ABSTRACT
RpoS (σS), the general stress response sigma factor, directs the expression of genes under a variety of stressful conditions. Control of the cellular σS concentration is critical for appropriately scaled σS-dependent gene expression. One way to maintain appropriate levels of σS is to regulate its stability. Indeed, σS degradation is catalyzed by the ClpXP protease and the recognition of σS by ClpXP depends on the adaptor protein RssB. Three anti-adaptors (IraD, IraM, and IraP) exist in Escherichia coli K-12; each interacts with RssB and inhibits RssB activity under different stress conditions, thereby stabilizing σS. Unlike K-12, some E. coli isolates, including uropathogenic E. coli strain CFT073, show comparable cellular levels of σS during the logarithmic and stationary growth phases, suggesting that there are differences in the regulation of σS levels among E. coli strains. Here, we describe IraL, an RssB anti-adaptor that stabilizes σS during logarithmic phase growth in CFT073 and other E. coli and Shigella strains. By immunoblot analyses, we show that IraL affects the levels and stability of σS during logarithmic phase growth. By computational and PCR-based analyses, we reveal that iraL is found in many E. coli pathotypes but not in laboratory-adapted strains. Finally, by bacterial two-hybrid and copurification analyses, we demonstrate that IraL interacts with RssB by a mechanism distinct from that used by other characterized anti-adaptors. We introduce a fourth RssB anti-adaptor found in E. coli species and suggest that differences in the regulation of σS levels may contribute to host and niche specificity in pathogenic and nonpathogenic E. coli strains.
IMPORTANCE Bacteria must cope with a variety of environmental conditions in order to survive. RpoS (σS), the general stress response sigma factor, directs the expression of many genes under stressful conditions in both pathogenic and nonpathogenic Escherichia coli strains. The regulation of σS levels and activity allows appropriately scaled σS-dependent gene expression. Here, we describe IraL, an RssB anti-adaptor that, unlike previously described anti-adaptors, stabilizes σS during the logarithmic growth phase in the absence of additional stress. We also demonstrate that iraL is found in a large number of E. coli and Shigella isolates. These data suggest that strains containing iraL are able to initiate σS-dependent gene expression under conditions under which strains without iraL cannot. Therefore, IraL-mediated σS stabilization may contribute to host and niche specificity in E. coli.
INTRODUCTION
Transcription in Escherichia coli is catalyzed by the RNA polymerase holoenzyme, which is composed of a core polymerase and a dissociable sigma factor. E. coli has one housekeeping sigma factor (σ70) and six alternative sigma factors (1). The core polymerase is unable to initiate transcription alone and requires an associated sigma factor to define promoter specificity and initiate transcription. RpoS (σS), the best studied of the alternative sigma factors, affects the expression of ~10% of the genes in E. coli K-12 either directly or indirectly (2). Many σS-dependent genes help to mitigate the effects of a variety of stressful conditions. To maintain appropriate levels of σS-dependent gene expression, the levels of σS and its activity are regulated at many levels: transcription, translation, stability, and association with the core polymerase (3). A widely appreciated manifestation of this regulatory network in laboratory-adapted E. coli is that the level of σS is low during logarithmic phase growth and increases substantially into and during stationary phase growth.
σS degradation is catalyzed by the ClpXP protease, and the recognition of σS by ClpXP depends on the adaptor protein RssB. A class of proteins known as RssB anti-adaptors was previously described in E. coli K-12 (4). These anti-adaptors (IraD, IraM, and IraP) interact with RssB and inhibit RssB activity under different stress conditions, thereby stabilizing σS. Interestingly, the anti-adaptors found in K-12 share no sequence identity and differ in the ways that they interact with RssB (5). RssB anti-adaptors occur in Salmonella and additional uncharacterized anti-adaptors in K-12 are hypothesized (6, 7).
Although σS levels are low during logarithmic phase growth and high during the stationary phase in K-12, elevated levels of σS during log phase growth are observed in some E. coli strains. In 2001, Culham et al. reported that two independent uropathogenic E. coli (UPEC) isolates (CFT073 and GR12) have comparable levels of σS during logarithmic phase and stationary phase growth (8) and in Shiga toxin-producing E. coli (STEC), there is a positive correlation between log phase σS levels and stress resistance (9). Additionally, it is appreciated that in K-12 and enterohemorrhagic E. coli (EHEC) strain O157:H7, σS contributes either directly or indirectly to gene expression during logarithmic phase growth (10, 11). These observations, combined with a larger body of knowledge that σS is needed for virulence in many, but not all, pathogenic bacterial species (12, 13), suggest that differences in the timing and magnitude of σS levels and activity may help to define host and niche specificity in both pathogenic and nonpathogenic bacteria.
We recently demonstrated that σS is needed by UPEC strain CFT073 to cope with phagocyte oxidase-mediated oxidative stress during urinary tract infection (13) and began to study the regulation of σS in this strain. Using a CFT073-based overexpression library, we identified an RssB anti-adaptor, heretofore named IraL to reflect that it inhibits RssB activity during logarithmic phase growth, leading to σS stabilization. Prior to this study, iraL was named ycgW. iraL is found in many Escherichia and Shigella isolates in addition to CFT073. Furthermore, although IraL and IraM, a previously characterized anti-adaptor from K-12, are homologs, they stabilize σS under different conditions and by different mechanisms.
RESULTS
Identification of a novel RssB anti-adaptor in E. coli CFT073.σS levels in CFT073 are comparable during the logarithmic and stationary growth phases, which contrasts with what is known about the regulation of σS levels in K-12 (8). To identify effectors of σS levels in CFT073, we constructed a transcriptional fusion of lacZYA to the promoter of a σS-dependent gene, katE, by using pPK7034 (14). σS was destabilized in CFT073 PkatE-lacZYA by deleting genes that code for homologs of RssB anti-adaptors that were characterized in K-12 (IraD, IraM, IraP), as determined by amino acid sequence identity. IraD, IraM, and IraP from K-12 share 74.6, 58.9, and 98.9% identity with their homologs in CFT073, YjiD, IraL, and YaiB, respectively. When CFT073 PkatE-lacZYA ΔyaiB ΔiraL ΔyjiD (σS-destabilized reporter strain) is plated onto MacConkey’s medium plus lactose, the colonies are less red than those of either the parent strain (CFT073 PkatE-lacZYA) or CFT073 PkatE-lacZYA ΔrssB but more red than those of CFT073 PkatE-lacZYA ΔrpoS (see Fig. S1 in the supplemental material). This indicates that there is less lacZ expression from the katE promoter in the σS destabilized reporter strain and that katE is σS dependent during growth under these conditions.
Figure S1
Copyright © 2014 Hryckowian et al.This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
To identify regulators of σS levels in CFT073, the σS-destabilized reporter strain was transformed with a pACYC184-based CFT073 genomic library and plated onto either MacConkey’s medium plus lactose or urine indicator agar. Urine indicator agar was designed for this study and was used as a surrogate for nutrient limitation and other stressful conditions present in the urinary tract (see Materials and Methods). A combined ~22,000 transformants were analyzed on these media. Transformants with LacZ activity higher or lower than that of pACYC184-containing transformants on either of these media were studied further. Among the transformants were clones containing homologs of genes known to regulate levels of σS in K-12, including genomic fragments with yaiB, iraL, and yjiD (see Table S2 in the supplemental material). We then noticed that the genomic context of iraL in CFT073 is different from that of its homolog in K-12, iraM, and that these two genes share 67.0 and 58.9% sequence identity at the nucleotide and amino acid levels, respectively (Fig. 1). We reasoned that IraL might act as an RssB anti-adaptor in CFT073 on the basis of sequence identity with IraM and its identification in our screen. We also hypothesized that the differences in sequence identity and genetic context between iraM and iraL contribute to differences under conditions under which these anti-adaptors stabilize σS.
Table S2
Copyright © 2014 Hryckowian et al.This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
iraL shares sequence identity with iraM from E. coli K-12 but differs in genetic context. (A) Visualization of regions of the K-12 and CFT073 chromosomes containing iraM and iraL, respectively. Block arrows represent annotated open reading frames from these two strains, and the number below each region indicates the chromosomal location. The region immediately upstream and containing the iraL start codon is detailed. The TSS of iraL (indicated by +1 and a leftward-pointing arrow) was determined by 5′ RACE. The predicted −10 and −35 sites are in bold, and the iraL start codon is underlined. (B) Nucleotide sequence alignment of the coding regions of iraL and iraM. Alignment was carried out with the ClustalW feature and the default parameters in MacVector 9.0.2. It was determined that these genes share 67% nucleotide sequence identity. Stars below the alignment denote regions of sequence identity. (C) Alignment of the amino acid sequences of IraL and IraM. Alignment was carried out with ClustalW as described above. It was determined that these polypeptides share 58.9% amino acid sequence identity. Stars below the alignment denote regions of sequence identity, while periods represent regions of weak similarity.
iraL TSS identification.We determined the transcription start site (TSS) of iraL via 5′ rapid amplification of cDNA ends (RACE) as described in Materials and Methods. Briefly, CFT073 was grown to mid-log phase and RNA was isolated. cDNA was made from this RNA sample with an iraL-specific primer (iraL-gsp1), the RNA was degraded, and a poly(C) tail was added to the 3′ end of the iraL cDNA that remained. By using an another iraL-specific primer (iraL-gsp2) and a primer that anneals to the poly(C) tail, the cDNA was amplified. Finally, a Sanger sequencing reaction was carried out with this amplified cDNA and iraL-gsp2. The 3′-most end of the reverse strand of iraL cDNA contains the poly(C) tail (see Fig. S2A in the supplemental material), indicating that the 5′ end of the iraL mRNA starts with the sequence 5′ ACA TCA CCA GCA AGG CAT AAA CAA GGA AAC CA 3′. On the basis of the similarity of the predicted −10 and −35 elements to the σ70 consensus promoter sequence (15), we suggest that the transcription of iraL is dependent on σ70 (Fig. 1A).
Figure S2
Copyright © 2014 Hryckowian et al.This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
iraL is found in many E. coli and Shigella isolates.Knowing that iraL is found in CFT073 but not K-12 and that iraM is found in K-12 but not CFT073, we decided to characterize the distribution of these genes in a larger population of isolates. To do this, we queried the NCBI GenBank database for fully sequenced genomes containing nucleotide sequences that match iraL or iraM and we developed a PCR-based assay to identify strains that have iraL or iraM. Primers were designed to regions of sequence divergence within the coding region of each of these genes (see Table S1 in the supplemental material) and were target specific (see Fig. S3 in the supplemental material). We assayed four available, previously described strain collections, the ECOR (16), Andreu prostatitis (17), Johnson urosepsis (18), and Mand STEC (9) collections. On the basis of these analyses, we observed that some strains have iraL alone (like CFT073), some have iraM alone (like K-12), some have both iraL and iraM, and others have neither iraL nor iraM (Fig. 2; see Tables S3 and S4 in the supplemental material). None of the laboratory-adapted strains tested have iraL. Additionally, as determined by Fisher’s exact test, iraL is found more frequently in UPEC (P = 0.0163), prostatitis (P = 0.0083), and Shigella (P = 0.0001) isolates than in commensals and is found less frequently in EHEC or STEC isolates than in commensals (P = 0.0323). Furthermore, the iraL promoter and its upstream regulatory region are present and well conserved among all of the fully sequenced iraL-containing isolates, all sharing ≥96% identity with the homologous region in CFT073 (see Fig. S2B in the supplemental material).
Figure S3
Copyright © 2014 Hryckowian et al.This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S1
Copyright © 2014 Hryckowian et al.This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S3
Copyright © 2014 Hryckowian et al.This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S4
Copyright © 2014 Hryckowian et al.This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Distribution of iraL and iraM among selected E. coli pathotypes. The coding sequences of iraL and iraM were queried against complete bacterial genomes in the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and several unsequenced strain collections were subjected to a PCR-based assay for iraL and iraM. Pathotypes with eight or more representatives are shown, with strains represented more than once consolidated into one entry. For the names and sources of all of the strains subjected to these analyses, including pathotypes with fewer than eight representatives, see Tables S3 and S4 in the supplemental material.
iraL affects the levels and stability of σS.To determine whether IraL affects levels of σS during growth in Luria-Bertani (LB) broth, immunoblot assays for σS were done with the total soluble protein isolated from logarithmic and stationary phase cells. In CFT073 ΔiraL and a Shigella sonnei ΔiraL mutant, the levels of σS were lower than in the wild type during logarithmic phase growth (Fig. 3A). To determine if IraL affects σS stability, cultures were grown to mid-log phase, protein synthesis was stopped by chloramphenicol (Cm) addition, and the total protein was sampled over time. The half-life of σS during the log phase in CFT073 is ~5 min, but its half-life is reduced to ~2.5 min in CFT073 ΔiraL. When iraL, under the control of its native promoter, is reintroduced into CFT073 ΔiraL at the att Tn7 site, the stability of σS is increased (Fig. 3B).
IraL affects the levels and stability of σS during log phase growth. (A) To assess the effects of IraL on σS levels, CFT073, CFT073 ΔiraL, S. sonnei, S. sonnei ΔiraL, K-12/pACYC184 (vector only), and K-12/pIraL (pACYC with iraL under the control of its native promoter) were grown to the log phase (LOG) or the stationary phase (STAT) and immunoblot assays for σS were carried out with 10 µg of total soluble protein from these samples. For accompanying Coomassie blue-stained gels that illustrate consistent loading of the protein samples, see Fig. S4 in the supplemental material. Marker (M) lanes contain the Precision Plus Protein Dual Color Standard that cross-reacts with the detection reagents used. (B) To assess the effects of IraL on σS stability, CFT073 and isogenic mutants were grown to the mid-log phase as described for panel A, protein synthesis was stopped with Cm, and samples of total protein were precipitated in 10% TCA at 3-min intervals after Cm treatment. Immunoblot assays were carried out with total protein normalized to the OD600, and σS levels were measured by densitometry. Data points represent the mean percentages of σS remaining relative to those at t = 0, and error bars represent ± the standard error of the mean of three replicates.
Figure S4
Copyright © 2014 Hryckowian et al.This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Additionally, when a plasmid containing iraL under the control of its native promoter (region containing the sequence corresponding to position −100 relative to the iraL TSS through the entire iraL coding region) is introduced into K-12, the levels of σS during logarithmic phase growth are higher than those in vector-only controls (Fig. 3A). For accompanying Coomassie blue-stained gels that illustrate the consistent loading of the protein samples examined in Fig. 3A, see Fig. S4 in the supplemental material.
IraL interacts with RssB.We hypothesized that IraL, as a suspected RssB anti-adaptor, should interact with RssB to stabilize RpoS. To test this hypothesis, we first performed copurification experiments (Fig. 4A; see Fig. S5A in the supplemental material). In these assays, an N-terminal His6 tag was added to IraL and an N-terminal calmodulin binding peptide (CBP) tag was added to RssB. The purification of His6-IraL on cobalt beads led to the copurification of CBP-RssB (Fig. 4A). Similarly, the purification of CBP-RssB on calmodulin beads led to His6-IraL copurification (see Fig. S5A). By this approach, we obtained evidence that, as expected, IraL interacts with RssB.
Figure S5
Copyright © 2014 Hryckowian et al.This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
IraL interacts with RssB. (A) IraL and RssB copurify. Strain AB054 was cotransformed with plasmids encoding CBP-RssB and His6-IraL, and cell lysates were prepared as described in Materials and Methods. Cobalt beads were used to precipitate CBP-RssB and its interacting partner(s), and immunoblot assays were carried out with protein and anti-CBP or anti-His6-peroxidase antiserum and visualized as described in Materials and Methods. (B) IraL interacts with full-length RssB and subdomains of RssB, as visualized on MacConkey’s medium. Overnight cultures of BTH101 coexpressing either T18-IraL or T18-IraM fusion protein and the T25-RssB or T25-RssB subdomain were spotted onto MacConkey’s medium plus maltose. Interaction of the proteins of interest is qualitatively shown as red coloration. (C) IraL interacts with full-length RssB and subdomains of RssB, as determined by β-galactosidase assay. Overnight cultures of BTH101 coexpressing either T25-IraL or T25-IraM and the T25-RssB or T25-RssB subdomain were subjected to a β-galactosidase assay as previously described. Bars on the graph represent mean β-galactosidase activities (Miller units [M.U]), and error bars represent the standard error of the mean of three replicates. WT, wild type.
To further characterize the interaction between IraL and RssB, we aimed to define the subdomain(s) of RssB that interacts with IraL. To this end, we used the bacterial two-hybrid method, as used previously to visualize interactions between RssB and the anti-adaptor proteins from E. coli K-12 (5). In this assay, the proteins of interest are fused to the two domains of adenylate cyclase (Cya) from Bordetella pertussis, T18 and T25. A positive interaction restores Cya activity in an E. coli cya mutant, and it can be detected on MacConkey’s medium or by β-galactosidase measurement (19, 20). By using this technique, we confirmed that IraL interacts with RssB (Fig. 4B and C; see Fig. S5B and C in the supplemental material). Interestingly, IraL interacts only with the N-terminal domain of RssB, which is an interaction pattern distinct from that of its homolog, IraM, which interacts with both the N- and C-terminal domains (Fig. 4B and C; see Fig. S5B and C in the supplemental material) (5). These data suggest that although IraL and IraM are homologs, they have diverged through evolution to stabilize RpoS under different conditions and by different mechanisms.
DISCUSSION
We and others observed that levels of σS in the logarithmic and stationary growth phases are comparable when CFT073 is grown in LB medium, which contrasts with what is known about the regulation of σS levels in laboratory-adapted strains of E. coli (8) (Fig. 3A). We rationalized that there may be differences in the regulation of σS between laboratory-adapted E. coli K-12 and UPEC strain CFT073 and sought to determine the genetic basis for this phenotype. We identified iraL in CFT073 after screening to change the levels of σS in a σS-destabilized reporter strain. We determined that IraL affects the levels and stability of σS, that IraL interacts with RssB in a manner that is different from that of other characterized anti-adaptors, and that iraL is found in many Escherichia and Shigella isolates.
iraL and a related gene, iraM, are located in different genetic contexts in CFT073 and K-12, respectively. In K-12, iraM is found on the e14 prophage; in CFT073, iraL is found on the potB genomic island; and neither of them is present in the other strain. The differences in location and sequence identity of these genes prompted us to examine other bacterial strains for the presence of iraL and iraM. By NCBI BLAST analysis, we determined that iraL and iraM are present in many fully sequenced Escherichia and Shigella strains (see Table S4 in the supplemental material) but are not found in the sequenced strains of other genera. Interestingly, the matches to iraL share ≥95% sequence identity over 100% of the coding region queried, with E values of ≤1 × 10−74. All matches to iraM are also conserved to this extent. Furthermore, there is no overlap between iraL or iraM matches. This observation of anti-adaptor sequence conservation led us to develop a PCR-based assay to detect iraL and iraM in unsequenced strain collections. By our BLAST- and PCR-based analyses, we found that many Escherichia and Shigella isolates have iraL. Additionally, iraL is significantly associated with some pathotypes of E. coli relative to commensal isolates (Fig. 2), suggesting that iraL-mediated σS stabilization may provide a fitness advantage in some pathotypes (UPEC, prostatitis, and Shigella isolates) but not others (EHEC or STEC isolates).
Previously, anti-adaptors were shown to be expressed under stressful conditions in K-12 and Salmonella (4). IraL is unique among the characterized anti-adaptors because it elevates σS during logarithmic growth in the absence of any additional stress. Some EHEC or STEC isolates have elevated levels of σS during logarithmic phase growth (9). Surprisingly, none of the strains described in this study by Mand et al. have iraL (Fig. 2; see Table S3 in the supplemental material). This supports the idea that there are one or more additional mechanisms by which to elevate the levels of σS during logarithmic phase growth and that these mechanisms, along with the specific environmental conditions that lead to their expression and activity, may be favored under conditions encountered by some pathotypes and not others.
We observed that when iraL and its upstream regulatory region are introduced into the att Tn7 site of CFT073 ΔiraL, σS stability is increased (Fig. 3B). Consistent with its role as an RssB anti-adaptor, we demonstrate that RssB-IraL interaction occurs and we provide evidence that IraL interacts with RssB differently than does IraM (Fig. 4; see Fig. S5 in the supplemental material). The observation that IraL and IraM interact differently with RssB will serve as the basis for future work in determining the residues that are important for anti-adaptor-mediated σS stabilization. Furthermore, differences in the modes of anti-adaptor-RssB interaction may provide for more finely tuned anti-adaptor-mediated regulation of σS levels under multiple stresses.
In Salmonella, an IraL- and IraM-like protein, RssC, has been identified (7). rssC and iraL share 49.3% identity and rssC and iraM share 47.4% identity, as determined by ClustalW alignment. On the basis of previous work in the Gottesman laboratory and BLAST analysis of sequenced bacterial strains, we determined that rssC is found only in Salmonella and that no Salmonella isolates have iraL or iraM (7) (see Table S4 in the supplemental material). This suggests that the iraL-iraM-rssC family of anti-adaptors may be evolutionarily labile to adapt to diverse bacterial hosts.
Our in silico analysis of iraL and its promoter revealed that the recently sequenced fatal urosepsis isolate JJ1886 (21) has two copies of iraL and its associated regulatory region (see Fig. S2B and Table S4 in the supplemental material). Given that there is a point mutation in the predicted −35 sequence of the JJ1886 iraL2 promoter (see Fig. S2B), it is not known if this copy of iraL is expressed in JJ1886 or in the other strains containing this mutation. Regardless, JJ1886 is the first example of a strain containing two copies of the same anti-adaptor, which further suggests that iraL may provide a fitness advantage under some of the environmental condition encountered by UPEC.
Though roles for σS during logarithmic phase growth are appreciated and it is suggested that elevated levels of σS during this growth phase may lead to an increase in stress resistance, this is the first example of an RssB anti-adaptor that elevates σS during logarithmic phase growth, as all other characterized anti-adaptors are induced under stress. We posit that IraL, a horizontally acquired effector that stabilizes σS during logarithmic phase growth, may affect σS-dependent gene expression during logarithmic phase growth and provide elevated levels of σS, which would allow increased σS-dependent gene expression when a stressful environment is encountered. Indeed, it is suggested that UPEC strains are more adaptable than fecal strains (22) and IraL-mediated σS stabilization may contribute to this resistance to changing environments. Because σS levels and activity are regulated at many levels, it may be that although levels of σS are high during log phase growth, levels of σS-dependent gene expression are kept low by one or more mechanisms. The regulation of IraL expression and activity and their impact on σS-dependent gene expression are active areas of investigation in our laboratories.
MATERIALS AND METHODS
Strains, plasmids, and oligonucleotides.The strains, plasmids, and oligonucleotides used in this study are listed in Tables S1 and S3. Cloning procedures were done with GoTaq and T4 DNA ligase from Promega and restriction endonucleases from New England Biolabs according to the manufacturers’ specifications. In-frame deletion mutants of E. coli CFT073 were constructed by using the Lambda Red mutagenesis protocol (23), which was modified to incorporate a generalized transduction step with ΦEB49 prior to pCP20-mediated antibiotic resistance cassette removal (24). Attempts to use this method to create the in-frame iraL deletion mutant of S. sonnei were unsuccessful. Therefore, the S. sonnei ΔiraL mutant strain was constructed by Lambda Red mutagenesis, as facilitated by pSIM6 (25), followed by pCP20-mediated antibiotic resistance cassette removal (23). This method of Lambda Red mutagenesis is suggested to be more effective than that of Datsenko and Wanner for recombineering of Gram-negative species that are divergent from K-12 (25). In support of this, our results suggest that pKD46 is ineffective as a tool in S. sonnei. The chromosomal PkatE-lacZYA fusion was constructed by cloning PkatE downstream of lacZYA with pPK7035 as described previously (14). Single-copy complementation of CFT073 ΔiraL was carried out by using a modification of the method of Bao et al. (26), which was used to insert iraL under the control of its native promoter into the chromosomal att Tn7 locus in CFT073. The region containing iraL and its promoter region (iraL plus the upstream region from −100 to +33 relative to the iraL TSS) was cloned into the pCIITn7K-a carrier plasmid (27) prior to conjugation and att Tn7 insertion into CFT073. The control strain (CFT073 ΔiraL Tn7::Kanr) contains an insertion of a kanamycin (Kan) resistance cassette at the att Tn7 site and was constructed as described above, except that no fragment was cloned into pCIITn7K-a prior to conjugation.
Media and growth conditions.All strains were grown in LB broth, on LB agar, on MacConkey’s medium (all from Difco), or on urine indicator agar. Urine indicator agar plates were made by supplementation of human urine plates (as described in reference 28) with filter-sterilized lactose and neutral red solutions added to final concentrations of 1 and 0.003%, respectively. These supplements were mixed into the urine-agar mixture immediately before it was poured. All strains were grown aerobically at 37°C and supplemented with the antibiotics Kan (40 µg/ml), carbenicillin (250 µg/ml), Cm (20 µg/ml), and ampicillin (Ap; 100 µg/ml), as applicable.
Overexpression library and screening for effectors of σS levels.Genomic DNA was isolated from cultures of E. coli CFT073 with the Promega Wizard Genomic DNA purification kit in accordance with the manufacturer’s instructions. The DNA was hydrodynamically sheared and size selected to approximately 5-kb fragments. The sheared DNA was filled in to create blunt ends with T4 DNA polymerase (New England Biolabs), and the fragments were then treated with T4 polynucleotide kinase in preparation for ligation. pACYC184 was isolated with the Qiagen Midiprep kit and digested with EcoRV (New England Biolabs). The sheared and blunted genomic DNA fragments were ligated into the digested vectors, transformed into NEB 5-alpha competent E. coli, and plated on LB broth plus Cm. Colonies were collected by flooding and swabbing the plates with LB broth plus 50% glycerol. The library contains ~40,000 clones (~40× coverage), 24 of which were sequenced to ensure that random fragments were present in each library (data not shown).
Identification of iraL TSS in CFT073.CFT073 was grown overnight in LB broth, subcultured (1:1,000) into 3 ml of fresh LB broth, and then grown to an optical density at 600 nm (OD600) of 0.3. The transcriptional profile of this culture was stabilized by adding 1 ml of the culture to an equal volume of RNAlater (Life Technologies). The mixture was vortexed and placed on ice. Cells were pelleted by centrifugation at 20,000 × g for 20 min at 4°C, and RNA was extracted from these cells with the TRIzol Plus RNA purification kit with on-column DNase treatment, according to the manufacturer’s specifications (Life Technologies). The Agilent Bioanalyzer 2100 and an RNA Pico chip were used to determine that the RNA was of adequate quality, according to the manufacturer’s specifications (Agilent Technologies). The 5′ RACE system kit was used according to the manufacturer’s specifications (Invitrogen) to map the TSS of iraL. cDNA was amplified with Taq polymerase (New England Biolabs). For the gene-specific primers (iraL-gsp1, iraL-gsp2, and iraL-gsp3) used in this protocol, see Table S1 in the supplemental material.
Assay for relative levels of σS.To assess levels of σS, the strains shown in Fig. 3A were grown overnight in LB broth, subcultured 1:1,000 in fresh LB broth, and grown to the mid-logarithmic phase (OD600 of 0.3 to 0.4) or for 24 h (stationary phase), and then total soluble protein was collected and subjected to Western blot analysis. Cells were collected in plastic bottles on ice containing phenylmethylsulfonyl fluoride (PMSF) and Cm or spectinomycin (final concentrations of 0.1 mM and 200 µg/ml in the cell suspension, respectively). Spectinomycin was used for K-12 strains containing pACYC184 and pWAM5035, which confer Cm resistance. After cells were pelleted by centrifugation at 6,700 × g for 15 min at 4°C, they were resuspended and washed once in resuspension buffer (10 mM Tris [pH 7.9], 1 mM EDTA, 5% glycerol, 0.1 mM PMSF). Cells were sonicated twice for 20 s each time to lyse them, and total soluble protein was collected after collection of the insoluble fraction by centrifugation at 20,000 × g for 20 min at 4°C. Levels of protein were measured in triplicate by the Bio-Rad protein assay, which is based on the Bradford method for protein quantification (29). Ten micrograms of total soluble protein was subjected to SDS-PAGE on a 4 to 20% Ready Gel Tris-HCl precast gel (Bio-Rad) in Tris-glycine buffer (25 mM Tris, 250 mM glycine, 0.1% SDS). Protein was electrophoretically transferred to a Hybond-ECL membrane (Amersham) at 40 V for 50 min in transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol). The blot was blocked with 5% skim milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween 20 [pH 7.6]) for 1 h, and then a 1:1,000 dilution of anti-σS monoclonal antibody (Neoclone) was added directly to the blocking solution and allowed to incubate on a rocker for 1 h. The membrane was then washed three times in TBST for 5 min at each washing. Subsequently, a 1:3,000 dilution of goat anti-mouse IgG antibody (Bio-Rad) was added and allowed to incubate for 30 min. The membrane was then washed with TBST as described above. Next, the ECL Prime Western blot detection reagent (Amersham) was applied to the membrane for 5 min of incubation, according to the manufacturer’s specifications. The blot was then removed from the detection reagent and imaged with Blue Ultra Autoradiography Film (ISC Bioexpress) and an X-Omat 2000A processor (Kodak). To ensure proper loading, duplicate gels were run and stained for total soluble protein with Coomassie blue.
σS stability assay.To assess IraL-dependent σS stability, cultures of CFT073 and isogenic mutants were grown overnight in LB broth, subcultured 1:1,000 in fresh LB broth, and grown to the mid-logarithmic phase (OD600 of 0.3 to 0.4). Protein synthesis was stopped with Cm (200 µg/ml), and the total protein was isolated over time following trichloroacetic acid (TCA) precipitation and Western blot analysis essentially as described previously (7), except that the total protein was precipitated in 10%, rather than 5%, TCA, and electrophoresis and Western blot assays were carried out as described above. ImageJ software was used to measure band intensity, and densitometry was used to compare the levels of σS at various time points after Cm addition to those at t = 0.
Bacterial two-hybrid system.We used the Cya-based bacterial two-hybrid system (19, 20). Plasmids containing proteins to be tested fused to the T18 and T25 domains of Cya from B. pertussis were cotransformed in BTH101 and plated on LB broth plates containing Ap (100 µg/ml) and Kan (50 µg/ml). Transformations were incubated at 30°C for 48 h. Cells were grown overnight in 3 ml of LB broth supplemented with Ap (100 µg/ml), Kan (50 µg/ml), and isopropyl-β-d-thiogalactopyranoside (IPTG; 0.5 mM) at 32°C. β-Galactosidase activity was determined with the standard assay (30). The values presented are means of at least three independent experiments. Two-microliter volumes of overnight cultures were also spotted onto MacConkey medium plus lactose containing 1% maltose.
IraL-RssB pulldown.Plasmids pACYC184-CBP and pACYC184-CBP-RssB were both cotransformed with plasmids pQE80L and pQE80L-IraL in K-12 iraM::Tetr (AB054) and selected on LB broth plates containing Ap (50 µg/ml) and Cm (25 µg/ml). The resulting transformants were grown overnight in LB medium, diluted into 50 ml of fresh LB medium containing Ap and Cm at an OD600 of ~0.01, and grown at 37°C. At an OD600 of ~0.7, cultures were induced for 1 h with 0.05% arabinose and 0.5 mM IPTG. Harvested cells were resuspended in 2 ml of IPP150 binding buffer (10 mM Tris-HCl [pH 8.0], 250 mM NaCl, 1 mM Mg acetate, 1 mM imidazole, 2 mM CaCl2, 0.1% Triton, 10 mM β-mercaptoethanol) for purification on calmodulin beads (Stratagene) or in 2 ml of buffer I (20 mM Tris-HCl [pH 8.0], 10 mM imidazole, 200 mM NaCl, 0.2% Triton, 10 mM β-mercaptoethanol) for purification on cobalt beads (Clontech). Cells were lysed with a French pressure cell and centrifuged at 8,228 × g for 15 min at 4°C. For each copurification assay, 50 µl of beads previously washed with buffer (IPP150 binding buffer for calmodulin beads and buffer I for cobalt beads) were incubated with 1.4 ml of extracts for 1 h at 4°C on a wheel. After incubation, the beads were washed five times with 1 ml of buffer (IPP150 binding buffer for calmodulin beads and buffer I for cobalt beads), resuspended in 50 µl of 2× SDS sample buffer (New England Biolabs), and heated for 10 min at 95°C with agitation. Samples were analyzed with Nu-PAGE 12% bis-Tris gels (Invitrogen, CA), transferred to nitrocellulose membrane, and probed with a 1:1,000 dilution of anti-CBP (Millipore) or anti-His6-peroxidase (Roche) antiserum. The blots were developed with the Lumi-Phos WB chemiluminescent substrate (Thermo Scientific) with the LAS-4000 Mini luminescent-image analyzer (Fujifilm).
Surveys for iraL and iraM in E. coli strains.To examine the distribution of iraL and iraM among E. coli isolates, we designed oligonucleotides to target dissimilar regions of these genes (see iraL/iraM survey primers listed in Table S1 in the supplemental material). These oligonucleotides were validated as target specific via colony PCR on CFT073, CFT073 ΔiraL, K-12, and K-12 iraM::Tetr (see Fig. S3 in the supplemental material) and subsequently used to survey the E. coli isolates listed in Table S3 in the supplemental material. After overnight growth on LB agar, isolated colonies were subjected to colony PCR with GoTaq according to the manufacturer’s specifications (Promega). PCR products were visualized via UV illumination after electrophoresis on 2% agarose gels and ethidium bromide staining.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health (NIH) grant R01-DK063250-07, and A.B. was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
We thank Susan Gottesman for constructive comments.
FOOTNOTES
- Received 7 March 2014
- Accepted 5 May 2014
- Published 27 May 2014
- Copyright © 2014 Hryckowian et al.
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