1 Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Professor Lineu Prestes, 1374, São Paulo-SP CEP:05508-900, Brazil
2 Department of Biochemistry, Tel-Aviv University, Tel-Aviv 69978, Israel
Correspondence
Beny Spira
benys{at}usp.br
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ABSTRACT |
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INTRODUCTION |
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ED and E
S recognize similar promoter sequences (Wise et al., 1996
; Espinosa-Urgel et al., 1996
; Gaal et al., 2001
; Lee & Gralla, 2001
) and in vitro studies have shown that many genes are transcribed by both E
D and E
S (Nguyen et al., 1993
; Tanaka et al., 1993
; Kusano et al., 1996
; Colland et al., 2000
; Bordes et al., 2000
), indicating that there is some overlap in promoter recognition by the two sigma factors. However, despite the similarities in promoter recognition in vitro, the two sigma factors are normally able to distinguish in vivo between
D- and
S-dependent promoters. No significant differences were found in the consensus sequence of the
D- and
S- dependent 35 elements (Becker & Hengge-Aronis, 2001
; Lee & Gralla, 2001
) except that the 35 region in
S promoters can be more degenerate than in
D promoters (Gaal et al., 2001
), suggesting that E
S interacts weakly or not at all with the 35 element. In vitro selection of an optimized
S promoter ended with identical consensus elements that agree with those of
D-dependent promoters, both in the 10 and 35 positions (Gaal et al., 2001
). However, a compilation of 41
S-dependent promoters has led to the consensus CTACACT at positions 13 to 7 (Lee & Gralla, 2001
) and another compilation of 56 promoters reached the consensus TG(n)02CYATACT (Lacour et al., 2003
). These studies have revealed that over 80 % of the natural
S-controlled promoters possess a cytosine at the 13 position (Espinosa-Urgel et al., 1996
; Becker & Hengge-Aronis, 2001
).
The PHO regulon of E. coli consists of more than 40 genes and operons whose transcription is induced under conditions of inorganic phosphate (Pi) starvation and that are related to the uptake and assimilation of Pi and phosphorylated compounds. The best characterized ones are phoA, phoE, the pst operon and the ugp operon, which encode, respectively, alkaline phosphatase (AP), the anion porin PhoE, the Pi transporter Pst and the glycerol-3-phosphate transporter Ugp. Apart from its role as a Pi-transporter, the Pst system also functions as a negative regulator of the PHO regulon, because most mutations in the pst operon lead to the constitutive synthesis of all PHO genes (Wanner, 1996). The promoters of the PHO genes display one or more consensus regulatory sequences known as PHO-boxes that replace the 35 element. Transcription is regulated by a two-component system that is composed of the proteins PhoB and PhoR. When the concentration of Pi in the medium decreases below a certain level, the sensor protein PhoR auto-phosphorylates and transfers the Pi group to the regulatory protein PhoB, which in turn binds to the PHO-boxes and allows transcription of the PHO genes by interacting with E
D (Wanner, 1996
; Makino et al., 1996
).
In preliminary experiments we have noticed that in rpoS mutants the expression of AP was considerably stronger than in the wild-type strain, implying that S is involved in the regulation of AP. Here we demonstrate that
S negatively affects the expression of phoA, phoB, phoE and ugpB, but not pstS. The competition between
S and
D for the core RNA polymerase is proposed to explain this differential effect of
S on the expression of the PHO genes.
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METHODS |
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PCR amplifications.
The rpoS fragment was amplified using genomic DNA extracted from strain MG1655 as template and the oligonucleotides rpoS+ (ATACTGCAGGCAGCAAAGGACAGG) and rpoS (CGTCGCGGCTGAAGCTTACAACAC). Bold letters indicate restriction sites. The DNA fragments used as probes for phoA, phoB, phoE, pstS and ugpB were amplified as above using the oligonucleotides phoA+ (CAGCATTCCTGCAGACGATAC) and phoA (GATCAAGCTTAATGTATTTGTACATGGAGAA), phoB+ (TCAAACACCTCAAGCGCGAG) and phoB (GCTCCAGTGCTTTACGCA), phoE+ (ACCTGGGGGCGTTGTATGAC) and phoE (TTGGTGCGATCTGAGTTGGTAT), pstS+ (CTTCCCTGCGCCGTGTATGC) and pstS (TCAGCGGAGATCAGTTTGGTGTT) and ugpB+ (GACGCGGTGCTGGAGTTCAATA) and ugpB (CCGCCCCTGGGTTTTTCTCATA), respectively.
Plasmid construction.
Plasmids pNP1 and pNP5 were constructed by digesting the rpoS PCR fragment with PstI and HindIII followed by ligation to the same sites of plasmids pKK223-3 and pACT3, respectively. Plasmid pBS11 was constructed by digesting a pst PCR fragment with DraI and BstYI followed by ligation to pKK232-8 digested with SmaI and BamHI.
Enzyme assays.
AP was assayed using p-nitrophenyl-phosphate (p-NPP) as substrate as described by Spira et al. (1995). AP-specific activity is represented by the increase in absorbance at 410 nm min1 (cell density)1. Catalase activity was measured qualitatively by mixing 50 µl cells (OD540=3·0) with 50 µl 3 % hydrogen peroxide and observing the appearance of bubbles caused by the release of O2. CAT assays were performed essentially as described by Shaw (1975)
. Cells were disrupted by sonication and protein concentration was determined by the method of Bradford (1976)
. The substrate was 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) together with acetyl-CoA and chloramphenicol in a total volume of 500 µl. The reaction was started by adding chloramphenicol at a final concentration of 0·1 mM to a cuvette containing 0·4 mg DTNB, 0·5 mM acetyl-CoA and cell extract. The absorbance increase rate at 412 nm was recorded. CAT activity was calculated as nmoles min1 (mg protein)1.
RNA extraction and Northern-blot analysis.
RNA was extracted by the guanidine thiocyanate method, as described by Chomczynski & Sacchi (1987). RNA (20 µg) was electrophoresed in a 1 % agarose gel containing 7 % formaldehyde for 3 h. The RNA was transferred to a nylon membrane by capillary action. Probes for phoA, phoB, phoE, pstS and ugpB were synthesized with [
-32P]dCTP by random primer labelling using the DNA fragments obtained by PCR, as described above. For synthesis of the rpoD probe, a 1·5 kb fragment digested from plasmid pRPOD with BamHI and SacI was used. The labelled probes were hybridized with the membranes at 42 °C for 1620 h and the membranes were exposed to X-ray films.
Site-directed mutagenesis and DNA sequencing.
Site-directed mutagenesis was performed by the circular mutagenesis method, using double stranded DNA templates and selection with DpnI, as described by Sambrook & Russell (2001). Plasmid pBS11, carrying a pstScat fusion, was used as a template for the PCR reaction. The oligonucleotides pstSmut+ (CTGTCACCTGTTTGTCTTATTTTGCTTCTCGTAGCCAACAAAC) and pstSmut (GTTTGTTGGCTAGGAGCAAAATAAGACAAACAGGTGACAG) contain the desired mutation (underlined). The product of the amplification was treated with DpnI and transformed into strains MG1655 and BS16. Both wild-type and mutated plasmids were sequenced in an automatic sequencer type ABI Prism 3100 Genetic Analyser (Applied Biosystems/Hitachi) to confirm the presence of the point mutation.
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RESULTS |
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DISCUSSION |
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In E. coli, S and the alarmone guanosine tetraphosphate (ppGpp) are the key factors that promote the transition from growth proliferation to stasis, where proteins related to protection against the deleterious effects of oxidation are expressed. This led to the suggestion that there is a trade-off between bacterial survival and proliferation, such that the expression of genes encoding proteins involved in cell growth is inhibited by factors that promote survival and vice-versa (Nyström, 2003
). The PHO regulon genes, whose function is to acquire and assimilate Pi in order to restore cell growth, are inhibited by
S whose main concern is with the expression of genes related to cell survival. The competition between
S and
D for the core RNA polymerase inhibits the
D-transcribed PHO genes either directly or indirectly via the
S-promoted inhibition of the positive regulator PhoB.
Unlike all other PHO genes tested, pstS was somewhat stimulated by the induction of S. In addition to its role in Pi transport the pst operon also serves as a negative regulator of the PHO genes (Wanner, 1996
). Moreover, its promoter carries a feature shared by many
S-promoters (Hengge-Aronis, 2000
), namely, the presence of a functional IHF binding site that helps elevate its expression and thereby reduce the expression of AP (Spira & Yagil, 1999
). The pst promoter is the only one of eight known PHO promoters that possesses a cytosine residue at the 13 position (Fig. 5
). Our results suggest that pst may be transcribed in vivo by both E
D and E
S, and that the other PHO genes are transcribed only by E
D. Is there a teleological reason for the differential behaviour of pstS in relation to the other PHO genes? Being a negative regulator of PHO, an increase in Pst expression would reduce the transcription of the other PHO genes that are driven by
D, thereby providing more RNA polymerase core enzyme to interact with
S. As a result,
S-dependent genes that are important to bacterial survival during stress could be more readily transcribed. In such a trade-off way, the controlled repression of the PHO genes by Pst might be beneficial for cell survival during prolonged Pi starvation periods. The negative effect of rpoS on gene expression as a result of
S competition against
D was already reported for other
D-transcribed systems. These include the glucose transport-related genes mal and mgl (Notley-McRobb et al., 2002
), the type 1 fimbrial genes fimA and fimB (Dove et al., 1997
), ompF (Pratt et al., 1996
), the stress-induced gene uspA (Farewell et al., 1998
) and several other genes that were found to be hyperexpressed in the absence of a functional
S (Xu & Johnson, 1995
; Farewell et al., 1998
).
Ruiz & Silhavy (2003) have recently shown that in a pstS mutant that causes PHO constitutivity
S is already strongly expressed in the exponential growth phase. The results shown in Fig. 3(b)
, where
S-dependent catalase activity was induced only upon entry into the stationary phase, suggest that even if
S is expressed at high levels in the exponential phase in PHO-constitutive mutants, it is not able to induce the synthesis of
S-dependent promoters. Also, there was no significant difference in the level of AP between the wild-type and the rpoS mutant during the exponential phase (Fig. 3a
). Kvint et al. (2000)
have demonstrated that
S-dependent promoters require ppGpp for induction in the stationary phase, but PHO-constitutive mutants present a low level of ppGpp in the exponential phase of growth (Spira et al., 1995
). Thus, if
S is present in the exponential phase, it is probably inactive.
In conclusion, we have shown that S negatively affects the expression of several PHO genes, but not that of the pst operon. We suggest that this effect is due to a competition between
S and
D for the core RNA polymerase. Since the PHO genes are transcribed by E
D, accumulation of
S in the cytosol during the starvation phase reduces their transcription. In contrast, pst, which is also a negative regulator of PHO, may be transcribed by both
S and
D. Through this mechanism the PHO regulon has evolved to maintain a trade-off balance between cell nutrition and cell survival during severe Pi-starvation stress.
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ACKNOWLEDGEMENTS |
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Received 27 February 2004;
revised 31 May 2004;
accepted 14 June 2004.
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