Journal of Bacteriology, June 2002, p . 3167-3175, Vol . 184, No . 12

An N-Terminally Truncated RpoS (^S) Protein in Escherichia coli Is Active In Vivo and Exhibits Normal Environmental Regulation Even in the Absence of rpoS Transcriptional and Translational Control Signals

K . Rajkumari¹ and J . Gowrishankar^1,2*

Centre for Cellular and Molecular Biology, Hyderabad 500 007,¹ Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500 076, India²

Received 10 August 2001/ Accepted 6 March 2002

Four conserved regions, numbered 1 to 4 beginning from the N-terminal end, have been identified in the ⁷⁰ family of proteins; some of these are further divided into distinct subregions (19) . Regions 2 and 4 are the most highly conserved among different members of the ⁷⁰ family, and these regions are involved in recognition of and binding to promoter DNA by the enzyme . On the other hand, subregion 1.1 appears to be conserved only in the housekeeping or indispensable proteins of different bacteria, as well as in ^S (see below) . In ⁷⁰, subregion 1.1 (comprising the segment from approximately residues 30 through 100) has been suggested to function as a mask in free protein for DNA-binding domains 2 and 4, so that the latter are exposed only when the factor is associated with the core enzyme, that is, the other subunits of the RNA polymerase holoenzyme (6) . Other studies have suggested that subregion 1.1 is also required for (i) the initial binding of ⁷⁰ to the core enzyme (10, 26); (ii) masking of other core-binding regions in the free ⁷⁰ subunit that are later involved in a holoenzyme interface (10); (iii) influencing the efficiency of transcription initiation, perhaps by constraining the holoenzyme to assess the fitness of a promoter by its -10 and -35 sequences (7, 40, 42); and (iv) imparting stability to the protein in vivo (42) . Consistent with these findings, mutant versions of ⁷⁰ with a deletion of subregion 1.1 confer a dominant-lethal phenotype in vivo (5, 34) .

In addition to ⁷⁰, E . coli cells possess a second primary sigma factor, RpoS or ^S (with 330 amino acid residues), that is encoded by rpoS and that is important for stationary-phase gene expression and survival (reviewed in references 12 and 14) . As a member of the ⁷⁰ family, ^S also has conserved regions 1 through 4 . The N-terminal stretch of 60 amino acid residues of ^S has been reported to share moderate sequence similarity with domain 1.1 of ⁷⁰ (19, 25), and both are also rich in acidic amino acid residues; however, the function of this region in ^S is not known . An alignment of these two segments of ^S and ⁷⁰ is shown in Fig . 1 .

 
The genes of the ^S regulon are induced under different environmental conditions, such as the stationary phase of growth, low pH, high osmolarity, or low incubation temperature, consequent to an elevation of the cytoplasmic concentration of ^S under each of these conditions (12, 14) . Cellular ^S content itself is determined by the interplay of a complex set of regulatory mechanisms that operate at the levels of rpoS transcription and translation as well as the stability of the protein .

We have found in this study that a mutant ^S protein which is missing its N-terminal 50 amino acid residues retains in vivo activity at ^S-regulated promoters . Our results obtained with strains expressing the mutant protein also suggest that environmental regulation of cellular ^S content may occur primarily at the posttranslational level .

 
Media and growth conditions. For routine experiments, Luria-Bertani (LB) medium (22) and glucose (Glu)-minimal A medium (22) were used as the nutrient and defined media, respectively, and the incubation temperature for growth was 37°C . MacConkey lactose agar was obtained from Difco . Cultures for ß-galactosidase assays were incubated with shaking (i) until mid-exponential phase (A₆₀₀ of 0.4 to 0.8) or until 1 h after entry into stationary phase; (ii) at 30 or 10°C; and (iii) in LB medium or low-osmolarity K-tryptone medium (27), the latter supplemented when necessary with NaCl to 0.3 M . Unless otherwise indicated, Ara induction experiments (11) were performed by supplementation of the growth medium with 0.2% Ara (or 0.2% Glu, as a control) . Concentrations of antibiotics used were as described earlier (20) .

Immunoblot analysis. The procedures used for electrophoresis of cell extracts on 10% polyacrylamide gels with sodium dodecyl sulfate, electroblotting to a polyvinylidene difluoride membrane, staining with Ponceau S, and sequential treatments with blocking reagent, primary antibody, alkaline phosphatase-conjugated secondary antibody, and chromogenic substrates were essentially as previously described (32) . As part of the protocol, 2.5 µl of rabbit anti-^S antiserum (kindly provided by Regine Hengge-Aronis) was preadsorbed at 4°C overnight with 1 ml of a sonicated cell extract (containing approximately 2 mg of protein) of either the rpoS::Tn10 strain RH90 or the rpoS strain RH100 in 50 mM Tris-Cl buffer (pH 8) containing 1 mM each EDTA and phenylmethylsulfonyl fluoride; following centrifugation at 12,000 x g for 20 min at 4°C, the supernatant was recovered and used in a total volume of 10 ml as the primary antibody preparation .

Other experimental techniques. The procedures used for phage P1 transduction (8), transposon tagging with Tn10dCm and genetic mapping (22), and experiments involving DNA manipulations (32) were as described previously . Three primers, 5'-GAACCAGTTCAACACGCT-3', 5'-ACCGAGGTAATGCGCTCGT-3', and 5'-CCGATGGGCATCGAC-3', which were specific, respectively, for the 5' end, the middle, and the 3' end of rpoS, were used for PCR amplification and DNA sequence determination of the rpoS locus . ß-Galactosidase assays were performed by the method of Miller (22); enzyme specific activity values are reported in Miller units .

 
We used classical genetic techniques (22) first to obtain a new transposon Tn10dCm insertion allele (designated zgc-912::Tn10dCm) 85% cotransducible with the locus in GJ829 that confers rho-modulated P1 -lac expression and then to map the chloramphenicol resistance (Cm^r) marker to the 62-min region of the chromosome (data not shown) . The Cm^r marker exhibited 85% linkage to rpoS, raising the possibility that the mutation in GJ829 that affects P1-lac expression is itself an rpoS allele .

The linked Cm^r marker also allowed us to construct, from wild-type strain GJ862, an isogenic pair of derivatives, GJ875 and GJ876, that carried, respectively, the mutant locus of GJ829 and its wild-type allele . PCR amplification of the rpoS gene from the two strains by using a pair of primers specific to the 5' and 3' ends of the gene (1.2 kb apart) indicated that the GJ829 mutation is associated with an insertion of 1.3 kb of DNA in the rpoS locus (data not shown) . DNA sequence analysis revealed it to be an IS10 insertion disrupting the rpoS open reading frame after codon 53 (Fig . 2), and the mutation was designated rpoS365::IS10 . The stock of strain GJ146 that had served as the immediate ancestor for GJ829 and GJ830 was also shown to carry the rpoS::IS10 mutation (data not shown); since GJ146 had been derived during several steps of Tet^s selection following successive transductions with Tn10 insertions (9), we presume that the IS10 transposition must have occurred subsequently, during routine strain maintenance, spontaneously, and unselected from one of the original Tn10-bearing loci in the chromosome . Incidentally, the site of rpoS365::IS10 insertion was identical to that for each of two different rpoS::Tn10 insertions oriented opposite one another and sequenced earlier (15) in strains RH90 and UM22 (Fig . 2), indicating that it is a hot-spot site for transpositions involving IS10 and its derivative composite transposons . Furthermore, data from an earlier study (27) indicated that the rho mutation does not restore proU P1-lac expression in strains carrying the rpoS359::Tn10 mutation of RH90 .

 
A plasmid (pETF) carrying the rpoS⁺ gene (39) was able to complement the rpoS::IS10 mutation for pHYD275-directed lac expression in the rho⁺ strain GJ829 (data not shown) .

Evidence for the synthesis of an ^S polypeptide with a deletion of its N-terminal 50 amino acids (^S1-50) in rpoS::IS10 rho mutants. There were two conceivable explanations, not mutually exclusive, for the above results . Transcription initiating from proU P1 of S . enterica is subject to premature termination (that is, attenuation), and this phenomenon is known to be Rho dependent (27, 28) . It was therefore possible that the rpoS::IS10 insertion mutation somehow accentuated the effect of Rho factor on transcription initiating from E . coli proU P1 as well . For example, even in an rpoS⁺ background, lacZ reporter gene expression from S . enterica proU P1 (on plasmid pHYD373) is absent in a rho⁺ strain (GJ862) and is stimulated in a rho mutant (GJ863) (27) (Table 2); in the rpoS::IS10 rho⁺ and rho derivatives (GJ829 and GJ830, respectively), lac expression from pHYD373 was virtually indistinguishable from that in the corresponding rpoS⁺ strains (Table 2) .

The second explanation (31) is that the rho mutation acted only to relieve the polarity effect of the IS10 insertion in rpoS . The location of the rpoS::IS10 insertion is such that (i) translation initiating from the start codon of rpoS will be terminated immediately within the insertion element after codon 53, and (ii) a potential start site for translation exists (with a polypurine stretch situated upstream of the putative initiation codon stretch to serve as a ribosome-binding site) proceeding outward from the other end of IS10 to produce an in-frame fusion of six codons of IS10 with codons 51 to 330 of rpoS (Fig . 2) . (The latter feature is absent from either of the Tn10 insertions at this same site in strains RH90 and UM22.) It was therefore possible that the rho mutation permitted transcription initiating from the rpoS promoter (and upstream promoters) to proceed through IS10 into the 3' end of the rpoS gene and that the N-terminally truncated mutant ^S polypeptide (hereafter referred to as ^S1-50) synthesized under these conditions contributed, either alone or in combination with the ^S N-terminal fragment from positions 1 to 53 [^S(1-53)] to proU P1 expression in vivo .

As a test of this second model, an immunoblot experiment was performed with anti-^S antiserum against cell extracts prepared from rho⁺ and rho derivatives of the rpoS⁺ and rpoS::IS10 strains (Fig . 3A) . An immunoreactive protein corresponding to ^S was present in the rpoS⁺ strains and was more prominent in the rho mutant (Fig . 3A, lane 4) than in the rho⁺ strain (lane 1), perhaps reflecting increased readthrough in the former from the promoter(s) for gene nlpD situated upstream of rpoS (17); the ^S band was absent in the rpoS::IS10 strains (lanes 2 and 3) as well as in the rpoS::Tn10 mutant control (lane 5) . On the other hand, a new protein cross-reacting with the anti-^S antiserum and whose faster migration was consistent with that for the postulated ^S1-50 polypeptide was detected only in the cell extract from the rpoS::IS10 rho derivative (Fig . 3A, lane 3) . These observations lent support to the suggestion that, in a rho background, an N-terminally truncated ^S protein was expressed from the chromosomal rpoS::IS10 allele .

 
^S1-50 expression from a heterologous promoter. As a further test of the model invoking a function for ^S1-50, we designed experiments to address the question of whether the putative IS10-encoded translation initiation signals are indeed proficient for the expression of the truncated protein from a heterologous inducible promoter in rho⁺ strains and, if so, whether the protein exhibits sigma activity in vivo . For this purpose, we used the thermostable high-fidelity Pwo DNA polymerase enzyme to amplify by PCR, from the chromosome of the rpoS::IS10 strain GJ875, an 0.85-kb DNA segment comprising the coding region of the putative ^S1-50 peptide along with the adjacent 43 bp from the end of IS10 . The DNA segment was then cloned downstream of the Ara-inducible promoter in plasmid vector pBAD24 (11), and the resulting plasmid was designated pHYD408 .

An immunoblot experiment with anti-^S antiserum was undertaken to demonstrate Ara-induced synthesis from the pHYD408 template of the N-terminally truncated ^S1-50 polypeptide (Fig . 3B) . As in the previous experiment, an immunoreactive protein corresponding to wild-type ^S was detected only in cells of the rpoS⁺ strain (Fig . 3B, lane 7) and was absent from those of the rpoS derivatives (lanes 6, 8, and 9) . Instead, a cross-reacting protein whose electrophoretic mobility was consistent with that expected for ^S1-50 was present in abundance in Ara-grown (Fig . 3B, lane 9) but not Glu-grown (lane 8) cells of the rpoS/pHYD408 derivative .

In experiments aimed at testing in vivo activity, we were able to show that plasmid pHYD408 could also direct substantial lac expression, specifically in an Ara-inducible manner, from the E . coli proU P1 promoter on pHYD275 in rho⁺ strains that carried the rpoS::IS10, the rpoS359::Tn10, or the rpoS mutation on the chromosome (Table 3) . These results provide additional support for the model invoking activity in vivo for the ^S1-50 protein and furthermore suggest that ^S(1-53) encoded by the chromosomal rpoS::IS10 allele may not be essential for this purpose (since considerable proU P1-lac expression was elicited in the rpoS background as well) .

 
Recognition of other ^S-dependent promoters by ^S1-50. With the aid of the respective promoter-lac operon fusions, we examined the in vivo activity of ^S1-50 (expressed either from the chromosomal rpoS::IS10 allele in a rho background or by Ara induction of rpoS rho⁺ strains carrying plasmid pHYD408) on the promoters of three other ^S-dependent genes, katE, osmY, and csiD . Although each of the two methods used to obtain the expression of ^S1-50 protein had their individual shortcomings—namely, a nonspecific reduction, associated with the pleiotropic rho mutation, of lac expression from promoters other than proU P1 even in control rpoS⁺ strains (Table 4) (see also reference 27) and considerable overproduction of the mutant protein in the Ara induction experiment—we reasoned that their potential confounding effects might not overlap .

 
The results from the experiments involving the rpoS::IS10 rho strains indicated that, unlike the situation with proU P1, the other three promoters were transcribed to the extent of only about 15 to 30% in the presence of ^S1-50 synthesized from the chromosomal allele, compared to their activities in the isogenic rho rpoS⁺ strains (Table 4) . Nevertheless, in vivo lac expression with ^S1-50 was higher than that in the corresponding rho rpoS strains . The Lac phenotypes of the different promoter-lac fusion strains (rho⁺ and rho, in combination with rpoS⁺, rpoS::IS10, and rpoS) on MacConkey lactose agar plates correlated well with the ß-galactosidase specific activities reported in Table 4 (data not shown) .

On the other hand, when ^S1-50 was overproduced in the rpoS rho⁺ strains by Ara induction from pHYD408, the level of expression of each of the three lac fusions was much higher and, at least for katE, comparable to that in the corresponding haploid rpoS⁺ rho⁺ derivatives (Table 4) . A substantial level of expression was also obtained for each of the lac fusions with Ara induction of pHYD408 transformants of rpoS::IS10 rho⁺ derivatives (data not shown) .

In the course of these studies, we observed that whereas the activities of other promoters, such as katE, in the pHYD408 derivatives (rpoS) were unaffected when the concentration of Ara as an inducer was varied between 0.2% (routinely used) and 0.02% (data not shown), the expression of csiD-lac was maximal at 0.02% Ara and then progressively declined with increasing sugar concentrations (Fig . 4) . Even in a control rpoS⁺ strain, GJ2785/pBAD24 (in which, as expected, the synthesis of ^S could occur without Ara) (Fig . 4), csiD-lac expression was progressively repressed by Ara at concentrations above 0.02% (Fig . 4) . It is known that the csiD promoter requires both ^S and cyclic AMP (cAMP)-cAMP receptor protein for its activity (21), and the present results therefore suggest (i) that the promoter is catabolite repressed in the rpoS⁺ strain at Ara concentrations above 0.02% and (ii) that a holoenzyme with the ^S1-50 subunit is also proficient for catabolite repression at this promoter .

 
Environmental regulation of ^S1-50. As mentioned above, it is believed that the regulation of cellular ^S levels by environmental variables, such as growth phase, temperature, and osmolarity, is achieved by a combination of transcriptional, translational, and posttranslational control mechanisms (12, 14) . On the other hand, Zgurskaya et al . (43) suggested that the increase in ^S content in stationary-phase cells may be accounted for solely by the increased stability of ^S, and Becker et al . (1) showed that the increase under these conditions occurs even in the absence of rpoS transcriptional or translational regulation .

In the present study, when we used the chromosomal rpoS::IS10 rho strain GJ830, we observed that lac reporter gene expression from proU P1 of E . coli or S . enterica was induced both by elevated osmolarity and by growth at 10°C and that the magnitude of such induction (4- and 10-fold, respectively) was comparable to that in the rpoS⁺ strains (Table 2) . Given that the cis regulatory sequences for native rpoS expression have been separated from the coding region of ^S1-50 by the IS10 insertion in GJ830, these results raised the possibility that environmental regulation of ^S1-50 synthesis could occur even in the absence of such sequences, but alternative explanations were not excluded by the data .

We therefore examined whether the cellular content of ^S1-50 could be regulated by the same environmental cues as those reported earlier for wild-type ^S (12, 14), even when the former is expressed by Ara induction from pHYD408, that is, from a template devoid of the transcriptional and translational controls of native rpoS . The intracellular concentrations of ^S1-50 were assayed both directly by immunoblotting with anti-^S antiserum (Fig . 5) and indirectly by determination of lac reporter gene expression from the proU P1 and katE promoters (Table 5) .

 
The data from the immunoblotting experiment indicated that the levels of ^S1-50 expressed following Ara induction in a rpoS strain increase progressively from the early exponential to the stationary growth phase (Fig . 5, lanes 2 through 4) and that the levels are also increased by growth under conditions of elevated osmolarity (Fig . 5, compare lanes 5 and 6) .

In the indirect assay (Table 5), lacZ reporter gene expression from the proU P1 and katE promoters was significantly higher in the stationary growth phase than in the exponential growth phase . The comparisons were done with cultures that had been subjected to Ara induction for both equal lengths of time and the same numbers of generations of growth . Similarly, the osmotic inducibility of both promoters was observed in derivatives expressing ^S1-50 from pHYD408 to the same extent as in the rpoS⁺ strain . Finally, both proU P1-lac transcription and katE-lac transcription directed by ^S1-50 were substantially higher in cultures grown at 10°C than in those grown at 30°C, and the magnitude of low-temperature induction was equivalent to that seen with wild-type ^S . The significance of these findings for the understanding of ^S synthesis and turnover mechanisms is discussed below .

The data presented in Table 2 suggest that chromosomally expressed ^S1-50 may be as proficient as wild-type ^S in its ability to initiate transcription from the E . coli and S . enterica proU P1 promoters . On the other hand, the in vivo activities of three other promoters, katE, csiD, and osmY, in the presence of ^S1-50 were only fractions (15 to 30%) of the values obtained with ^S (Table 4) .

Two alternative explanations, not mutually exclusive, are possible for the latter finding . The first is that it represents a defect in ^S activity associated with the N-terminal truncation (that can be overcome at least partially by increased expression of the mutant protein from plasmid pHYD408) . With a K173E mutant of ^S, Becker et al . (1) had earlier analogously demonstrated normal activity at one promoter (csiD) and three- to fivefold reduced activities at several others (osmY, bolA, and otsB) . The second explanation is that of a confounding effect of the rho mutation on some ^S-dependent promoters . As reported earlier (27) and as is also clear from the data in Tables 2 and 4, even in an rpoS⁺ background the rho mutation has opposite effects on expression from proU P1 (increase) and expression from the csiD, osmY, or katE promoter (decrease) .

In vitro experiments to assess the binding affinity of ^S1-50 for core RNA polymerase and to determine the transcription activity of the reconstituted enzyme at the various promoters will help address these questions . Catabolite repression at csiD was preserved under conditions where the promoter was active in vivo with ^S1-50, suggesting that an interaction of RNA polymerase bearing the mutant subunit with cAMP-cAMP receptor protein is normal (21) .

^S1-50 and environmental regulation of ^S-specific promoters. As mentioned above, the intracellular ^S concentration varies according to the growth conditions and is regulated by different mechanisms, including those that operate at the steps of rpoS transcription, rpoS translation, and ^S proteolysis (12, 14) . There also exists substantial evidence for the notion that regulation of the intracellular ^S content explains the regulation of expression of genes of the ^S regulon by environmental variables, such as osmolarity (13, 16), growth phase (16, 41), and temperature (36), or by factors such as Hfq (23, 24) and ClpXP (33) .

In the present study, therefore, the first suggestion that ^S1-50 may exhibit normal environmental regulation even in the absence of the rpoS transcriptional and translational control signals came from the data in Table 2, which showed that osmotic and cold induction of proU P1 was unaffected in the chromosomal rho rpoS::IS10 strain (in which the rpoS expression control sequences, although still retained in cis, are separated from the coding region for ^S1-50 by IS10) .

This possibility was then validated in experiments in which ^S1-50 was designed to be expressed from a plasmid construct (pHYD408) that completely lacked the rpoS transcriptional and translational control sequences . Although Ara-induced synthesis of ^S1-50 occurred at higher-than-physiological levels in these experiments, environmental regulation by growth phase, low temperature, and high osmolarity was demonstrated by direct immunoblotting and indirect reporter gene expression assays . The nature and extent of regulation so identified for ^S1-50 are similar to those described earlier for wild-type ^S (12, 14) .

Since normal environmental regulation of ^S1-50 could be demonstrated even in the absence of the transcriptional and translational control elements of wild-type rpoS, our data suggest that alternative mechanisms, such as those affecting proteolytic degradation of ^S, may be the primary determinants for regulation under these conditions . The ^S1-50 protein retains the ^S turnover element, which has been implicated in the regulation by RssB of ClpXP-catalyzed degradation of the protein (1) .

Although regulation at the level of ^S proteolysis was suggested earlier as one of several mechanisms involved in osmotic and stationary-phase induction of the cognate ^S-specific promoters (12), its relative importance in either of these phenomena had so far not been defined . Nor had such a mechanism been postulated to participate in the phenomenon of low-temperature induction, since previous studies had described roles for only DsrA RNA and Hfq protein in the stimulation of rpoS translation during low-temperature growth (18, 30, 36, 37) .

In summary, therefore, three major conclusions that have emerged from the present investigation are that (i) the N-terminal region of ^S is not essential for in vivo sigma activity on at least one promoter (proU P1), although its removal leads to an apparent partial reduction of activity at three other promoters; (ii) environmental regulation of the ^S regulon is largely unaffected even in the absence of cis elements needed for the transcriptional and translational control of rpoS; and (iii) the N-terminal region is also not necessary for the posttranslational regulation of ^S .

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What Is Prokaryote?, What Is Biofilter?, What Is Growth Medium?, What Is Bioengineering?, What Is Fermentation?, s, Microbiology, a, Microbes, i, Microorganism, c, Microbe, r, Bacterium, n, Pseudomonas aeruginosa, i, Antibiotics, r, Antimicrobial, i, Water treatment, r, Clostridia, i, Cholera, r, E coli O157, c, Thermophile, i, Escherichia coli, s, Acinetobacter, s, Candida albicans, c, Escherichia coli, c, Antibiotics, o, Bacteroides, s, Meningococcus, o, Escherichia coli, a, Antimicrobial, s, Staphylococcus, a, Escherichia coli, c, Salmonella typhimurium, a, Pseudomonas aeruginosa




 

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