gene regulation in bacteria is largely driven by the switch between sigma factors bound to the core rnA polymerase. Escherichia coli possesses seven sigma factors being each one responsible for the identification of a different set of promoter, and consequently, for the transcription of a spe-cific set of genes (Ishihama, 2000). σ70 and σS are the main sigma factors in E. coli (Paget and Helmann 2003; Hengge-Aronis 2002). σ70 (also known as σD) is responsible for the transcription of the majority of E. coli genes, particularly those related to bacterial growth, nutrient assimilation and metabolism. rpoS (also known as σS or σ38) is the second most important sigma factor. It recognizes promoters asso-ciated with stationary phase survival and the general stress response (Hengge-Aronis 2002). The rpoS gene is highly polymorphic (Atlung et al. 2002; Ivanova et al. 1992), and such variation is known to confer on bacteria different phe-notypes regarding nutritional competence and stress resist-ance (notley-Mcrobb et al. 2002). Zambrano and col-leagues have described that wild-type E. coli cells grown in batch cultures for long periods of time tend to accumulate mutations in the rpoS locus, enhancing their fitness and their ability to compete with bacteria from younger cultures; this phenotype was designated gASP (growth Advantage in Stationary Phase) (Zambrano et al. 1993; Finkel 2006). Subsequent studies have shown that wild-type strains tend to accumulate mutations in rpoS or in genes that positively regulate rpoS whenever the population is under nutrient lim-itation for prolonged periods (notley-Mcrobb et al. 2002; Maharjan et al. 2006; Wang et al. 2010, Spira et al. 2011). A very common mutation in E. coli strains is an amber stop codon at position 97–99 of rpoS OrF (Atlung et al. 2002; Subbarayan and Sarkar 2004a, b). Starting from an alterna-tive translation start site at position 157, a truncated rpoS
Abstract The rnA polymerase associated with rpoS transcribes many genes related to stationary phase and stress survival in Escherichia coli. The DnA sequence of rpoS exhibits a high degree of polymorphism. A C to T transition at position 99 of the rpoS OrF, which results in a premature amber stop codon often found in E. coli strains. The rpoSam mutant expresses a truncated and par-tially functional rpoS protein. Here, we present new evi-dence regarding rpoS polymorphism in common laboratory E. coli strains. One out of the six tested strains carries the rpoSam allele, but expressed a full-length rpoS protein owing to the presence of an amber supressor mutation. The rpoSam allele was transferred to a non-suppressor background and tested for rpoS level, stress resistance and for the expression of rpoS and sigma70-dependent genes. Overall, the rpoSam strain displayed an intermedi-ate phenotype regarding stress resistance and the expres-sion of σS-dependent genes when compared to the wild-type rpoS+ strain and to the rpoS null mutant. Surprisingly, overexpression of rpoSam had a differential effect on the expression of the σ70-dependent genes phoA and lacZ that, respectively, encode the enzymes alkaline phosphatase and β-galactosidase. The former was enhanced while the latter was inhibited by high levels of rpoSam.
H. F. galbiati · n. P. Taschner · B. Spira (*) Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. lineu Prestes, 1374, São Paulo, SP CeP: 05508-900, Brazile-mail: [email protected]
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protein with 277 amino acids is produced by this mutant (Subbarayan and Sarkar 2004a). This protein, designated rpoSam (or σam), is 53 amino acids shorter than the wild-type rpoS, and it is able to recognize promoters and to transcribe rpoS-dependent genes albeit less efficiently. The relevance of the rpoSam allele can be highlighted by its widespread distribution among E. coli lab strains (Atlung et al. 2002), which led previous studies to propose that rpoSam should correspond to the ancestral version of rpoS in K-12 strains (Subbarayan and Sarkar 2004c; Jishage and Ishihama 1997).
The PHO regulon consists of more than 40 genes and operons related to the uptake and assimilation of Pi and phosphorylated compounds. Among the best-characterized PHO genes are phoA and the pst operon, which, respec-tively, encode the enzyme alkaline phosphatase (AP) and a high-affinity Pi-transport system (Wanner 1996). The pro-moters of the PHO genes display one or more consensus regulatory sequences known as PHO boxes that replace the -35 region. Transcription of the PHO genes is regulated by the two-component system PhoB/Phor. When the con-centration of Pi in the external medium decreases below 4 µM, the sensor protein Phor auto-phosphorylates and transfers the Pi group to PhoB, which in turn binds to the PHO boxes and interacts with σ70 initiating the transcrip-tion of the PHO genes (Makino et al. 1993; Wanner 1996). We have previously reported that with the exception of the pst operon, rpoS negatively affects the transcription of the
PHO genes, due to the competition between rpoS and σ70 for the core rnA polymerase (Taschner et al. 2004, 2006).
Here, we show that a common K-12 strain, C600, carries both the rpoSam allele and an amber suppressor mutation. When transferred to a non-suppressor background, rpoSam confer on the bacterium a moderate level of stress resist-ance and expression of rpoS-dependent genes. It is also shown that rpoSam plays a differential role in the expres-sion of σ70-dependent genes.
Methods
Bacterial strains, plasmids and media
The strains and plasmids used in this study are described in Table 1. lB is the standard complex medium (Miller 1992); Medium A is a semi-rich Pi-limited medium (Pi concen-tration ≈ 0.2 mM) (levinthal et al. 1962). The strains and plasmids used in this study are described in Table 1.
Construction of strains
Transfer of chromosomal markers was performed by P1 transduction essentially as described (Miller 1992). The rpoSam allele was introduced into Mg1655 by two sequential transductions. First, the zfi3251::Tn10 marker was transduced from strain BS110 (rpoS+ zfi3251::Tn10)
Table 1 Strains and plasmids used in this study
Strains genotype references
Bl21 dcm ompT hsdS(rB-mB-) gal Daegelen et al. (2009)
Mg1655 E.coli K 12 wild-type Blattner et al. (1997)
nP3 C600 rpoS::Tn10 This study
nP1233 Mg1655 rpoS::Tn10 rssB::Km This study
W3110 E.coli K 12 wild-type Hayashi et al. (2006)
r015 MC4100 (osmY::lacZ) Kmr Weichart et al. (1993)
Mg1655 rpoSam Mg1655 zfi3251::Tn10 (rpoS+ from C600) This study
C600 rpoS1655 C600 zfi3251::Tn10 (rpoS+ from Mg1655) This study
Plasmids genotype references
pDHY272 proU-lacZ Dattananda et al. (1991)
pnP5 rpoS+ pACT3 Taschner et al. (2006)
pnP10 C600 rpoS (rpoSam) pACT3 This study
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to strain C600 (rpoS has a 66 % co-transduction rate with zfi3251::Tn10; Barth et al. 1995). Colonies were selected for resistance to tetracycline and screened for high AP activity (similar to C600 AP level) to ensure the rpoSam allele was not replaced by the co-transduction of zfi3251::Tn10 and rpoS+. The resulting strain was then used as a donor for the co-transduction of rpoSam and zfi3251::Tn10 into Mg1655. The transductants were selected for tetracycline resistance and screened for high AP activity. Other phenotypic tests, such as glycogen stain-ing with an iodine solution (Hengge-Aronis and Fischer 1992) and catalase activity (Mulvey and loewen 1989), both stronger in rpoS+ than in rpoSam strains, confirmed the status of rpoS in the transductants. Finally, the rpoS allele in the new strain was confirmed by DnA sequenc-ing of the region surrounding codon 33 of rpoS OrF. The osmY::lacZ chromosomal fusion was transferred from strain r0151 into Mg1655 derivatives by P1 transduction. Transductants were selected for kanamycin resistance. The rssB::Km mutation was transduced from strain MC4100BS rssB::Km (Spira et al. 2011).
Sequence analysis of rpoS
A 1.2-Kb fragment containing the entire rpoS OrF was amplified by PCr using primers rpoS429F (5′–ggAACAA CAAgAAgTTAAgg) and rpoS1730r (5′-gTATgggCgg TAATTTgAC). PCr products were purified from the gel with the Wizard DnA purification system (Promega). DnA was sequenced in an automatic sequencer type ABI Prism 3100 genetic Analyser (Applied Biosystems/Hitachi, Warrington, UK) using oligo rpoS9274r (5′-CAgACCAC gATTgCCATAAC).
enzymatic assays
Alkaline Phosphatase (AP) was assayed as previously reported (Spira et al. 1995). Cells were grown in Medium A, and p-nitrophenyl-phosphate (pnPP) was used as a substrate. The reaction was stopped by the addition of 0.25 M na2HPO4 and the AP-specific activity was calculated according to the equation A410 × time (min)−1 × cell density (OD600)
−1. β-galactosidase assays were performed as described (Miller 1992). Ortho-nitrophenyl-β-d-galactopyranoside (OnPg) was used as a substrate. Miller units were calculated follow-ing the equation A420 × 1000 x min−1 × OD−1
600.
Stress assays
Bacterial viability was tested under three different stress conditions: high osmolarity, oxidative stress and cold stress. In all cases cells were grown overnight at 37 °C in lB, washed twice in 0.9 % naCl and diluted to a final
concentration of 3000 cells/ml. For the oxidative stress, cells were incubated for 20 min in the presence of 6 mM H2O2 at room temperature. About 100 μl aliquots were col-lected every 5 min and spread on lB agar. For the osmotic stress, the bacterial suspension was exposed to increasing concentrations of naCl (1, 2, 3, 4 and 5 M) for 4 h at room temperature after which 100 μl samples were plated onto lB agar. For the cold stress assay cells were kept at 4 °C for 5 days, 100 μl aliquots were collected every 24 h and plated as above. The survival rate in all cases was estimated by colony counting.
Immunoblot
For the detection of the non-truncated rpoS (Fig. 2a) pro-teins were extracted from bacteria grown overnight in lB. For the detection of the truncated rpoS, bacteria grown overnight in medium A supplemented with 1 mM Pi were diluted 50-fold in non-supplemented medium A and grown for 4–5 h until they reached the Pi-starvation phase (OD600 ~ 0.9). The entrance into the Pi-starvation phase was determined by the increase in AP activity. equal amounts of cells (equivalent to 1 OD600) were then resuspended in 0.1 ml application buffer (0.5 M Tris/HCl, 2 % SDS, 5 % 2-mercaptoethanol, 10 %, v/v, glycerol and 0.01 % bromo-phenol blue). Samples were boiled for 10 min and proteins were resolved in a 12.5 % denaturing polyacrylamide gel. After electrophoresis, proteins were transferred to a nitro-celullose membrane (Protran, Perkin-elmer life Sciences) using the Trans-Blot SD Semi-dry Transfer Cell apparatus (Biorad). A blocking step with 5 % skim milk was per-formed and the membrane was incubated with 2,000-fold diluted monoclonal anti-rpoS antibody (Santa Cruz) and 20,000-fold diluted peroxidase conjugated anti-mouse Igg (Pierce). The Super Signal West Pico kit (Pierce) was used to detect the rpoS bands as recommended by the manufac-turer and the membrane was exposed to X-ray films.
Competition assay
Strains Mg1655 (lacZ::Tn5) and Mg1655 rpoSam (zfi::Tn10) were grown in non-supplemented medium A or in medium A + 1 mM Pi for 10 days at 37 °C under agita-tion. Samples were taken daily, diluted and plated on lB plates supplemented with tetracycline (15 mg/ml) or kana-mycin (50 mg/ml). CFU number was assessed and plotted. The rpoS phenotype of the strains was confirmed by testing the colonies for catalase activity.
Statistical analysis
All statistical analyses were done using graphPad Prism 5 for Windows. The standard error of the mean was
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calculated according to the formula SeM = SD/√n, where SD is the standard deviation (Cumming et al. 2007). Com-parison between two strains was done with Student’s t test. Statistical analysis between more than two strains was done by One-way AnOVA with Dunnett’s post test. The number of asterisks (*, ** or ***), respectively, represent p < 0.05, p < 0.01 and p < 0.001.
Results
rpoS status and AP activity in six wild-type E. coli strains
Previous work in our laboratory has shown that the level of AP in Pi-starved and non-starved E. coli cells varied according to the status of rpoS in those strains (Taschner et al. 2004; Spira and Ferenci 2008). We used the relation between AP and rpoS levels to investigate the status of rpoS in five commonly used laboratory strains: the K-12 strains C600, CSH109, MC4100, Mg1655, W3110 and the B strain Bl21. The rpoS::Tn10 mutation was intro-duced into each of these strains by P1 transduction. The AP activity of the wild-type strains and of their respec-tive rpoS::Tn10 mutants was determined in Pi-starved cultures. An intrinsic variability in the level of AP among the different wild-type strains was observed (Fig. 1). The AP level ranged from 0.9 e.U. (CSH109) to 4.0 e.U. (C600). The AP activity in most strains fluctuated around 1.5 e.U. With the exception of C600, disruption of rpoS enhanced AP activity by at least twofold. The increase
in AP indicates that Bl21, CSH109, MC4100, Mg1655 and W3110 carry functional rpoS alleles. Transcription of the σ70-dependent gene phoA is negatively affected by rpoS in these strains (Taschner et al. 2004). All wild-type strains presented high catalase activity (not shown) indi-cating that all, including strain C600, express a functional rpoS protein, while the rpoS::Tn10 mutants were cata-lase-negative, as expected.
DnA sequencing of the rpoS OrF of the six wild-type strains showed they carry almost identical sequences, except for one or two differences at position 97–98 (codon 33) of rpoS OrF (Table 2). In most strains codon 33 encodes either glutamine or leucine, but in C600 an amber stop codon was found. A TAg codon at this posi-tion was observed in other E. coli strains (Atlung et al. 2002; Subbarayan and Sarkar 2004c; Belin 2003; Yoshida et al. 2002). Strains that carry the rpoSam allele produce low amounts of a truncated rpoS protein and express less efficiently rpoS-dependent genes (Atlung et al. 2002;
Fig. 1 Differential effect of rpoS on the AP activity of six E. coli strains. The wild-type strains and their respective rpoS::Tn10 mutants were grown overnight in medium A (low Pi) and assayed for AP activity. The AP enzyme units were calculated according to the formula e.U. = A410 OD−1
600 min−1. each bar represents the mean (±SeM) of three independent experiments. *, ** and ***represent the statistical significance (p < 0.05, p < 0.01 and p < 0.001, respectively) of the difference between the wild-type strain and its respective rpoS::Tn10 mutant
Table 2 rpoS polymorphism in six E. coli strains
Strain Codon 33 AA residue
Bl21 TTg leucine
C600 TAg Amber
CSH109 TTg leucine
MC4100 CAg glutamine
Mg1655 CAg glutamine
W3110 CAg glutamine
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Subbarayan and Sarkar 2004a; Vijayendran et al. 2007; Kabir et al. 2004). Interestingly, our W3110 stock car-ries a CAg codon, present in only one out of five differ-ent W3110 stocks hitherto tested (Subbarayan and Sarkar 2004c).
C600 produces a full-length (38 kDa) version of rpoS
Strain C600 carries a supE44 (glnV44) allele (http://cgsc.biology.yale.edu/cgi-bin/sybgw/cgsc/Strain/11195) that suppresses amber mutations. given the high level of pol-ymorphism found among E. coli lab stocks (Spira et al. 2011; Chen et al. 2004; Jishage and Ishihama 1997), we tested whether strain C600 from our collection can sup-press amber mutations. lambda phage strains that cannot replicate due to the presence of amber mutations in the O gene (erdile and Inman 1984) and thus do not form plaques in a non-suppressor (Su−) host were applied to C600, Mg1655 (Su−) and CSH110 (Su+) bacterial lawns. Table 3 shows that the phage strains lambda Y998 and lambda 1323 that carry amber mutations formed plaques on CSH110 and
C600, but not on Mg1655. Phage lambda Y1 that does not carry an amber mutation in the O gene grew on all three bacterial lawns. These results confirmed that C600 is an amber suppressor strain.
next, a Western-blot analysis of rpoS in C600 and Mg1655 was conducted. Bacteria were grown overnight in lB and assayed for the presence of rpoS using anti-rpoS monoclonal antibodies. Figure 2a shows that C600 expresses a 38 kDa (long) version of rpoS identical to that of the rpoS+ strain Mg1655. It is worth noticing that Mg1655 and C600 display similar levels of rpoS.
The rpoSam allele in the non-suppressor strain Mg1655
To study the behavior of the rpoSam allele in a non-sup-pressor background, the rpoSam allele was transferred to strain Mg1655. The resulting strain, Mg1655 rpoSam, was assayed for rpoS by immunoblotting of an over-night culture, but a band corresponding to the truncated rpoS could not be detected. To improve the likelihood of detection of rpoSam another growth strategy was employed; overnight bacteria were diluted and grown in non-supplemented medium A until they entered the Pi-starvation phase, characterized by a strong increase in AP activity (not shown). Proteins were then extracted and immunoblotted as above. The rpoSam protein could not be observed in a wild-type background (not shown). A rssB::Km mutation was then introduced into strain Mg1655 rpoSam because inactivation of the chaper-one rssB significantly enhances rpoS stability (Zhou
Table 3 Suppression of amber mutations
a Values represent PFU/ml
λY1 λY998 λ1323
C600 2.4 × 109a 6.0 × 108 4.0 × 109
CSH110 2.4 × 109 1.6 × 109 >1010
Mg1655 4.0 × 109 – –
A
B
Fig. 2 Western-blot analysis of rpoS. equal amounts of proteins were extracted, resolved by SDS-PAge and probed with a mono-clonal anti-rpoS antibody. A. Bacteria were grown overnight in lB medium; Mg and C600 represent different culture batches of the wild-type strains Mg1655 and C600. B. Bacteria were grown in medium A until they entered the Pi-starvation phase; immunoblots were performed with the following strains: A, Mg1655 rssB::Km; B, Mg1655 rpoSam; C, Mg1655 rpoS::Tn10; D, Mg1655 rpoS::Tn10 rssB::Km transformed with pnP10 (prpoSam); e, Mg1655
rpoS::Tn10 rssB::Km transformed with pnP10 supplemented with 1 mM IPTg. Numbers represent the place of the molecular weight markers on the gel; long rpoS, 38-kDa rpoS protein; truncated rpoS, 30-kDa rpoSam. The labels ‘non-specific’ and ‘degradation,’ respectively, represent a non-specific protein that cross-reacted with the rpoS antibody and the degradation products of rpoS. The auto-radiogram was overexposed to permit the visualization of the poorly expressed rpoSam protein in lane B
et al. 2001; Hengge 2009). A faint band of 30 kDa cor-responding to the truncated rpoS could be observed only in an overexposed film in the rssB::Km background (Fig. 2b, lane B). To increase the cellular level of the truncated rpoS, rpoSam was cloned under the control of the Ptac promoter (plasmid pnP10) and transformed into Mg1655 rpoS::Tn10 carrying the rssB::Km muta-tion. The intensity of the rpoSam band was stronger in this strain (lane D) and was further increased by the addition of IPTg (lane e). These results suggest that in a non-suppressor background, such as in strain Mg1655, the truncated form of rpoS is expressed albeit at low levels. Several low MW bands visible in lane A (Mg1655 rssB::Km) plausibly correspond to degra-dation products of the original 38 kDa rpoS protein. The 38 kDa band present in lane e (pnP10 in Mg1655 rpoS::Tn10 rssB::Km + IPTg) is likely to result from the readthrough of the amber codon (see “Discussion” section).
effect of rpoSam on stress
rpoS is the key master regulator of the stress response in E.coli allowing cells to switch from exponential or restricted growth to a maintenance and survival lifestyle (Hengge-Aronis 2002; Kolmsee and Hengge 2011). The wild-type strain Mg1655 expresses moderate amounts of rpoS and it is fairly resistant to environmental stresses (Spira et al. 2008). To test the level of protection provided by the rpoSam allele in this strain, bacterial survival was assessed by challenging cells to three different stressful conditions: high osmolarity, oxidative stress and cold.
resistance to high osmolarity was assayed in strains Mg1655, Mg1655 rpoS::Tn10 and Mg1655 rpoSam by exposing the bacteria to increasing concentrations of naCl for 4 h. The resistance level was estimated by CFU counting of the surviving bacteria. The wide range of naCl concentrations used in this study (1 to 5 M) is in agreement with the reported capability of E. coli to face extremely high salt concentrations
a
c
b
Fig. 3 effect of rpoSam on stress resistance. Bacteria were incubated for a 4 h in the presence of 1–5 M of naCl; b 20 min in the pres-ence of 6 mM H2O2 and c 5 days at 4 °C. Mg1655 (filled square),
Mg1655 rpoS::Tn10 (filled triangle) and Mg1655 rpoSam (filled cir-cle). Bacterial survival was determined from plate counts. Each point represents the mean ± SeM of three independent experiments
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(Hrenovic and Ivankovic 2009; How et al. 2013). Figure 3a shows that Mg1655 survival was not disturbed even at the highest naCl concentration while the rpoS::Tn10 mutant displayed a strong dose-dependent decrease in survival rate. Mg1655 rpoSam was less sensitive than the rpoS::Tn10 mutant, but not as resistant as the wild-type strain.
rpoS is also involved in the protection against oxidative stress by promoting the synthesis of catalase and other pro-teins that neutralize reactive oxygen species (loewen and Triggs 1984; Chiang and Schellhorn 2012). Survival of the Mg1655 derivatives was challenged by exposing the bacte-ria to 6 mM H2O2. Figure 3b shows that Mg1655 was only mildly affected by H2O2 while both Mg1655 rpoS::Tn10 and rpoSam strains displayed a low survival rate. This sug-gests that the rpoSam allele is unable to confer on Mg1655 the ability to produce sufficient catalase to degrade H2O2, in accordance with previous reports (rajkumari and gow-rishankar 2002; Subbarayan and Sarkar, 2004a). The low survival rate of rpoSam cells exposed to oxidative stress is also corroborated by the fact that this strain displayed low levels of catalase activity (not shown).
E.coli is a mesophile organism with optimal growth temperature around 37 °C. A shift from 37 °C to 10–15 °C progressively slows growth rate and at 8 °C cell growth is halted (Jones et al. 1987; Yamanaka 1999). Under these conditions, bacterial survival is hampered and cryoprotec-tion is at least partially conferred by rpoS (Vidovic et al. 2011). To test the effect of low temperature on bacterial survival the Mg1655 derivatives were exposed to 4 °C dur-ing the course of 5 days. Figure 3c shows that Mg1655 was unaffected by the low temperature conditions while the rpoS::Tn10 mutant presented 80 % mortality at the end of the 5-day incubation. Mg1655 rpoSam showed an
intermediate behavior with high resistance until 72 h fol-lowed by a drastic decline in the survival rate between 72 and 96 h. Overall, the stress assays showed that the rpoSam allele confers on E. coli an intermediate level of resistance against environmental stresses.
effect of rpoSam on the expression of rpoS- dependent genes
A bacterial strain with modest levels of stress resistance is likely to be partially deficient in the expression σS-dependent genes. To test this assumption, the effect of the rpoSam allele on the transcription of proU and osmY, two σS-dependent genes and that are associated with osmoregulation in E. coli (Yim and Villarejo 1992) was evaluated. In an osmotic upshift, many cellular functions are impaired and most transporters are inhibited; however, osmosensory transporters that medi-ate the uptake of K+ or osmoprotectants are activated (Wood 2011). High concentrations of K+ induce proU, an operon that encodes a high-affinity betaine transport (Sutherland et al. 1986). Betaine is an osmoprotectant that restore turgor pressure. Plasmid pDHY272 that carries a proU-lacZ tran-scriptional fusion was transformed into the different Mg1655 derivatives, which were subsequently grown overnight and assayed for β-galactosidase (β-gal) activity. Figure 4 shows that the activity of the proU promoter in the rpoS+ strain was ten times as high as in the rpoS::Tn10 mutant. The rpoSam strain showed a moderate enzyme level, being ~40 % lower than that of the wild-type strain but still 7 times higher than the activity displayed by the rpoS::Tn10 mutant.
The expression of osmY increases in response to an osmotic upshift and to a reduction in growth rate (Yim et al. 1994; Weber et al. 2006). To analyze the effect of rpoSam on osmY, the chromosomal fusion osmY-lacZ (Weichart et al. 1993) was transduced into the Mg1655 set of strains. Bacteria were grown overnight in lB medium and assayed for β-galactosidase. The osmY-lacZ activity was 5.3 times higher in Mg1655 than in the rpoS::Tn10 or rpoSam strains. Unlike proU, it is apparent that rpoSam is unable to promote the transcription of osmY (Fig. 4).
The level of catalase was also assessed by observing the bubbling of bacterial patches flooded with H2O2. Mg1655 rpoSam produced considerably less bubbling than the wild-type strain (not shown), indicating that catalase production in this strain is crippled. In summary, the results of these assays indicate that the low survival of the rpoSam strain is related to the humble expression of rpoS-dependent genes.
The effect of rpoSam on σ70-dependent genes
The phoA gene that encodes AP is a σ70-dependent gene whose transcription is negatively affected by high levels of rpoS (Taschner et al. 2004; Spira and Ferenci 2008 and
Fig. 4 effect of rpoSam on proU and osmY transcription. Strains Mg1655, Mg1655 rpoS::Tn10 and Mg1655 rpoSam transformed with plasmid pDHY272 (proU-lacZ) or transduced with the osmY-lacZ fusion were grown overnight in lB and assayed for β-gal activity. Each bar corresponds to the average of six independent assays ± SeM. ** and ***represent the statistical significance (p < 0.01 and p < 0.001, respectively) of the difference between Mg1655 and the respective strain
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see Fig. 1). To test the effect of the rpoSam allele on phoA expression, the activity of AP was assayed in Mg1655, Mg1655 rpoS::Tn10 and Mg1655 rpoSam. Bacteria were grown overnight in medium A (low Pi) and assayed for AP activity (Fig. 5). The level of AP in Mg1655 rpoSam and in Mg1655 rpoS::Tn10 was very similar, being 3.5-fold higher than that of the rpoS+ parent strain. This result is consistent with the notion that rpoSam expressed from a single chromosomal copy does not effectively compete with σ70. To test whether higher levels of rpoSam would reduce AP activity, the rpoSam allele was cloned in a plas-mid under the control of Ptac (plasmid pnP10). pnP5 (prpoS+) and pnP10 were transformed into Mg1655 and the transformants were assayed for AP in the presence of IPTg. Surprisingly, not only that overexpression of rpoSam did not inhibit AP activity, but there was a significant incre-ment in AP in the pnP10 transformant. This unexpected result suggests that rpoSam plays a positive role in phoA transcription. Introduction of pnP5 into Mg1655 reduced AP activity, as previously shown (Taschner et al. 2004).
To test whether the positive effect of rpoSam on AP can be extended to other σ70-dependent genes, the expres-sion of endogenous β-galactosidase, encoded by lacZ, in the Mg1655 set of strains was analyzed. Bacteria were grown overnight in lB medium supplemented with 0.1 mM IPTg to induce the lac operon and assayed for β-galactosidase. Figure 5 shows that as expected the level of β-gal was higher in Mg1655 rpoS::Tn10 than in the wild-type strain. β-gal activity in Mg1655 rpoSam was indistinguishable from that of the rpoS::Tn10 mutant. In contrast to AP, overexpression of either rpoS+ (pnP5) or
rpoSam (pnP10) strongly inhibited β-gal activity. This indicates that high levels of rpoSam do inhibit the expres-sion of an ordinary σ70-dependent gene, such as lacZ.
The rpoSam strain displays a gASP phenotype
Bacteria that express low levels of rpoS or that show a par-tial loss of rpoS function display a gASP phenotype and outcompete rpoS+ strains when incubated for long periods in the stationary phase or when continuously grown under nutrient-limited conditions (notley-Mcrobb et al. 2002; Zambrano et al. 1993). The rpoS amber mutant has also been shown to have a survival advantage over rpoS+ strains when co-cultured for several days in lB medium (Sub-barayan and Sarkar 2004b). To test whether rpoSam confers a gASP phenotype under Pi-limitation, strains Mg1655 and Mg1655 rpoSam were co-cultured in medium A and in medium A supplemented with Pi. Figure 6 shows that although the rpoS+ strain had a 4-log advantage over its isogenic rpoSam at the beginning of the curve, in both media rpoSam eventually outcompeted rpoS+. The rpoSam strain took over the culture under Pi-limitation and under Pi-excess after 7 and 8 days, respectively. These results confirm that the rpoSam strain displays a gASP phenotype that is particularly stronger under Pi-limitation.
Discussion
The specificity of promoter recognition in bacteria is given by the replacement of σ factors attached to the core rnA
Fig. 5 effect of rpoSam on endogenous AP and β-gal activity. Mg1655, Mg1655 rpoS::Tn10, Mg1655 rpoSam and Mg1655 transformed with pnP5 (rpoS from Mg1655) or pnP10 (rpoSam from C600) were grown overnight in lB and assayed for AP (dark col-umns) and β-gal activity (light columns). The AP enzyme units were calculated according to the formula e.U. = A410 OD−1
600 min−1. Each bar correspond to the average of six independ-ent assays ± SeM. *, ** and ***represent the statistical significance (p < 0.05, p < 0.01 and p < 0.001, respectively) of the difference between Mg1655 and the respective strain
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polymerase, each one being responsible for the transcrip-tion of a specific set of genes (Ishihama 2000). σ70 and σS, the main sigma factors in E.coli (Paget and Helmann 2003; Hengge-Aronis 2002), compete for the catalytic core of rnA polymerase (Maeda et al. 2000), and this com-petition generates a trade-off in which an inverse relation between growth and survival is observed (nystrom 2003). This antagonist relation gives more interesting insights when rpoS polymorphisms are considered, acknowledg-ing that variations in rpoS levels have direct influence on this balance (King et al. 2004). A frequent rpoS polymor-phism is characterized by the presence of an amber stop codon at the position 33 of rpoS OrF, resulting in a trun-cated (Atlung et al. 2002; gowrishankar et al. 2003; raj-kumari and gowrishankar 2002; Subbarayan and Sarkar 2004b) and partially functional protein (Subbarayan and Sarkar 2004a). As an example, the E. coli genetic stock center (CgSC) has 114 strains carrying the rpoSam allele, though most are derived from a single laboratory (B. Wan-ner’s lab) ancestral strain (http://cgsc2.biology.yale.edu/Mutation.php?ID=43666). The present study aimed to characterize some common E. coli laboratory strains in respect to their rpoS allele and to understand the benefits
and drawbacks in carrying the rpoSam allele in a non-sup-pressor background. It was shown that translation of the truncated form of rpoS from the distal alternative ATg is not very efficient, resulting in low levels of rpoSam that sometimes resembles the behavior of an rpoS null mutant (Subbarayan and Sarkar 2004b). The results presented here provide evidence that the rpoSam allele provides in most cases an intermediate phenotype which may be advanta-geous under certain circumstances, such as during competi-tion with other strains in a phosphate-limited environment.
The fact that the truncated form of rpoS could not be detected in the suppressor strain C600 could be due to the complete suppression of the amber mutation. Indeed, a recent report has shown that suppression efficiency by supE44 at the stationary phase is 92.68 % (Singaravelan et al. 2010). However, given the difficulty in detecting the rpoSam band even when overexpressed from a strong promoter, it is more likely that its absence in the suppres-sor background was due to the poor level of translation of rpoSam from a single chromosomal copy (Fig. 2 and Sub-barayan and Sarkar 2004a). Amber mutations can be sup-pressed even in the absence of the supE44 allele, albeit only with a 2.4 % efficiency (Singaravelan et al. 2010). It means that 2.4 % of the total rpoS protein present in the Mg1655 rpoSam strain are of the long (38 kDa) type. This fact may explain the presence of a 38-kDa band when the rpoSam allele was overexpressed (lane e, Fig. 2b). However, this level of suppression, though significant could not account for the observed behavior of the rpoSam strain, implying that most of the effect regarding stress resistance and gene expression was caused by the truncated rpoSam.
When present at a single chromosomal copy, rpoSam did not inhibit the expression of lacZ, a σ70-dependent gene. However, overexpression of rpoSam strongly reduced β-gal activity suggesting that rpoSam is able to effectively associate with the core rnA polymerase and to compete with σ70. This also indicates that the low-rpoS status dis-played by Mg1655 rpoSam (low resistance to stress and low expression of rpoS-dependent genes) is likely caused by the low cellular concentration of rpoSam rather than to a mechanical disadvantage of rpoSam, such as poor bind-ing to the core rnA polymerase. given the fact that phoA is a σ70-dependent gene and as such is inhibited by the competition between σ70 and rpoS for the core rnA poly-merase (Taschner et al. 2004), the positive effect of rpoSam overproduction on AP activity was unexpected. Our inter-pretation for this result is that e σSam can recognize the promoter of phoA and transcribe the gene. Similarly, over-production of rpoD that encodes σ70 also enhances AP expression (Taschner et al. 2004). The absence of 53 amino acids at the n-terminal may increase the promiscuity of rpoS, which only then is able to interact with PhoB at the phoA promoter and to facilitate the transcription of phoA.
Fig. 6 Competitive fitness of strains Mg1655 and Mg1655 rpoSam co-cultured continuously in medium A +Pi a and medium A −Pi b. The initial concentration of each culture was 108/ml for the rpoS+ and 104/ml for the rpoSam strain. growth curves are representative results of two independent experiments
The n-terminus of rpoS is the least conserved portion of the protein and its function is not clear. In σ70 this part of the protein corresponds to the 1.1 region who mediates the interaction between the sigma factor and the enzyme core (gruber et al. 2001) and masks the promoter-binding domain of the free σ70 (Dombroski et al. 1993). Based on some evidences, such as faster electrophoretic mobility of both σ70 and rpoS proteins deleted of their 1.1 region, it was suggested that the 1.1 region of rpoS may be struc-turally and functionally analogous to the 1.1 region of σ70 (gowrishankar et al. 2003). Though no direct evidence for the function of this region in rpoS is available, it is clear that the n-terminus is not essential for rpoS function, as shown here and elsewhere (Atlung et al. 2002; rajkumari and gowrishankar 2002; Subbarayan and Sarkar 2004c).
The effect of rpoSam on the expression of proU and osmY was not uniform. It is apparent that even low levels of rpoSam are sufficient to elicit proU transcription. On the other hand, PosmY was severely impaired in the rpoSam strain, possibly because the truncated form of rpoS is less proficient in binding to the osmY promoter. Similar results were observed by other authors (rajkumari and gow-rishankar 2002; Vijayendran et al. 2007). The inability of rpoSam to transcribe osmY could also explain the partial survival rate observed in Mg1655 rpoSam under osmotic stress. Though the response to osmotic stress requires the activation of several genes, osmY was shown to be partic-ularly important when naCl is the stressful agent (Weber et al. 2006).
One of the rpoS-dependent proteins expressed upon entering stationary phase is catalase, which promptly degrades hydrogen peroxide (loewen and Triggs 1984). Mg1655 rpoSam showed a moderate level of catalase (Atlung et al. 2002; Subbarayan and Sarkar 2004b and this study), which clearly was not sufficient to provide protec-tion against oxidative stress.
The truncated allele showed an intermediate behavior regarding cold stress. It has been shown that a strain carry-ing the rpoSam allele produces less trehalose, which is of primary importance for viability at 4 °C (Vijayendran et al. 2007; Kandror et al. 2002). This could explain the sensi-tive profile of Mg1655 rpoSam toward low temperatures. likewise, low levels of protection conferred by rpoSam in bacteria exposed to other adverse conditions, such as nitro-gen starvation and resistance to high hydrostatic pressure have been reported (Kabir et al. 2004; robey et al. 2001). It is important to note that rpoS is under the influence of several inputs that regulate its cellular concentration; thus protein levels are vulnerable to variations even in strains with identical rpoS alleles (Ferenci 2005; Ferenci et al. 2011). This implies that even slight differences in strain background could alter rpoS or rpoSam levels, impacting consequently on gene regulation and cell protection.
In conclusion, we showed that the K-12 strain C600 car-ries the rpoSam allele, but due to a suppressor mutation, it expresses only the full-length form of rpoS. When the rpoSam was transferred to a non-suppressor background, low levels of a truncated rpoS could be observed. In this genetic background rpoSam conferred on the bacterium an intermediary pattern of stress resistance and expression of rpoS-dependent genes. Using endogenous lacZ expres-sion it was shown that rpoSam efficiently competed with σ70 only when overexpressed. Interestingly, overexpression of rpoSam increased the level of AP suggesting that the truncated rpoS may interact with PhoB and carry out the transcription of phoA and possibly of other PHO regulon genes as well. The rpoSam strain displayed a strong gASP phenotype particularly under Pi-starvation.
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