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Characterization of the Osmoregulated Escherichia Coli Prou Promoter and Identification of Prov As a Membrane-Associated Protein

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Characterization of the Osmoregulated Escherichia Coli Prou Promoter and Identification of Prov As a Membrane-Associated Protein Molecular Microbiology (1989) 3(11), 1521-1531 Characterization of the osmoregulated Escherichia coli proU promoter and identification of ProV as a membrane-associated protein G. May, E. Faatz, J. M. Lucht, M. Haardt, The prop-encoded transport system has a iow affinity for M. Bolliger^ and E. Bremer* giycine betaine and is present in the cytopiasmic mem- Department of Biotogy. University of Konstanz, PO Box brane (Milner et ai. 1988). The protZ-encoded transport 5560, D'775O Konstanz. FRG. system has a high affinity for glycine betaine and is binding-protein-dependent (May ef ai. 1986; Higgins et ai, 1987a; Barron efa/., 1987;foranoverview, see Ames, Summary 1986). Analyses of the cloned proU region from £ coli The Escherichia coli proU operon encodes a high- have demonstrated that this iocus consists of at ieast affinity, binding-protein-dependent transport system three genes (proV. proW, and proX) that are organized in for the osmoprotectant gtycine betaine. Expression of an operon (Gowrishankar et ai, 1986; Faatz et ai, 1988; proU is osmoregulated, and transcription of this Dattananda and Gowrishankar, 1989). From the recently operon is greatly increased In cells grown at high determined proU DNA sequence {Gowrishankar, 1989), osmoiarity. Characterization of the proU operon and the products of these genes have been deduced. The proV its promoter provrded results similar to those pub- gene encodes a hydrophiiic protein {M, ^ 44162) with lished elsewhere (Gowrishankar, 1989; Stirling et a/., homology to the energy-coupling component of binding- 1989). The previously identified proU601 mutation, protein-dependent transport systems. A hydrophobic which leads to increased prot>expression both at low- polypeptide (M, = 37619) is encoded by proW and is and high osmolarity, is a G to A transition in the thought to be located in the cytoplasmic membrane. The Pribnow box of the proU promoter, which increases proX gene encodes the periplasmic glycine betaine-bind- the homology of the -10 region to the consensus ing protein (GBBP; Mr = 33729). sequence of E. coli promoters. Using an antiserum Expression of the proU operon is strongly stimulated at raised against a ProV-^-gaiactosidase hybrid protein, the level of transcription by increases in medium osmo- we have identified ProV as a protein associated with larity (Gowrishankar, 1985; Cairney etai, 1985b; Dunlap the cytoplasmic membrane. This cellular location is and Csonka, 1985; Barron ef ai, 1986; Gutierrez et al., consistent with its proposed role as the energy- 1987). Uptake of K"^ and the concomitant synthesis of coupling component of the ProU transport system. glutamic acid are the cell's primary adaptive response to an increase in medium osmolarity (Epstein, 1986). The strength of proU transcription appears to be linked to the Introduction intracellular accumulation of K* -gtutamate (Epstein 1986; The Gram-negative bacteria Escherichia coli and Sutherland etai. 1986; Higgins etai, 1987b; Ramirez ef Salmonella typhimurium can adapt to environmental con- ai, 1989). However, the mechanism by which K^-g!uta- ditions of high osmoiarity by a variety of mechanisms, one mate influences proU expression is unclear. One poss- of which is the synthesis or uptake of osmoprotective ibility is that the effect of K^-glutamate is mediated by a organic compounds, the so-called compatible solutes reguiatory protein. Alternatively, accumulation of K*- (see Strom et ai, 1986; Epstein, 1986; Wood, 1988; glutamate could influence the initiation of prot-/transcrip- Csonka, 1989). Glycine betaine is among the most tion directly by modifying RNA polymerase activity, its effective compatible solutes (Le Rudulier et al., 1984). in £ interaction with the proU promoter, or the DNA structure of CO//and S. typhimurium. there are two transport systems the proL/regulatory region. Recent evidence suggests an for this substrate; ProP and ProU (Perroud and Le important role for DNA topology in proU transcription Ruduiier, 1985; Cairney etai, 1985a. b; May etai, 1986). (Higgins et ai, 1988a), but there is no definitive proof that changes in DNA structure mediate the osmotic control of proL/expression. To analyse the ProU system further and determine the Received 9 May, 1989; revised 18 July, 1989. 'Present address: Mikro- mechanism by which osmolarity regulates proL/transcrip- biologisches Institut, ETH Zurich. Schmelzbergstrasse 7, CH-8008 Zurich. Switzerland. "For correspondence. Tel. (7531) 882042. tion, we have sequenced the proU regulatory region and 1522 G.May etal. kb Fig. 1. Physical maps of plasmids used. 1? 13 Restriction maps of plasmids carrying the proU region or prol/-/acZ fusions: only restriction sites Soli relevant for this study are indicated. The BamHI EcoRVSspI Sspp l Ssrf BamHI chromosomat material carried by the various pOS25 I [EcoRV plasmids is shown. The extent of the proU operon and its direction of transcnption are proV indicated by arrows. Chromosomal material 3' to Sdl the proU operon was removed in plasmid pOS49 by Ba/31 digestion, and the deletion endpoint EcoRVSspI Sspl Pvul 5spl EcoRV was determined by restriction analysis with a posw U A .1 H margin of error of ±300bp. A defined deletion using £coRV was then introduced to remove the chromosomal material 5' to proU. Plasmid 5spl Pvul Sspl Pvul pOS40 carries the 3' part of the proUoperon, posw JI I allowing gene expression under lacPO (hatched box) control. Plasmids pOS7 and pOS13 canry tecZ fusions to different genes of the proU operon. Hatched bars represent the '/acZand locY- pOS7 lacY genes; solid bars represent the terminal 117bp from the Send of phage Mu. The zig-zag Soil line indicates the hybrid protein encoded by the EcoRI EcoRV' 'l»(pro(J-/acZ)hyb2 fusion: the ammo- and Pvul JacZ pOS13 carboxy-termini are indicated by N and C, respectively. For plasmid pOS13, the locations of proV prow the first two genes {proVand proW) of the proU operon are indicated. Plasmid pOS13 can-ies some material from phage \ to the left of the fcoRV site; the extent of this matenal is not known. have defined the proU promoter by mutant analysis and G-C base pair at position 1582 bp within pral/was fused to mapping of the transcriptional initiation site. In addition, the 117bp DNA segment from the MuS region, which is we have used an antiserum raised against a ProV- present at the fusion joints of all A.p/acMu-generated tacZ [i-galactosidase hybrid protein to identify the cellular protein fusions (Bremer ef at.. 1984). The reading frames of location of the proV gene product. the proV and the 'tacZ genes are correctly aligned, permitting the synthesis of a large (150kD) ProV- p-galactosidase hybrid protein (May etai, 1986). These Results data provide experimental support for the proV reading i • frame proposed by Gowrishankar (1989). In plasmid Nucleotide sequence of the 5'-region of the proU pOS13, the MuS region was fused to the A-T base pair at operon position 3124bp in proX. the structural gene for GBBP. However, the reading frames of the proX' and '/acZgenes The DNA sequence of the proU regulatory region and the are not properly aligned, resulting in an out-of-frame first two genes, proV and proW, of the proU operon was fusion. This explains why the expression of the <!> {proU- determined using DNA fragments from plasmids pOS7 tacZ) hybi 1 fusion is very low compared to that of proX iproV-lacZ) and pOSI 3(proX-/acZ) (Fig. 1). We have (May etai, 1986; Faatz etai, 1988). established the DNA sequence of a 3124bp segment that begins at the EcoRV site proximal to the proL/operon and extends to the proX-/acZfusion joint in pOS13 (Fig. 1). This Immunotogicat identification of the proVgene product sequence (data not shown) is identical to that recently reported by Gowrishankar (1989) and is therefore not The ProV-p-galactosidase hybrid protein was used to discussed here. We found only one minor difference: the Identify the ProV protein and its cellular location. We G-C base pair at position 40bp in Gowrishankar's se- purified this hybrid protein from the l(proU)600 strain quence is not present in our sequence (see Fig. 4). As a EF047(pOS7) and raised an antiserum against it. In result, the nucieotide positions we use here are one Western blotting experiments, this antiserum reacted with number lower than the corresponding positions in his the purified hybrid protein and with the hybrid protein sequence. We also determined the DNA sequence of the present in whole-celt extracts of strain EF047(pOS7) (Fig. (p (proU-tacZ) hyb2 and <l> (proU-tacZ) hybi 1 fusion joints 2C, lanes 1 and 3). No specific reaction was seen with in plasmids pOS7 and pOS13, respectively. In pOS7, the whole-cell extracts prepared from the ^(proU)600 strain Characterization of the proU promoter of Escherichia coli 1523 mtnioells had been radiolabelled prior to sodium dodecyl sulphate-poiyacrylamide gel eiectrophoresis {SDS-PAGE} and Western blotting; thus a direct correlation could be shown between the immunologically detected bands on the Western blot and the bands detected by autoradio- graphy of this blot {compare Fig. 2C. lanes 8 and 9, with Fig. 2D). The apparent molecular weight of the immuno- logically reacting protein is in agreement with the molecu- lar weight of ProV (M, ^ 44162) that was deduced from the 1 2 3 ^ S 6 7 8 12 proV DNA sequence {Gowrishankar. 1989; this study). Our results, therefore, characterize ProV as a protein associated with the cytoplasmic membrane. We reinvestigated the immunological cross-reaction of the *(prol/-/acZ)hyb2-encoded ProV-p-galactosidase hybrid protein with an antiserum raised against purified GBBP {Barron et ai, 1987). Our ProV-p-galactosidase -ProV antiserum did not react with purified GBBP (Fig. 2C. lane 2), and a rabbit antiserum raised against the purified GBBP ^-GBBP (see the Experimental procedures) did not react with the purified ProV-p-galactosidase hybrid protein {Fig.
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