The yicJI operon of the common genetic backbone of Escherichia coli codes an α-xylosidase and a transporter of the galactosides–pentoses–hexuronides : cation symporter family. In the extraintestinal pathogenic E. coli strain BEN2908, a metabolic operon (frz) of seven genes is found downstream of the yicI gene. It was proved that frz promotes bacterial fitness under stressful conditions. During this work, we identified a motif containing a palindromic sequence in the promoter region of both the frz and the yicJI operons. We then showed that these two operons are cotranscribed, suggesting a functional relationship. The phenotypes of frz and yicJI deletion mutants were compared. Our results showed that although the yicJI operon is not essential for the life of E. coli, it is necessary for its fitness under all the growth conditions tested.
The yicI and yicJ genes are part of the common genetic backbone of Escherichia coli. The analysis of sequenced E. coli genomes indicates that these two genes form an operon. In E. coli K-12 substrain MG1655, the yicJI operon is located between the yicH and the tRNA selC locus (Fig. 1). YicI is a family 31 α-glycosidase proved to be a hexameric α-xylosidase with low α-glucosidase activity. Its substrate specificity suggests that it is involved in the degradation of oligosaccharides containing the α-1,6-xylosidic linkage, like isoprimeverose, which constitutes a part of xyloglucan (Okuyama et al., 2004; Lovering et al., 2005). The yicJ gene located beside yicI encodes a protein that belongs to the galactosides–pentoses–hexuronides : cation symporter family (Poolman et al., 1996). It is hypothesized that this transporter takes an oligosaccharide then metabolized to xylose and glucose by YicI (Okuyama et al., 2004).
We previously identified a carbohydrate metabolic operon (frz) that is highly associated with extraintestinal pathogenic E. coli (ExPEC) strains. The frz operon codes for three subunits of a phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS) transporter of the fructose subfamily, for a transcriptional activator containing PRD domains (PTS regulatory domains), for two type II ketose-1,6-bisphosphate aldolases, for a sugar-specific kinase [repressor, ORF, kinase family (ROK)], and for a protein of the cupin superfamily. We proved that frz promotes bacterial fitness under stressful conditions, such as oxygen restriction, the late stationary phase of growth, or growth in serum or in the intestinal tract. Furthermore, we showed that frz is involved in adherence to and internalization in human type II pneumocytes, human enterocytes, and chicken liver cells by favoring the ON orientation of the fim operon promoter and thus acting on the expression of type 1 fimbriae, which are the major ExPEC adhesins. Both the PRD activator, FrzR, and the metabolic enzymes encoded by the frz operon are involved in these phenotypes (Rouquet et al., 2009). As the effects of the Frz components depend on the composition of the growth medium, it was hypothesized that the Frz system senses its environment to allow the expression of genes implicated in type 1 fimbriae synthesis and in the protection of the bacteria from the particular environmental stresses encountered during both nutritional deprivation (late stationary phase of growth) and oxygen restriction. Microarray experiments that allowed the identification of several genes whose expression is significantly modified in the frz mutants strengthen this hypothesis (our unpublished data).
Sequencing of the genomic environment of the frz operon in the ExPEC strain BEN2908 indicated that it is located between the yicH and the yicI genes of E. coli and PCR experiments showed that it is separated by only the yicI and the yicJ genes from the tRNA selC locus (Rouquet et al., 2009). An in silico crossover between two direct repeats identified in the intergenic regions of yicH-ORF8frz and yicI-ORF1frz allows the deletion of the frz operon from the genome of strain BEN2908 and the conservation of 53 base pairs from the intergenic regions between the yicH and the yicI genes. These 53 base pairs are alignable (58% identical nucleotides) with the yicH-yicI intergenic region of the commensal E. coli K-12 substrain MG1655 (Rouquet et al., 2009). The data described above, the fact that the G+C content of the frz operon (48.7%) is close to the G+C content (50.8%) of the entire E. coli genome, and the absence of insertion element remnants (integrase or transposase genes or plasmidic replication origin) indicate that the frz operon was probably not acquired by horizontal transfer by commensal E. coli strains, but, rather, was initially present in the ancestor of commensal and ExPEC strains and was then deleted during evolution in most of the commensal strains. The frz operon is highly associated with E. coli clonal groups B2 and to a lesser extent with group D, two phylogenetic groups in which the majority of ExPEC strains are clustered (Moulin-Schouleur et al., 2007; Rouquet et al., 2009). Interestingly, group B2 is considered by some to be the first E. coli group to emerge (Lecointre et al., 1998). It is thus plausible that the frz operon and the yicJI operon that are transcribed in the same direction as the frz operon and that also code for proteins involved in carbohydrate metabolism form a unique functional metabolic unit in the most primitive E. coli strains.
In this work, we evaluated the putative functional relation between the frz and the yicJI operons of an ExPEC strain, and we found that the yicJI operon is involved in the fitness of the bacteria.
Materials and methods
The ExPEC strain BEN2908 is a nalidixic acid-resistant derivative of strain MT78 isolated from the trachea of a chicken with a respiratory tract infection (Dozois et al., 1994). BEN2908 belongs to the phylogenetic cluster B2-1 (Moulin-Schouleur et al., 2007). Strain BEN2908Δfrz is a mutant of strain BEN2908 in which the entire frz operon was deleted and replaced with a kanamycin resistance cassette. The deletion procedures conserved the putative transcription terminators of yicH and yicI (conservation of 43 and 75 nucleotides just downstream of the stop codons of yicH and yicI). The construction of strain BEN2908Δfrz was described earlier (Rouquet et al., 2009).
Bacterial growth conditions
Strains were routinely grown in Luria–Bertani (LB) broth [10 g L−1 tryptone (Becton-Dickinson & Company), 5 g L−1 yeast extract (Becton-Dickinson & Company), 10 g L−1 NaCl, pH 7.4] at 37 °C with agitation and on LB agar plates (1.2% agar). Unless otherwise stated, nalidixic acid (30 μg mL−1) and kanamycin (50 μg L−1), each at the indicated concentration, were used when necessary. For co-cultures of strain BEN2908 and its ΔJI isogenic deletion mutant (containing a kanamycin resistance cassette) or strain BEN2908 and its Δfrz isogenic deletion mutant, each strain was first separately incubated overnight in 5 mL of LB broth containing nalidixic acid at 37 °C with aeration. Strains BEN2908 and BEN2908ΔJI or BEN2908 and BEN2908Δfrz were then inoculated in equivalent numbers in 10 mL of LB containing nalidixic acid. These co-cultures were incubated in 50-mL Erlenmeyer flasks at 37 °C for 72 h. The contents of the Erlenmeyer flasks were then mixed and 10-fold dilutions of the co-cultures were plated on LB agar containing nalidixic acid and incubated at 37 °C. All the colonies from one of the nalidixic acid LB plates (at least 100 colonies) were then picked on LB agar plates containing kanamycin and on LB agar plates containing nalidixic acid. The proportion of the kanamycin-resistant strains was then evaluated. For co-cultures of BEN2908 and its ΔJI isogenic deletion mutant or of BEN2908 and its Δfrz deletion mutant in chicken serum or in IF0 minimal medium (100 mM NaCl, 5 mM NH4Cl, 2 mM NaH2PO4·H2O, 0.25 mM NaSO4, 0.05 mM MgCl2, 1 mM KCl, 30 mM triethanolamine-HCl, pH 7.3) containing 5 mM as a sole carbon source, a similar protocol was followed, but the overnight cultures were first centrifuged at 4000 g for 10 min. Bacteria were then washed three times with phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 2 mM KH2PO4, 10 mM Na2HPO4, pH 7.4) or IF0 and resuspended in the same volume of PBS or IF0 before being inoculated either in chicken serum (Sigma-Aldrich) previously decomplemented by 30 min of incubation at 56 °C and containing nalidixic acid or in IF0.
Nucleic acid manipulations
Standard DNA manipulation techniques were carried out as described by Sambrook & Russell (2001). Plasmid and E. coli chromosomal DNA were purified using the Nucleobond PC100 and Nucleospin tissue kits according to the manufacturer's protocol (Macherey-Nagel). For the extraction of total RNA, bacterial cells taken in the mid-exponential phase of growth were first treated with RNA Protect (Qiagen). The stabilized RNAs were then extracted using an RNA Pure Yield kit (Promega). Bacteria were transformed by electroporation following the method of Tung & Chow (1995). For Southern blot hybridization, DNA restriction fragments were subjected to electrophoresis and transferred to a Hybond-N+ membrane (Amersham, GE Healthcare Life Sciences). Probes were labeled with peroxidase, and hybridized DNA fragments were revealed using an enhanced chemiluminescence kit (RPN3000; Amersham Pharmacia Biotech), as described by the manufacturer.
Amplification of DNA sequences by PCR
Unless otherwise stated, PCR amplification was performed in a mixture with a 50-μL total volume containing 1 μM of the forward and reverse primers, 200 μM of each dNTP (Finzyme, Ozyme, France), and 1.25 U of Taq DNA polymerase (New England Biolabs Inc.) in a PCR buffer containing 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 20 mM Tris-HCl, pH 8.8 (New England Biolabs Inc.). Amplifications were performed in a Perkin-Elmer thermocycler (GeneAmp 9700; Applied Biosystems) with the following temperature program: one cycle of 45 s at 95 °C; 30 cycles of 45 s at 95 °C, 60 s at temperature 5 °C lower than the average Tm values of the primers, and 1 min kb−1 at 72 °C; and finally, one cycle of 10 min at 72 °C.
Reverse transcriptase (RT)-PCR
RT-PCRs were performed on RNAs purified during the exponential phase of growth, as described previously (Gilot et al., 2000). In brief, after treatment with DNase I, total RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Invitrogen) and the reverse primers of interest (Yici-as, caccagggcagtaaagcgctct; C4488-5as, ccagccattgctcaagtaaacgtaaa; C4488-6as, tgataaagtagcgttctgacaattt). After the inactivation of the reverse transcriptase, the reaction mixtures were used for PCR amplification of the cDNA with the reverse primers and appropriate forward primers (Yici-s, ggataatgaaatggtggtctatgctgc; Yici-s2, cagcacgtactggcaacacga or RI88-Yici-P, ggcttcttaatctgtgataagggt) as described above for the amplification of DNA sequences by PCR. Control RT-PCRs, excluding reverse transcriptase, were performed to check for DNA contamination of the RNA preparations.
Construction of the BEN2908ΔJI∷kan mutant
The JI operon was deleted from the chromosome of strain BEN2908 using the red recombination procedure (Datsenko & Wanner, 2000). Briefly, the JI operon was replaced with a kanamycin resistance cassette that was generated by PCR using primers with extensions that are homologous to regions adjacent to the sequences to be inactivated. The kanamycin resistance cassette was obtained by PCR amplification from plasmid pKD4, using the primers RI-yicJ-P1 (CAAGAATCATAAATTAATAACCAGATATCGGAATATTCG CTCTCGCAGGGGTGTAGGCTGGAGCTGCTTCG) and RI-yicI-a (ATTTACCGGATA CGACACAAAACCATTCGTATCCGGCATTCTTCAATAGAAGAGCGCTTTTGAAGCTGGG). The 5′ extensions (underlined in the primer sequences) of the RI-yicJ-P1 and RI-yicI-a primers are homologous to 50 nucleotides immediately upstream of the start codon of yicJ and to 50 nucleotides immediately downstream of the stop codon of yicI, respectively. The deletion procedure thus conserved the complete intergenic regions between selC and yicJ and between yicI and the frz operon. The replacement of the JI operon was confirmed by PCR using the primer pairs C4488/skana (acaatagtcgtatattcccttcgagg/caacctgccatcacgagatt) and askana1/RI-YicJ-selC (cagatagcccagtagctgacatt/ggcgcattatagctacttccttga), which allows the detection of left and the right junctions between the bacterial chromosome and the kanamycin resistance cassette, respectively. PCR with the primer pairs C4488/RI-YicJ-selC allowed the amplification of a 1991-bp DNA fragment, confirming the integration of the complete kanamycin resistance cassette. Southern blots of EcoRV- or SspI-digested DNA of the mutants with a probe that was generated by PCR amplification of the kanamycin resistance gene (primers Cat51, gtgtaggctggagctgcttc and Askana2, ccgaagcccaacctttcata) revealed a 3956- and a 1502-bp DNA fragment, respectively. This indicated that the kanamycin cassette was also not illegitimately inserted into another part of the genome.
Nucleotide sequence accession number
The sequence of the E. coli strain BEN2908 JI region has been deposited in the EMBL database under accession numbers FR667153, FM253092, and AY857617.
Results and discussion
Comparative analysis of the selC-yicJ and the yicI-ORF1frz intergenic regions
To determine whether common DNA motifs putatively involved in the regulation of the yicJI and the frz operons are present in the yicJI and frz intergenic regions, we first completed the sequence of the yicJI region of the ExPEC strain BEN2908. As in other sequenced E. coli genomes, the BEN2908 yicJ gene is separated from the yicI gene by only nine nucleotides. Correctly spaced σ70−10 and σ70−35 putative promoter sequences and a putative ribosome-binding site were identified upstream of the start codon of the yicJ gene (Fig. 2a). A functional σE promoter of the yicJI operon was identified upstream of the tRNA selC locus of E. coli K-12 substrain MG1655 (Fig. 1; Rhodius et al., 2006). This sequence is not present in strain BEN2908 due to the integration of a pathogenicity island at this site (Chouikha et al., 2006). A signal structure proposed by Laikova to be the XylR-binding site was also found between the putative ribosome-binding site and the σ70−10 promoter sequence, suggesting, as it was proposed, that the yicJI operon is a member of the xylose regulons (Fig. 2a; Laikova et al., 2001). This motif was not found in the yicI-ORF1frz intergenic region. Finally, a motif containing a palindromic sequence was found in the identified promoter sequence of the yicJI operon and three nucleotides upstream of the σ70−35 promoter sequence of the frz operon (Fig. 2b). This motif is correctly spaced to be a binding site for a regulator involved in the transcription of the two operons. Works are in progress in our laboratory to determine whether it represents an FrzR-binding site.
The yicJI operon is cotranscribed with the frz operon
The identification of a common motif in the yicJI and frz intergenic regions prompted us to test whether the two operons are cotranscribed. We previously identified a hairpin structure containing a G+C-rich region in the yicI-ORF1frz intergenic region (294 nt) of strain BEN2908. This RNA secondary structure begins 13 nucleotides after the stop codon of yicI and is followed by a polyU sequence. These features indicate the presence of a rho-independent transcription terminator. We also identified σ70−10 and σ70−35 putative promoter sequences beginning 54 and 76 nucleotides upstream of ORF1frz, respectively (EMBL accession number FM253092).
To determine whether the yicJI and the frz operons are cotranscribed, RT-PCR experiments were performed with a reverse primer localized at the 5′ end of ORF1frz and a forward primer localized at the 3′ end of the yicI gene. The functionality of the ORF1frz identified promoter was also tested by RT-PCR using a reverse primer localized at the 5′ end of ORF1frz and a forward primer localized between the identified σ70−10 ORF1frz promoter sequence and the start codon of ORF1frz. As a control, transcription of the yicI gene was tested by RT-PCR with reverse and forward primers, both localized in the yicI gene. Figure 3 indicates that some transcripts of yicI pass through the transcriptional terminator identified in the yicI-ORF1frz intergenic region and form cotranscripts with frz genes (lanes 2 and 4). The stronger amplification of ORF1frz transcripts by RT-PCR with primers localized in ORF1frz and between its promoter and start codon than with primers localized in yicI and in ORF1frz suggests that the ORF1frz promoter identified is functional (Fig. 3, lanes 4 and 6).
Phenotypic analysis of the yicJI deletion mutant
The identification of a similar DNA motif in the yicJI and frz promoter regions and of yicJI and frz cotranscripts suggests that these two operons could be involved in the same physiological pathway. We thus compared the phenotypes of yicJI and frz deletion mutants under diverse growth conditions previously used to reveal the role of the frz operon (Rouquet et al., 2009). We first noticed that the yicJI mutant formed smaller colonies than the wild type and the Δfrz strains on LB-agar plates. We then compared the growth of the wild-type strain, the Δfrz mutant, and the ΔJI mutant during agitated and static cultures in LB-medium. Whereas the growth curves of the wild-type and of the Δfrz mutants were similar under both conditions, the ΔJI mutant was affected in its ability of adaptation to the stationary phase of growth (OD600 nm of the ΔIJ mutant culture is 1 or 0.7 U lower than that of the wild-type strain after 72 h of agitated or static growth, respectively; Fig. 4). We reported previously that the frz operon is involved in the survival mechanism of BEN2908 during the late stationary growth phase in LB medium and in serum. Indeed, during co-cultures under oxygen-restricted conditions (static cultures), the wild-type strain BEN2908 outcompeted the BEN2908Δfrz strain during the late stationary growth phase, but not during the exponential growth phase. This phenotype is strongly affected by oxygenation, as it is not revealed when the co-cultures are agitated (Rouquet et al., 2009). We thus tested the survival ability of the ΔJI mutant under these co-culture conditions, and we found that its fitness is strongly affected during the late stationary phase of growth, even when the co-cultures are highly agitated (Fig. 5, A–C). As the effect of the Frz system on the survival ability of the bacteria during the late stationary phase of growth was found to depend on the composition of the culture medium, we analyzed the survival ability of the ΔJI mutant during static co-cultures with the wild-type strains in minimal media in which the fitness of the Δfrz mutant is not or only slightly affected (d-glucose, d-fructose, d-sorbose, and d-psicose). In contrast to the Δfrz mutant, the survival ability of the ΔJI mutant is strongly affected during the late stationary phase of growth in all these minimal media (Fig. 5, D–G). As isoprimeverose was found to be a substrate of YicI, we also tested the survival ability of the ΔJI and the Δfrz mutants during static co-cultures with the wild-type strain in a minimal medium containing this sugar as a sole carbon source. Again, the fitness of the ΔJI mutant was strongly affected during the late stationary phase of growth (6.2 ± 1.0% of mutant in the population after 7 days of co-culture), whereas that of the Δfrz mutant was not (53.7 ± 1.6% of mutant in the population after 7 days of co-culture).
In conclusion, although the phenotypes of the Δfrz and the ΔJI mutants are not completely similar, both frz and yicJI metabolic operons are involved in the fitness of the bacteria and are cotranscribed through molecular mechanisms that could involve the FrzR activator and phosphoryl group transfer. Indeed, the level of phosphorylation of PTS components, such as those of the Frz system, depends on the presence of their substrates in the environment of the bacteria and has a direct impact on the modulation of phosphoryl group transfer to PRD domains of PRD regulators, such as the FrzR activator. Changes in the phosphorylation level of these regulators can alter the expression of operons encoding PTS transporters and PRD protein-regulated genes carrying out diverse cellular functions of the bacteria (Deutscher et al., 2006). The FrzR activator could act similarly by being involved in the regulation of both the frz and the yicJI operons.
Although the yicJI operon is not essential for the life of E. coli, our results indicate that it is necessary for its fitness under all the tested growth conditions. The molecular mechanisms by which the YicJ and YicI proteins are involved in the fitness of the bacteria and particularly in its capacity to survive during the late stationary phase of growth are actually unknown. However, some metabolic enzymes were described to also play a regulatory role by binding to DNA and RNA, by being involved in mRNA degradation, or by sequestering transcriptional regulators (Morita et al., 2004; Loughman & Caparon, 2006; Domain et al., 2007; Commichau & Stülke, 2008; Commichau et al., 2009). Similarly, the YicI glycosidase, which is devoid of predicted nucleic acid-binding sites, might be involved both in the metabolism of oligosaccharides containing α-1,6-xylosidic linkage and in the interaction with protein(s) involved in the fitness of the bacteria during the late stationary phase of growth. This model is now being tested in our laboratory.
This work was supported by the Era-NET PathoGenoMics European program (grant ANR-06-PATHO-002-01) and by the Institut Fédératif de Recherche 136 ‘Agents transmissibles et Infectiologie’ (France). G.R. was supported by a grant of the Fondation de la Recherche Médicale (Fin de thèse – scientifique).