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1 Institute of Microbiology and Epizootics, Free University Berlin, Philippstraße 13, D-10115 Berlin, Germany
2 Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
3 Institute for Genetics, University of Cologne, Zülpicher Straße 47, 50674 Cologne, Germany
Correspondence
Ganwu Li
liganwu{at}iastate.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the sequence of yjjQ and its flanking region is AM184164.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). To elucidate the pathogenesis of APEC, we adapted signature-tagged transposon mutagenesis to a 5-week-old chicken model and identified 28 virulence-associated genes (Li et al., 2005). Among them, yjjQ encodes a putative transcriptional regulator since sequence analysis revealed that it harbours a helix–turn–helix (HTH) DNA-binding domain shared by all members of the LuxR family regulators (Chirwa & Herrington, 2003; Crater & Moran, 2001; Pruneda-Paz et al., 2004). The presence of yjjQ has been reported for commensal and various pathogenic bacteria such as enterohaemorrhagic E. coli (EHEC), uropathogenic E. coli (UPEC), Shigella and Salmonella by genomic sequencing. However, the function of YjjQ remains unknown.
To compete and survive in a changing environment, bacteria have developed elaborate global regulation networks to sense and respond to stimuli from outside. For example, guanosine tetraphosphate (ppGpp) downregulates rRNA synthesis during nutritient starvation (Chatterji & Ojha, 2001); the glutamate decarboxylase system together with AR1 and AR3 (arginine decarboxylase system) are jointly responsible for defence against acid stress (Foster, 2004); the challenge of oxidative stress is met by the OxyR and SoxR/S system (Storz & Imlay, 1999), while heat-shock proteins respond to elevation in temperature (Gophna & Ron, 2003); alternative sigma factors such as RpoS, RpoE and RpoH play an important role in bacterial survival by directing the transcription of genes involved in starvation survival and resistance to oxidative, acid, thermal and osmotic stress (Kazmierczak et al., 2005; Loewen & Hengge-Aronis, 1994). In addition, Fur affects iron uptake by binding to the promoter region of iron-regulated genes with ferrous iron as a cofactor, thus repressing the expression of these genes (Hantke, 2001). The virulence and pathogenesis of medically important organisms are also regulated by these regulators, and in recent years great progress has been made in identifying those that coordinate expression of virulence genes (Byrne & Swanson, 1998; Johnson et al., 2006a; Lucas et al., 2000; Mellies et al., 2006; Pizarro-Cerda & Tedin, 2004). Perhaps not surprisingly, such regulators are almost as numerous as the environmental signals to which virulence gene expression has been found to respond (Dong et al., 2005; Lucas & Lee, 2001; Sturme et al., 2005), and many of them remain to be identified. In this study we aimed to elucidate the possible function of YjjQ as a regulator. We show that YjjQ is involved in virulence of APEC by regulating iron uptake.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Oligonucleotide primers are given in Table 2. Growth of bacterial cultures was performed at 37 °C in Luria–Bertani (LB) broth, M9 medium or modified low-phosphate-glucose-salts medium (LPM) which lacks KCl (Sambrook & Russell, 2001; Seputiene et al., 2003). When necessary, antibiotics were added at the following concentrations: ampicillin, 50 µg ml–1; kanamycin, 50 µg ml–1; nalidixic acid, 30 µg ml–1; chloramphenicol, 25 µg ml–1.
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In vitro competition and in vivo tests in chickens.
For in vitro competition assays cultures of mutant and wild-type strain were mixed in a ratio of 1 : 1, incubated in LB broth for 4 h at 37 °C and then plated onto media with or without kanamycin. For in vivo competition assays (Li et al., 2005), 5-week-old female specific pathogen-free chickens (Lohmann Selected White Leghorn; Lohmann Tierzucht) were inoculated with 108 c.f.u. of this mixture (in a dose of 0.5 ml) by intratracheal application. Chicks were provided with food and water ad libitum. After 24 and 48 h of infection, the spleen, heart, liver, lungs and kidneys of five chickens were collected, weighed and homogenized, and serial dilutions were plated on LB medium with and without kanamycin for selection of mutants or total bacteria, respectively. A competitive index (CI) was calculated for the mutant by dividing the output ratio (mutant/wild-type) by the input ratio (mutant/wild-type).
For individual chicken tests, 108 c.f.u. of the wild-type or mutant strain were inoculated into the trachea of 20 chickens. After 24 h and 48 h, 10 chickens were euthanized and examined for macroscopic lesions. The spleen, pericardium, anterior lobe of the liver, lungs and kidneys of 10 chickens were collected, weighed and homogenized. Bacterial loads were determined by plating serial dilutions of the homogenates on selective LB agar medium.
Statistical comparisons were performed by using Student's t test to determine the difference between two groups. A P value of <0.05 was considered significant.
DNA manipulation and data analyses.
DNA manipulation and transformation were performed according to standard methods (Ausubel, 1994). All restriction and DNA-modifying enzymes were purchased from Roche Molecular Biochemicals. Sequence analysis was done by AGOWA GmbH, Berlin, Germany, by using the chain-termination sequencing technique and an ABI model 373A DNA sequencer. Sequence data and promoter analyses were performed by applying the HUSAR sequence analysis package from the German Cancer Research Centre (DKFZ) in Heidelberg, Germany, and Neural Network Promoter Prediction software.
Expression studies
Construction of a LacZ-negative mutant strain.
An insertion mutation in the lacZ gene of IMT5155NalR was constructed by using suicide plasmid pGP704 (Herrero et al., 1990). The lacZY fragment was amplified by PCR with primers 1529 and 1530. Underlined bases in the sequences listed in Table 2
indicate restriction cutting sites of BglII and SalI. The recombinant plasmid of pGP704 with the lacZY fragment was transformed into E. coli strain S17
pir. Mating was performed between S17pir/pGP704 and the recipient strain IMT5155NalR. The conjugants were selected on LB agar plates with 100 µg ampicillin ml–1, 30 µg nalidixic acid ml–1 and 1 mM X-Gal. One white colony was picked up as a LacZ-negative strain and designated IMT5155 lacZ–.
Construction of lacZ transcription fusions.
PyjjQ–lacZ fusions were constructed by cloning PCR fragments containing terminal EcoRI and BamHI sites to EcoRI- and BamHI-restricted pRS551 (Simons et al., 1987). Primers 1781 and 1782 (Table 2) were used for amplification of fragment PyjjQ1 encompassing the yjjQ upstream region from position –398 to +28, relative to the translation start codon (accession no. AM184164). Cloning of this fragment resulted in plasmid pPYJJQ1. Fragment PyjjQ2 encodes the yjjQ upstream region from position –660 to +28 and was constructed by using primers 1780 and 1782. The plasmid carrying this cloned PyjjQ2 was named pPYJJQ2. Both plasmids were used to transform IMT5155 lacZ–, resulting in recombinants IMT5155P1 and IMT5155P2.
β-Galactosidase assays.
These assays were carried out as described by Miller (1972). E. coli strains were grown overnight in LB broth at 37 °C and the cultures were then diluted 100-fold into fresh medium and grown to an OD600 of 1.0. One-millilitre volumes of these fresh cultures were diluted into 50 ml LB, LB with 2 or 4 % NaCl, LB with 500 or 100 µM H2O2, M9 minimal medium with 0.4 % glucose as sole carbon source, and modified LPM of pH 7.0 or 4.5. Samples for photometric quantification and for β-galactosidase assays were taken every hour and tests were performed in triplicate.
Mutant generation and BglJ overexpression plasmid construction.
The yjjQ gene was replaced with a chloramphenicol-resistance gene by using the red recombinase system (Datsenko & Wanner, 2000). The chloramphenicol acetyltransferase gene was amplified from plasmid pKD3 by PCR using primers 2376 and 2377 (Table 2). Underlined bases in the sequences indicate sequence homology to the yjjQ flanking region. The PCR product was gel purified and electroporated into M18E10 containing the red recombinase expression plasmid, pKD46. Following electroporation, the samples were incubated at 28 °C for 1 h in SOC broth (Sambrook & Russell, 2001) and plated on LB agar containing chloramphenicol for overnight growth at 37 °C to select for transformants and to induce the loss of pKD46. The bglJ deletion mutant KEC361 was generated from wild-type strain IMT5155 using the same strategy except that primers 2376 and 2377 were used (Table 2). The mutant was confirmed by PCR and Southern blotting.
The BglJ overexpression plasmid pKEAP1 carries bglJ under control of the tac promoter. This pACYC-derived plasmid also carries the lacIq gene and a kanamycin-resistance gene (Paukner, 2007). Further, in E. coli K-12 induction of pKEAP1-encoded bglJ relieves the H-NS-mediated repression of the bgl promoter confirming that BglJ is expressed (Paukner, 2007). Plasmid pKEAP1 was transformed into wild-type strain IMT5155 to give IMT5155P3.
One-dimensional SDS-PAGE.
Cells of IMT5155 wild-type, the yjjQ transposon-insertion mutant M18E10 and the 
yjjQ mutant KEC363 were obtained from overnight cultures in LB and from cultures grown in M9 medium with 0.4 % glucose for 6 h. Cells were harvested by centrifugation, washed with phosphate-buffered saline (PBS) and resuspended in 200 µl lysis buffer containing 9 M urea, 2 % CHAPS (Sigma Aldrich), 70 mM dithiothreitol (Sigma Aldrich), and 2 % ampholyte (pH 2.0–4.0; Amersham Biosciences). The resuspended cell pellet was six times frozen in liquid nitrogen, thawed and mixed. Then it was ultrasonicated six times for 20 s during a 30 min incubation at room temperature. Cell debris was removed by centrifugation, and the protein concentration in the aqueous supernatant was quantified photometrically. Ten-microgram samples of protein were separated on 15 % SDS-PAGE gels by standard methods (Ausubel, 1994) and silver staining was carried out as described by Tsai & Frasch (1982).
Proteomic analysis.
Proteins from wild-type IMT5155 and mutant M18E10 cultured in M9 minimal medium were extracted as described above. Protein samples (300 µg) from both strains were separated by 2D-PAGE according to the method of Hochstrasser et al. (1988). Briefly, proteins were separated in the first dimension according to their isoelectric point (pI) by isoelectric focusing in a pH gradient ranging from 3.0 to 10.0. In the second dimension, the proteins were separated according to their molecular masses by SDS-PAGE and visualized by Coomassie blue. Cellular proteins of each tested strain were isolated in at least three separate experiments, and 2D-PAGE of each protein preparation was repeated at least three times. The 2D gels used for comparison analysis were digitized at a resolution of 150 d.p.i. using a PowerLook 2100XL with transparency adaptor. 2D image analysis was performed using the Proteomweaver software (Definiens).
Only proteins that could be reproducibly and clearly found to increase or decrease at least 2.5-fold in abundance, based on the intensity of the protein spots, were further analysed. The following procedures were commercially finished by the Proteome Factory (Berlin, Germany). Analysis of protein spots excised from 2D gels was performed by in-gel tryptic digestion. The generated peptides were applied to an Agilent 1100 nanoLC system with trap column online-coupled to an ion trap mass spectrometer Esquire3000plus (Bruker). The MS-MS data were searched against the NCBInr protein database using the Mascot search engine.
RNA preparation and real-time RT-PCR.
To restrict iron availability the cation-exchange resin Chelex 100 (Bio-Rad) was added to liquid media (batch method according to the supplier's manual) and glassware was soaked overnight in 10 % HCl and then rinsed extensively in double-distilled deionized water. Ca2+ and Mg2+ were added to Chelex-treated LB to final concentrations of 0.2 and 0.1 M. The wild-type strain IMT5155, its yjjQ mutants M18E10 and KEC363, bglJ deletion mutant KEC361 and BglJ-overexpressing strain IMT5155P3 were grown in treated LB medium to the early exponential phase (OD600 1.0) and total RNA was isolated using the High Pure RNA isolation kit (Roche Diagnostics). Treatment of total RNA with DNase and analysis on formaldehyde gels was performed as described by Puopolo et al. (2001). RNA concentrations were determined using a NanoDrop 1000 apparatus (NanoDrop Technologies), and 1 µg total RNA was reverse transcribed in triplicate using random hexamers and M-MLV reverse transcriptase (Promega). Real-time PCR was done using the SYBR green PCR Master Mix (Applied Biosystems) according to the manufacturer's guidelines. Reactions (10 µl) were performed using 1 µl cDNA template per reaction. A 7900HT Fast Real-time PCR machine (384-head) (Applied Biosystems) was used. PCR conditions consisted of an initial incubation step for 10 min at 95 °C, followed by 40 cycles for 30 s at 95 °C, 30 s at 59 °C, and 30 s at 72 °C. Melting curve analyses were performed after each reaction to ensure amplification specificity. Differences (n-fold) in transcript were calculated using the relative comparison method, and amplification efficacies of each primer set were verified as described by Schmittgen et al. (2000). RNA levels were normalized by using the housekeeping gene dnaE as control. Primers used for real-time RT-PCR analysis are listed in Table 2.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The capacity of mutant M18E10 to compete for growth in chicken tissues with wild-type strain IMT5155 (O2 : H5 : K1) was compared by in vivo and in vitro competition assays. The mutant showed moderately attenuated growth in lung and kidney, with competitive indices (CI) of 0.031 and 0.021 (P<0.01), respectively; whereas in liver, spleen and heart, M18E10 was strongly out-competed by the wild-type (CI 0.0037, 0.001 and 0.0015, respectively, P<0.001). In contrast, the in vitro CI was approximately 0.30 (P<0.05).
Mutant M18E10 and wild-type IMT5155 were further tested individually in chickens by imitating the natural route of infection and administering a dose of 108 c.f.u. into the trachea of 5-week-old specific pathogen-free birds. All 10 birds that were challenged with the wild-type strain developed severe bilateral airsaculitis, moderate pericarditis, marked pulmonary congestion, severe hypertrophy and congestion of the spleen, and moderate perihepatitis covered by a thick yellow fibrous membrane. In contrast, M18E10 caused only moderate airsaculitis, mild pericarditis and pulmonary congestion, slight hypertrophy of the spleen and mild perihepatitis without yellow fibrous membrane. The capacity of M18E10 and the wild-type strain to colonize internal organs was studied by determining colony numbers in selected organs 24 and 48 h after infection. Both strains colonized the avian lung, heart, liver, spleen and kidney. Significantly, after 24 h maximum colonization of the lung as well as of the heart was observed for the wild-type, while M18E10 colonized only the lung at a maximum level. Minimal colony counts were obtained in liver (3.46x102 c.f.u. for M18E10) and spleen (8.61x105 c.f.u. for wild-type). After 48 h the results differed to some extent, with both the wild-type and the mutant strain colonizing the kidney maximally. Except in the kidney, c.f.u. levels were much lower at 48 h than at 24 h after infection, and minimum levels were seen in the liver for both strains. However, on average the M18E10 mutant strain was reisolated at levels 10–1000 times lower than the wild-type strain from all internal organs at both time points (Fig. 1).
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). The products were sequenced commercially at AGOWA and all sequence data have been submitted to the EMBL database under accession number AM184164. Sequence analyses revealed that the upstream gene of yjjQ is yjjP, encoding a putative outer-membrane protein. Between yjjQ and yjjP, a 557 bp non-coding region is present which may harbour promoter and regulator elements for yjjQ as predicted by the Neural Network Promoter Prediction software. The gene immediately downstream of yjjQ is the regulator gene bglJ. BglJ, when constitutively expressed, activates expression of the β-glucoside operon, bgl (Giel et al., 1996; Madhusudan et al., 2005). The 5' end of bglJ shows a 42 bp overlap with the 3' end of yjjQ. In the yjjQ mutant strain M18E10, the Mini-Tn5 transposon was inserted 358 bp downstream of the yjjQ start codon.
The 726 bp yjjQ nucleotide sequence of E. coli IMT5155 is highly similar to that of E. coli strains MG1655, UPEC CFT073 (O6 : H5) and EHEC EDL933 (O157 : H7), and Shigella flexneri (98–99 %) (Blattner et al., 1997; Hayashi et al., 2001; Welch et al., 2002), while there is lower similarity to that of Salmonella Typhimurium (67 %) (McClelland et al., 2001). Analysis of the amino acid sequence suggested that yjjQ encodes a putative protein of 242 amino acids which harbours a helix–turn–helix (HTH) DNA-binding domain shared by all regulators of the LuxR family, suggesting that yjjQ encodes a transcriptional regulator protein.
Transcription of yjjQ is induced in M9 medium and mutant M18E10 shows reduced growth in this medium
To further characterize this putative regulator, we studied its expression under different environmental conditions, expecting that these results would provide clues for further functional analysis. Two fragments of the putative 5' regulatory promoter region of yjjQ were cloned and fused with promoterless lacZ, resulting in P1yjjQ : : lacZ, which encodes a fragment from –398 to +28, relative to the translation start, and P2yjjQ : : lacZ, encoding a fragment from –660 to +28 of the yjjQ promoter region. Expression analysis of transformants of strain IMT5155 lacZ– carrying these two fusions revealed that P1yjjQ : : lacZ expression was approximately two times higher than that of P2yjjQ : : lacZ under all conditions tested, including LB, M9, LB with 4 % NaCl, LB at pH 5, and LB with 500 µM H2O2 (P<0.01). These results suggested the existence of a negative regulatory element for yjjQ expression within the –398 and –660 region. Analysis of the promoter activity directed by P1yjjQ showed that the activity was enhanced about twofold in M9 (2200 units) compared to LB medium (1000 units) (P<0.01).
As the transcription of yjjQ was shown to be upregulated in M9 medium, we further compared growth of the M18E10 mutant and wild-type in this medium. Interestingly, the mutant grew extremely slowly in M9 with 0.4 % glucose as the sole carbon source. While the wild-type reached an OD600 of 1.7 after 7 h, M18E10 reached an OD600 of only 0.2 after the same period of time (Fig. 2). In contrast, the growth rates of wild-type and mutant strain in LB medium were nearly the same, with the mutant showing slightly slower growth during the exponential phase (data not shown). Since the transposon-insertion mutation could not be complemented, we constructed a yjjQ deletion mutant. The phenotypes of the wild-type strain IMT5155, the transposon-insertion mutant strain M18E10 and the yjjQ deletion mutant KEC363 (Table 1) were compared. As expected, the yjjQ-deleted mutant KEC363 could hardly grow in M9, like the original mutant M18E10 (Fig. 2). To rule out the possibility that the insertion of the mini-Tn5 exerts a polar effect on the downstream gene bglJ, we further constructed the bglJ deletion mutant KEC361 and BglJ-overexpressing strain IMT5155P3. No difference of growth in M9 was observed between these two new strains and the wild-type strain. These results corroborate our conclusion that the phenotypic changes in the mutants were due to loss of yjjQ function and not to effects on the downstream overlapping sensor-regulatory BglJ system (Fig. 2).
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). Due to the fact that growth differences between M18E10 and the wild-type strain were most obvious in M9 medium with glucose as the sole carbon source, extracted proteins from both strains grown in M9 were also examined by SDS-PAGE. In this analysis more than 10 differing protein bands were seen between wild-type and mutant strain, including ones representing both upregulated and downregulated proteins (Fig. 3a). Furthermore, the transposon-insertion mutant M18E10 and the yjjQ deletion mutant KEC363 showed a similar expression profile when grown in M9 (Fig. 3b). The expression profiles of the bglJ deletion mutant KEC361, BglJ-overexpressing strain IMT5155P3 and the wild-type grown in M9 were also compared; no obvious difference was detected (results not shown). These results suggest that the observed differences between wild-type and mutant strain grown in M9 were indeed caused by mutation of the yjjQ gene.
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) and were subjected to further analyses by nanoLC-ESI-MS(n). Two of the spots (14 and 25) possessed only low identification scores, while for two others (16 and 18) MS analysis did not yield an unequivocal identification. Among the remaining 21 protein spots, 14 were consistently enriched from the wild-type and 7 from the mutant strain (Table 3).
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): SitA is a periplasmic iron-binding protein which is encoded in a four-member operon that mediates manganese and iron transport (Hantke, 2001; Sabri et al., 2006; Zhou et al., 1999). The second downregulated protein, ChuA, is involved in haem uptake in EHEC, UPEC, APEC (Li et al., 2005; Torres & Payne, 1997; Welch et al., 2002) and several other bacterial pathogens. The TonB-dependent outer-membrane protein FyuA is encoded by the ferric yersiniabactin uptake system (Pelludat et al., 1998), and the putative function of IreA is a siderophore receptor associated with ExPEC (Russo et al., 2001). SitA, ChuA and FyuA were identified as unique protein spots in wild-type IMT5155 cultured in M9 medium, while IreA was approximately 10 times more abundant in this strain in comparison to mutant M18E10.
Several enzymes involved in amino acid synthesis were also identified as proteins repressed in the yjjQ mutant. TyrB and AroA are related to synthesis of aromatic amino acids (Ingraham et al., 1987), with the former representing an aminotransferase that is involved in the last step of biosynthesis of both tyrosine and phenylalanine. The 5-enolpyruvylshikimate-3-phosphate synthase AroA is a key enzyme in the aromatic amino acid biosynthetic pathway in micro-organisms. 2D signals of both enzymes from wild-type were about threefold stronger than those of mutant M18E10. Similarly, IlvD (dihydroxy acid dehydratase), which catalyses dehydration reactions of 
,β-dihydroxy acids to yield the -ketoacid precursors of isoleucine and valine (Ingraham et al., 1987), was shown to be about five times more abundant in the wild-type compared to the mutant strain. Succinyltransferase DapD was targeted as a unique protein spot from the wild-type. This enzyme is involved in the succinylase pathway synthesizing DL-DAP, which is the direct precursor of L-lysine and an important constituent of the cell wall peptidoglycan (Fuchs et al., 2000). To check whether addition of amino acids would improve the growth of mutants in M9, 0.4 % Casamino acids (Sigma) was added. Indeed, the growth of both mutant strains M18E10 and KEC363 recovered to the level of wild-type in M9 with Casamino acids.
In addition, several proteins involved in molecular chaperoning (DnaK) (Slepenkov & Witt, 2002), acid tolerance response (YfiD) (Stancik et al., 2002), LPS core region biosynthesis (RfaE) (Valvano et al., 2000), transcriptional regulation and other functions were identified. Interestingly, YbeJ, a putative glutamate/aspartate periplasmic binding and transport protein (Tremoulet et al., 2002) was represented by one of the five unique spots from the wild-type. While most of the identified proteins migrated as single spots on 2D gels, YfiD possessed two spots with different abundances less than 0.2, consistent with a wild-type bias for this protein.
Among the nine mutant-enriched protein spots, two were found to be unique for the yjjQ mutant, suggesting that these proteins may be repressed by YjjQ (Table 3). Two of them are directly or indirectly linked to amino acid synthesis: Asd is an aspartate-semialdehyde dehydrogenase involved in lysine biosynthesis and was identified as a unique spot in mutant M18E10. The abundance of YjgF in M18E10 was about 3.4 times greater than that in the parental strain; this protein is involved in isoleucine biosynthesis (Schmitz & Downs, 2004).
LpxD is an acyltransferase participating in lipid A biosynthesis while AceA is an isocitrate lyase (Rieul et al., 1988; Vaara & Nurminen, 1999). Both LpxD and AceA were more than four times more abundant in mutant M18E10 when compared to the wild-type. TolC is a membrane channel tunnel protein involved in enterobactin transport and multi-drug efflux (Bleuel et al., 2005). This protein serves as an outer-membrane channel, and it was three times more abundant in mutant M18E10. Finally, FliC was identified as a further unique protein spot in mutant M18E10. The flagellar filament is composed of a single protein flagellin and most E. coli have a flagellin gene fliC (Wang et al., 2003). However, when motility of mutant strains M18E10 and KEC363 and the wild-type was compared in LB medium with 0.3 % agar, no difference was observed. This suggests that the regulation of fliC by yjjQ might occur only under specific conditions.
Expression of iron-uptake genes is downregulated at the level of transcription
Because YjjQ is a putative transcriptional regulator, the regulation of the targeted iron-associated genes was confirmed by real-time RT-PCR at the level of transcription. Since the yjjQ mutant grew poorly in M9 medium and the cell density may be not sufficient to exhaust all the iron present in the medium, the iron-regulated genes may not be induced. To avoid overinterpreting the results of proteomic analysis, the total RNA was isolated from yjjQ transposon-insertion mutant strain M18E10, deletion mutant strain KEC363 and wild-type strain cultured in LB depleted of iron, instead of M9. Results of real-time RT-PCR showed that expression of all of four identified iron-uptake genes, ireA, fyuA, chuA and sitA, was downregulated in both transposon-insertion mutant strain M18E10 and yjjQ deletion mutant strain KEC363 when cultured in LB without iron. The maximum downregulation was observed for chuA (10.6-fold in M18E10 and 9.7-fold in KEC363); while the expression of fyuA was downregulated only 4.6-fold in strain M18E10 and 4.3-fold in strain KEC363. The mRNA abundances of sitA and ireA in the mutant M18E10 strain were about 6.6-fold and 5.4-fold, respectively, lower than those in the wild-type. Similarly, the mRNA abundances of sitA and ireA in strain KEC363 were 6.2 and 4.9-fold lower than those in the wild-type. Statistical significance was calculated on Ct values (Schmittgen et al., 2000) with a paired t test, and P values were all <0.0001. To determine if the downstream overlapping gene bglJ plays any role in the regulation of iron-uptake genes, we also compared the mRNA level of ireA, fyuA, chuA and sitA in bglJ deletion mutant KEC361, BglJ-overexpressing strain IMT5155P3 and the wild-type strain. No significant difference was found.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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yjjQ mutant and the insertion mutant was reduced in M9 medium. Proteomic analysis revealed that the iron-uptake receptors SitA, FyuA, ChuA, and IreA were downregulated in the yjjQ mutant. Furthermore, their regulation occurs at the transcription level, as confirmed by real-time RT-PCR. We believe that this is the first characterization of a yjjQ mutant with defects in APEC virulence, apparently due to impaired iron uptake.
It was not possible to do a yjjQ complementation test, although great efforts were made. Fragments of yjjQ and yjjQ-bglJ were cloned into plasmid pKESK22 under control of promoter Ptac and plasmid pBAD18-Cm under control of promoter PBAD. No evidence was found by SDS-PAGE that YjjQ was expressed when the plasmids thus obtained were transformed into transposon-insertion yjjQ mutant M18E10 and deletion mutant KEC363 respectively and then induced by arabinose or IPTG. Correspondingly, no trans-complementation of phenotypes was observed in in vitro tests in these plasmid-complemented mutant strains. The same was true when yjjQ and yjjQ-bglJ with their putative promoters (including 400 bp upstream sequence) were cloned into a low-copy plasmid derived from pACYC184. The failure of the complementation might be due to a clone/expression problem with yjjQ. Because the in vitro results were ambiguous, the in vivo virulence of those strains was not checked. To rule out a polar effect of the transposon insertion or another possible mutation, a yjjQ deletion mutant, a deletion mutant in the immediately downstream gene bglJ and a BglJ overexpression strain were generated. In vitro phenotypes such as growth in M9, protein expression profile and mRNA abundance of iron-uptake receptors were checked and none of the results hinted at any polar effect or a second mutation. Giel et al. (1996) previously showed in E. coli K-12 that a Tn10 insertion in yjjQ caused BglJ to be constitutively expressed, thus activating expression of the β-glucoside operon, bgl. However, deletion of the downstream gene bglJ from the transposon-insertion mutant M18E10 (IMT5155 yjjQ : : mini-Tn5 bglJ : : Cm or M18E10 bglJ : : Cm) did not abolish the phenotype of defective growth in M9 (data not shown). These results corroborate our conclusion that the phenotypic changes in the mutants were probably due to loss of yjjQ function.
Insertional mutation of yjjQ led to virulence attenuation of APEC IMT5155 in vivo and showed that mutant M18E10 was attenuated 30- to 1000-fold in internal organs. Likewise, individual tests demonstrated that the bacterial loads of the mutant in each organ were much lower than that of the wild-type. Among the proteins that were shown to be repressed or downregulated in mutant strain M18E10, four are parts of previously described iron-uptake systems and their regulation by YjjQ has been confirmed at the transcription level using real-time RT-PCR. The acquisition of iron is crucial for bacterial survival and infection, since this metal is necessary for a series of cellular functions and the concentration of free iron is exceedingly low in host tissues. Therefore bacteria have developed several strategies to sequestrate this essential element (Perkins-Balding et al., 2004) and iron-acquisition systems have been associated with bacterial virulence especially for bacteria causing septicaemia (Janakiraman & Slauch, 2000; Li et al., 2005; Russo & Johnson, 2000; Schaible & Kaufmann, 2004; Torres & Payne, 1997). One of the downregulated genes, sitA, is part of the four-member sit operon which is included within SPI1 of Salmonella Typhimurium and is believed to be located on a pathogenicity island in Shigella flexneri as well (Runyen-Janecky et al., 2003; Zhou et al., 1999). Previous studies have shown that the Sit system is required for full virulence of Salmonella Typhimurium and that a sitB mutant of APEC strain IMT5155 is markedly attenuated for survival in the chicken as demonstrated by in vivo competition assays (Janakiraman & Slauch, 2000; Li et al., 2005). Only recently, Sabri et al. (2006) demonstrated that, unlike other SitABCD metal-type transporters identified in Salmonella Typhimurium and Shigella flexneri, the sitABCD operon of APEC strain 
7122 is located on a virulence plasmid and not only mediates transport of iron and manganese but also is involved in oxidative stress defence of this pathogen. The sitABCD operon was also identified on large plasmids in two other APEC strains of serotypes O1 and O2 (Johnson et al., 2006b, c), and moreover IMT5155 was confirmed to harbour two copies (chromosomal and plasmid) of particular operons (unpublished data from our laboratory). Duplication of the sit operon in this pathogen may hint at an enhanced capacity to transport manganese or iron. The particular relevance of this metal-acquisition locus for APEC is furthermore displayed by its high prevalence (Ewers et al., 2007), and the fact that this system is more commonly detected in clinical isolates than in strains from healthy poultry or environmental samples (Rodriguez-Siek et al., 2005a, b). Likewise, the second downregulated gene encoding the membrane receptor for yersiniabactin, FyuA, is significantly more often detected in outbreak isolates than in isolates from healthy layers (Rodriguez-Siek et al., 2005a, b). The third protein identified and belonging to this category is ChuA, which is an outer-membrane acceptor involved in haem uptake in a series of bacterial pathogens, e.g. EHEC, UPEC, Neisseria meningitidis and Shigella dysenteriae (Mills & Payne, 1995; Torres & Payne, 1997; Welch et al., 2002). The ability to use haem/haemoglobin as iron source has been implicated as an important determinant of virulence and it is assumed that these compounds provide the requisite amounts of iron necessary for the expression of virulence factors such as haemolysins, proteases or cytolysins (Lee, 1995). Furthermore, ChuA has been confirmed as playing a role in the pathogenesis of APEC (Li et al., 2005) and 52.1 % out of 436 APEC strains were shown to harbour this gene (Ewers et al., 2007). The fourth identified protein, IreA, is a novel iron-responsive element first identified in ExPEC and its amino acid sequence has 48–56 % similarity to that of previously identified siderophore receptors. Its expression was upregulated extensively in human urine, in human ascites and in human blood relative to expression in LB medium (Russo et al., 2001) and it was recently identified in a novel pathogenicity island in an APEC strain (Kariyawasam et al., 2006b). The ireA gene is predominantly present in APEC rather than in avian commensal E. coli (Kariyawasam et al., 2006a).
The master regulator of bacterial iron homeostasis is Fur, which can directly bind to the promoter region of iron-regulated genes with ferrous iron as a cofactor, thus repressing the expression of these genes (Hantke, 2001). In certain cases, Fur indirectly regulates iron-uptake systems by regulating three types of activators: two-component signal transduction systems, AraC-like regulators of the synthesis of siderophores and their uptake systems, and extracytoplasmic function (ECF) sigma factors (Hantke & Braun, 1998). Our results showed that YjjQ probably regulates iron-uptake receptors SitA, FyuA, ChuA and IreA, and its regulation has been confirmed at the transcription level by real-time RT-PCR. Whether YjjQ plays a role in Fur regulation or vice versa requires further study.
Several proteins with other functions were identified by proteomic analysis, such as transcriptional regulation, LPS and outer-membrane protein biosynthesis, energy metabolism, molecular chaperoning and amino acid synthesis. The putative regulatory function of YjjQ in those phenotypes requires further study. EHEC, UPEC, Shigella and Salmonella, as well as APEC, also contain a yjjQ gene. Future studies will hopefully lead to a more comprehensive understanding of YjjQ pathogenesis in these important human pathogens.
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ACKNOWLEDGEMENTS |
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Edited by: D. L. Gally
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REFERENCES |
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), M18E10 (
), KEC363 (x), KEC361 (
) and IMT5155P3 (
) in M9 medium with glucose as the sole carbon source; the values presented are the means and standard errors (not shown where smaller than symbols) of at least three independent experiments. KEC361 and IMT5155P3 grew as well as the wild-type in M9 (group 1), while M18E10 and KEC363 could hardly grow in M9 (group 2). The differences between group 1 and group 2 were significant (P<0.01).
