions, deletion of fnr did not affect kgtP expression while loss of arcA upregulated kgtP expression about fivefold. Under anaerobic conditions, deletion of either fnr or arcA caused upregulation of kgtP expression (4-fold and 700-fold increase, respectively). Deletion of both fnr and arcA led to a greater increase in kgtP expression than individual deletions of fnr and arcA. These results indicate that both fnr and arcA negatively modulate kgtP expression. Further, in LMP10 strain, anaerobiosis repressed kgtP expression about 40-fold, as compared to aerobic expression, whereas in LMP10Δfnr and LMP10ΔarcA mutants, anaerobiosis repressed kgtP expression 10-fold and 4-fold, respectively (Figure ). These results clearly demonstrate that in either fnr or arcA mutant, anaerobic repression of kgtP was partially relieved compared with that in their parental strain LMP10, suggesting both fnr and arcA are involved in the anaerobic repression, with ArcA being a major player.

Roles of Loss of fnr or arcA leads to different effects on kgtP and c5038 expression. Various strains were grown aerobically or anaerobically in M9 medium containing KG as the sole carbon source. TMAO was used as electron acceptor anaerobically. Expression of c5038-lacZ or kgtP-lacZ in different strains was represented by β-galactosidase activity. (B) Non-radioactive EMSA studying the binding of ArcA to kgtP promoter regions. (C) Non-radioactive EMSA studying the binding of FNR to kgtP promoter regions. Gel-extracted PCR products of kgtP promoter region and cat (ChlR gene) coding region were used as probes. Purified His6-ArcA or His6-(FNR)2 fusion protein was added in different concentrations in each reaction mixture as indicated. DNA fragments were stained with SYBR green. AP, acetyl phosphate.

We next tried to understand the mechanisms by which ArcA and FNR repress the expression of kgtP. Sequence analyses of promoters repressed by ArcA or FNR suggest that ArcA and FNR inhibit gene expression through competitive binding to –10 or/and –35 region, thereby blocking occupancy of RNA polymerases (Myers et al., 2013; Park et al., 2013). Using a recently developed binding consensus sequence of ArcA ([GT][TA][TA][AG][AC][AT][AT][AT][AT][AT], each pair of letters in each bracket indicate two most frequent bases in that position, with the first being the most frequent) (Park et al., 2013), we were able to identify one perfect match (GTTGCATAAT) that overlaps the putative –35 region and two imperfect matches close to the perfect one (Figure ). Similarly, using a recently developed binding consensus sequence of FNR ([TA][TC][GA][AC][TC]nnnn[AG][TA][CT][AG][AC]) (Myers et al., 2013), we were able to identify a perfect match (ATGATatgcAACAA) that lies between –10 and –35 region. To test whether ArcA and FNR directly binds to the promoter region of kgtP, EMS assays were carried out. DNA fragments with sizes around 230 bp containing the predicted sites were prepared. Purified His6-tagged ArcA and FNR variant (FNRD154A) proteins (Shan et al., 2012) were obtained by nickel affinity chromatography. As shown in Figure , the DNA probe of kgtP promoter region can be shifted by ArcA protein, but the negative control probe cannot be shifted. With the same amounts of protein, the presence of acetyl phosphate appears to not affect binding of ArcA to DNA under our testing conditions. Also, FNR recombinant protein can shift the DNA probe of kgtP, but not the control probe (Figure ). These results indicate that ArcA and FNR can bind to the promoter region of kgtP directly. Altogether, these data suggest that ArcA and FNR likely repress kgtP expression through binding to promoter region and occluding the occupancy of RNAP.

For c5038, deletion of either fnr or arcA or both resulted in a significant reduction in c5038-lacZ expression in comparison to that in the LMP10 strain (P < 0.01), suggesting both fnr and arcA positively modulate c5038 expression. We next used EMSA to test if ArcA directly associates with DNA probe of c5038 promoter region. The results show that ArcA protein cannot shift the DNA probes (Supplementary Figure S3), raising the possibility that ArcA may not regulate c5038 expression directly. Taken together, these data indicate that FNR and ArcA stimulated c5038 but repressed kgtP expression.

Response of c5038 and kgtP Expression to Different Stimuli and the Role of CRP

In contrast to c5038, what stimuli induce kgtP’s expression is still unclear. To test if the expression of kgtP is influenced by the addition of KG, we measured and compared the expression levels of kgtP during bacterial growth in M9 (Glyc), M9 (Glyc and KG), and M9 (KG), respectively. Surprisingly, kgtP expression in all three media is comparable (Figure ), indicating that kgtP expression does not respond to the presence of KG. We next tested and compared the expression of kgtP in LB rich medium, M9 (Gluc), and M9 (Glyc). The results showed that the order of kgtP expression levels is as follows: M9 (Glyc) > M9 (Gluc) > LB.

Response of kgtP expression does not respond to the presence of KG. Bacteria were grown aerobically or anaerobically in different media containing the indicated carbon and energy source. TMAO was used as an electron acceptor for anaerobic tests. Gluc, glucose; Glyc, glycerol. (B) Role of CRP in kgtP expression. Bacteria were grown in M9 containing glycerol as sole carbon source. Expression of kgtP-lacZ was indicated by β-galactosidase activity. Bars represent means ± SEM from three independent experiments performed in triplicate.

The cAMP-CRP complex is a global regulatory element that induces a number of genes involved in the utilization of carbons in the absence of glucose (Botsford and Harman, 1992; Kolb et al., 1993), including glycerol catabolic regulon (Iuchi et al., 1990). We therefore examined whether CRP contributes to the regulation of kgtP expression. As shown in Figure , kgtP expression in the strain lacking crp was downregulated, as compared to the parental strain LMP10. This result indicates that CRP positively regulates kgtP expression. Collectively, these data suggest that kgtP might be responsive to the carbon state of the cell, and be used for carbon scavenging.

Roles of Different Alternative Sigma Factors in the Expression of c5038 and kgtP

KguR involved in direct activation of c5038 was predicted to belong to the category of bacterial enhancer-binding proteins (bEBPs) which interact with σ54 factor (Bush and Dixon, 2012), raising the possibility that c5038 is a new member of the σ54 regulon. The “A,” –30 site relative to the start codon was identified to be the TSS of c5038 (Figure ). Further analysis of the promoter region of c5038 did reveal a motif that is highly similar to the σ54 binding consensus “–24 to –12” region (Figure ), suggesting that c5038 transcription might depend on sigma54. To assess the contribution of rpoN (the gene coding for sigma54) in c5038 expression, we compared the expression levels of c5038 in LMP10 with that in LMP10ΔrpoN. As shown in Figure , deletion of rpoN abolished the KG induction of c5038 expression, as compared to parental strain LMP10. To ensure that no unwanted mutation was responsible for such phenotype, a complementation study was performed by introducing a copy of plasmid-borne rpoN controlled by its putative native promoter. The result showed that with rpoN reintroduced, the expression level of c5038 was rescued.

Role of alternative σ Loss of RpoN affects kgtP and c5038 expression differently. Various strains were grown aerobically (kgtP) or anaerobically (c5038) in M9 medium containing glycerol (kgtP) or KG (c5038) as the sole carbon source. TMAO was used as electron acceptor anaerobically. Expression of c5038-lacZ or kgtP-lacZ in different strains was represented by the β-galactosidase activity. e.v., empty vector; prpoN, complementation plasmid carrying rpoN. (B) Non-radioactive EMSA studying the binding of RpoN to promoter regions. PCR products of glnA (right three lanes) and c5038 (left four lanes) promoter regions were used as probes. Purified His6-RpoN fusion protein was added in different concentration in each reaction mixture as indicated. DNA fragments were stained with SYBR green. (C) Growth of the wild-type (WT) and rpoN deletion mutant. Bacteria were cultured in M9 minimal medium supplemented with KG as the sole carbon source and TMAO as an electron acceptor. Samples were taken at the indicated time points, and their OD600 was measured. Each point in the growth curve represents the average measurement from triplicate experiments. (D) The role of rpoS in kgtP expression in different growth phases. Bacteria were grown aerobically in M9 medium containing glycerol as the sole carbon source. Expression of kgtP-lacZ was represented by the β-galactosidase activity.

Sigma54 protein was shown to be capable of binding to target promoter regions independent of core RNA polymerase (Shingler, 2011). To evaluate if σ54 protein directly associates with c5038 promoter region, EMSA was used to study the binding of σ54 proteins to DNA molecules. σ54 was over-produced in E. coli as His6-RpoN fusion protein, which was then purified to homogeneity against nickel column. glnA promoter region, which was previously shown to be directly associated with RpoN (Huo et al., 2006), was included as a positive control; on the other hand, xylA promoter region, a member of σ70 regulon (Song and Park, 1997; Desai and Rao, 2010), was included as a negative control. As we expected, His6-RpoN fusion protein can shift P and P DNA probes, but not the P (Figure ; Supplementary Figure S4) probe. Altogether, these data indicate that c5038 is a new member in the RpoN regulon.

Because RpoN is required for c5038 expression which is important for growth of CFT073 on KG, we then examined the growth kinetics of the WT and ΔrpoN mutant in M9 (KG) containing abundant nitrogen source NH4Cl under anaerobic conditions. The results demonstrate that ΔrpoN mutants had a longer lag phase and grew significantly slower than their parental WT strain (P < 0.001) (Figure ). These data suggest that RpoN contributes to anaerobic utilization of KG, possibly through regulating c5038 expression.

In contrast to c5038, deletion of rpoN had no effect on kgtP expression (Figure ). We have shown earlier that kgtP expression remains high in M9 (Glyc), which is considered to be a carbon- and amino acid-limited environment. RpoS, also known as sigma38 factor, is a stress-responsive global regulator, which is induced under stress conditions such as carbon, phosphorus, nitrogen, or amino acid scarcity (Notley and Ferenci, 1996). Thus, rpoS was deleted from LMP10 strain, and its effects on kgtP expression were determined. Unexpectedly, loss of RpoS increased kgtP expression by about twofold, irrespective of growth phase (Figure ). Therefore, RpoS is a negative modulator of kgtP expression.

c5038 But Not kgtP Contributed to UPEC Fitness In vivo

Now that c5038 and kgtP were demonstrated to play differential roles in vitro, we next tried to investigate if loss of c5038 or kgtP affects UPEC fitness in vivo. Mice were challenged transurethrally with a 1:1 mixture of WT and a mutant, followed by recovering bacteria from tissues 48 h post inoculation and calculating competitive indices. Note that when inoculated in LB medium with 1:1 ratio, any two strains contained in the inoculums were recovered equally in numbers during exponential growth phase, indicating that they were equally fit in vitro. Figure showed that the c5038 mutant was significantly outcompeted by the WT in both bladder and kidneys (P-value equals 0.0005 and 0.0458, respectively). In vivo complementation experiments were performed to confirm that the mutation is not polar and the disadvantage is not caused by a secondary mutation (Figure ). The stable low-copy pGEN plasmid was used as it was shown to be well maintained in CFT073 up to 48 h even in the absence of antibiotic pressure. The Δc5038 mutant carrying empty vector displayed the expected competitive disadvantage in bladder and kidneys when co-challenging mice with the WT containing pGEN (P-value equals 0.0161 and 0.0342, respectively) whereas Δc5038 harboring a copy of c5038 on pGEN plasmid was equally fit as WT (pGEN). Therefore, reintroduction the c5038 gene into the c5038 mutant can potentially complement the negative effect on colonization caused by mutation of c5038.

Fitness of Bacteria were recovered from mice transurethrally challenged with a 1:1 mixture of two strains. Colonies on LB plates and LB plates containing the indicated antibiotics were enumerated. Competitive indices at 48 hpi were determined by dividing the ratio of two strains recovered by the ratio of the same two strains in the inoculum. Each group contains 10 or 6 mice, and experiments were performed at least twice. Bars represent the medians, and a Wilcoxon signed rank test was used to determine the significance of CI against a hypothetical median of 1. The threshold for statistical significance was a P-value < 0.05. ∗P < 0.05; ∗∗∗P < 0.001. (A) In vivo competition of Δc5038::KanR/WT, ΔkgtP::ChlR/WT, and Δc5038/kgtP::ChlR/Δc5038. (B) In vivo complementation study of c5038.

In contrast to the c5038 mutant, the kgtP mutant colonized murine urinary tracts as well as the WT (Figure , CI was not significantly different from the hypothetical value 1). To rule out the possible masking effect of c5038 on kgtP, we tested the effects of mutating kgtP in Δc5038 genetic background on fitness in vivo. Deletion of kgtP in Δc5038 background had no impact on colonization of mice by CFT073, further corroborating that kgtP is not important for colonization of mice by UPEC. Taken together, these results suggest that c5038 but not kgtP is important for UPEC fitness in vivo.

Phylogenetic Analysis of c5038 and kgtP

In light of distinct roles of C5038 and KgtP in KG utilization and in vivo fitness of CFT073, their phylogenetic trees were constructed in order to understand their evolutionary routes (Escherichia spp. strains were excluded). Comparing the two phylogenetic trees (Supplementary Figures S5A,B), it is evident that the two transporters have different patterns: KgtP is clustered with many E. coli close relatives whereas C5038 is clustered with only two of them, with others being Pseudomonads and Burkholderiales. These data strongly indicate that C5038 and KgtP have distinct evolutionary trajectories, supporting our suggestion that c5038 was acquired through horizontal gene transfer.

Discussion

Bacterial pathogens usually possess more than one system/factor to execute certain cellular function critical for pathogenicity (Brussow et al., 2004). In UPEC, multiple fimbrial genes and iron acquisition systems exist and contribute to UPEC’s fitness in different host niches and environments (Garcia et al., 2011; Spurbeck et al., 2011). In this study, we described multiple aspects of transcriptional regulation of two transporters, C5038 and KgtP, and effects of their regulation on physiological roles played by the two transporters. The expression of c5038 was induced solely by KG under anaerobic conditions, which was mediated by ArcA and FNR regulators responsible for adaptation to low-oxygen environments. Thus, it can be expected that possession of c5038 can significantly increase UPEC’s growth on KG anaerobically (Figures ). Phylogenetic analysis supports that c5038 was acquired through horizontal gene transfer. Further, c5038 is highly prevalent in UPEC strains (78%), but very lowly in diarrheagenic E. coli strains (0%) (Cai et al., 2013). These findings prompted us to reason that C5038 is a specialized protein that carries out important functions for UPEC under in vivo conditions. During infection by UPEC, host sites such as renal proximal tubule cells could provide abundant KG (Martin et al., 1989; Pritchard, 1995) while oxygen tension is low (Melican et al., 2008). Indeed, loss of c5038 led to reduced fitness during colonization of murine urinary tracts by UPEC (Figure ). Although the c5038 mutant was outcompeted less than twofold, it is plausible that under our testing conditions, C5038 did not exert effects to its full potential. Acquisition of genes or factors that promote fitness of bacterial pathogens is a recurring theme. For example, in response to tetrathionate, genomic island-encoded loci ttrABC provide a growth advantage for Salmonella by utilizing tetrathionate anaerobically as an electron receptor produced by inflamed guts (Price-Carter et al., 2001; Winter et al., 2010).

The expression of kgtP is not inducible by KG and appears to be constitutively expressed in K-12 strain, implying that kgtP is expressed at similar levels in different environments (Seol and Shatkin, 1992). We did show here that kgtP expression in CFT073 does not respond to the addition of KG, but its expression was high in M9 (Glyc) and low in M9 (Gluc) and LB. This was consistent with previous KG accumulation assays demonstrating that maximal intracellular concentration of KG was achieved for bacteria grown on glycerol, followed by those on Gluc and LB (Seol and Shatkin, 1992). Because one of the major stresses E. coli face during growth on glycerol is limited carbon availability, it is tempting to hypothesize that kgtP senses carbon availability of bacterial cells and that importing KG could scavenge carbons (Yan et al., 2011) and help improve carbon status, considering the role of KG in providing carbon skeletons for both TCA cycle (tricarboxylic acid cycle) and amino acid metabolism. Supporting data is that the expression level of kgtP was higher in stationary phase when carbon sources are mostly depleted (Supplementary Figure S6), and was regulated by CRP and stress sigma factor σS. However, more biochemical evidence is needed to further address that hypothesis. Similar to KgtP in K-12, KgtP in UPEC str. CFT073 is also required for aerobic growth using KG as the sole carbon source. On the contrary, KgtP only played a minor role in bacterial growth on KG under anaerobic conditions (Figure ). Expectedly, loss of KgtP did not result in any competitive disadvantage in the animal model. Overall, we for the first time revealed kgtP is subject to regulation by oxygen tension, which was mediated by ArcA and FNR.

The TCA cycle is a bi-functional pathway that generates ATP during the catabolic process and supplies skeletons in the anabolic process of biosynthesis (Kim and Gadd, 2008). As a result, transport, assimilation, and conversion of intermediates in TCA cycle are subject to regulation in response to oxygen availability (Nakano et al., 1998; Kim and Gadd, 2008). For four-carbon (C4)-dicarboxylates in TCA cycle including succinate, fumarate, and malate, E. coli possess both aerobic and anaerobic transporters, namely, DctA (Davies et al., 1999) and DcuABC (Zientz et al., 1996; Golby et al., 1998). Similar to DctA, KgtP requires protons to import its substrate-KG (Seol and Shatkin, 1992). Similar to DcuABC which exchanges their substrates with succinate, we also found that overexpression of c5038 led to decreased succinate assimilation, suggesting that C5038 likely exported succinate (data not shown). To ensure optimal assimilation of KG, KgtP as a proton symporter needs to be inhibited in low-oxygen environments in order to save energy, whereas C5038 can function in a substrate-product exchange fashion which is less energy-consuming and thus highly desirable. Indeed, our data show that FNR and ArcA co-repress kgtP but co-activate c5038 expression, emphasizing roles of FNR and ArcA in promoting a coordinated response to KG in anaerobic environments. Likewise, for C4-dicarboxylate transporters, DctA is repressed by ArcA under anaerobic conditions (Davies et al., 1999) while Dcu system is activated by FNR under anaerobic conditions (Engel et al., 1992). Conceivably, possessing functional alternatives with optimized regulatory patterns for importing KG should greatly promote UPEC’s fitness in changing environments. To our knowledge, for the first time, we reveal bacteria carry both aerobic and anaerobic transporter systems for KG. The conformity between C4- and C5-dicarboxylates transporters makes us speculate that such transport systems and regulatory patterns might be widely existed for other important metabolites in bacteria.

Since FNR activates arcA expression under anaerobic conditions (Compan and Touati, 1994), it raises a question of whether FNR can affect kgtP expression through ArcA. Our data showed that in the absence of arcA, mutation of fnr can still increase the expression of kgtP by about twofold, indicating that FNR can regulate kgtP independent of ArcA. ArcA represses kgtP expression to a higher degree than FNR does, this can be at least partially attributable to the fact that ArcA can potentially bind to three putative binding sites, whereas FNR can only bind to one site. By binding to more than one site, ArcA has a potential to form multimer (Pellicer et al., 1999; Jeon et al., 2001); in contrast, FNR usually works as a dimer (Lazazzera et al., 1996; Jervis and Green, 2007). In the arcA mutant, kgtP expression in aerobiosis was higher than that in anaerobiosis, this may be due to that (1) fnr can still repress gene expression in anaerobiosis when arcA is absent, (2) there are other activating elements (probably cAMP-CRP) that function better under aerobic conditions, or (3) the metabolic changes caused by arcA deletion.

Like TCS response regulator KguR and inducer KG, sigma54 factor is also essential in activating c5038 expression, thereby adding one more level of control in the existing regulatory network for orchestrating c5038 expression. It is well established that members of sigma54 regulon are primarily involved in nitrogen metabolism, although exceptions exist (Shingler, 2011). The inclusion of sigma54 in c5038 regulatory network is expected to have great biological significance as nitrogen availability and KG accumulation are inversely correlated (Reyes-Ramirez et al., 2001; Yan et al., 2011). Recent studies have suggested that UPEC can encounter environments with limited nitrogen source during infection (Snyder et al., 2004). Therefore, it is likely that expression of c5038 helps accumulate KG and further to adapt to nitrogen-limited urinary tracts (Snyder et al., 2004; Hagan and Mobley, 2007). Notably, RpoN coding for sigma54 factor and a bEBP regulator MifR were both required for the expression of KG transporter PA5530 (Lundgren et al., 2014) in Pseudomonas aeruginosa, highlighting the possibility of commonly using a similar mechanism to link KG and nitrogen metabolism. By contrast, sigma54 factor does not contribute to kgtP expression, further distinguishing it from c5038.

In summary, we employed bioinformatic, biochemical, and genetic tools to study two transporters involved in KG utilization, demonstrating that C5038 is likely an anaerobic KG importer but KgtP is an aerobic KG importer. Their distinct roles can be at least in part attributed to distinctive transcriptional regulatory patterns. The development of these two systems should provide adaptive advantages for UPEC in various environments.

Author Contributions

WC, SY, and HZ conceived and designed the experiments; WC, YY, and XC performed the experiments; WC and HZ analyzed the data; WC, SY, and HZ contributed reagents/materials/analysis tools; WC, SY, and HZ wrote the paper. All authors contributed, read and approved the final manuscript.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.