Molecular Microbiology (1989) 3(11), 1521-1531

Characterization of the osmoregulated Escherichia coli proU 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 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 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 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 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 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 (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. 2A, lane 12 12 1). The anti-GBBP serum specifically detected the glycine betaine-binding protein in whole-cell extracts and peri- Fig. 2. Immunological detection of the GBBP and ProV proteins. Proteins were separated by SDS-PAGE, transferred to sheets of plasmic shock fluids of osmotically induced cultures of the nitrocellulose and probed with antiserum against GBBP (A) or the proU^ strain EF038 (Fig. 2A, lanes 2, 5, and 6). GBBP was ProV-p-galactosidase hybrid protein (C). Autoradiographs (8 and D) absent from extracts of the MprotJ)600 strain EF047 were obtained from the Western blots of radiolabelled protein of minicells. (pOS7) and from uninduced cultures of strain EF038 {Fig. A. purified ProV-p-galactosidase hybrid protein (1), purified GBBP (2), 2A, lanes 3 and 4). Thus, GBBP is not immunologically whole-cell extracts from strains: EF047(pOS7) grown tn MMA with 0.3M related to the ProV-p-galactosidase hybrid protein. We NaC! (3), EF038 grown in MMA (4), EF038 grown in MMA with 0.3M NaCI (5); periplasmic fraction from EF038 grown in MMA with 0.3 M NaCI also tested the reaction of extracts from minicells carrying (6); extracts from minjceils radiolabelled in the presence ot 0.3 M NaCI pOS49 (proV^ proW* proX*) or pOS13 {proV^ proW carrying plasmid pOS13 (7) or pOS49 (8). The solid triangle indicates the position of GBBP, and the asterisk marks the position of the presumed proX-tacZ) (Fig. IA) with the anti-GBBP serum. GBBP GBBP precursor. could be detected in minicells carrying pOS49 but not in B. Autoradiograph of lanes 7 and 8 from the Western blot shown in (A). those harbouring pOS13 {Fig. 2A. lanes 7 and 8), as The open triangle marks the position of the ProW protein; the arrowhead expected from the genetic organization of the proU indicates the position ot the pOS13-encoded hybrid protein. C. Lanes 1 to 5 as in (A); cytoplasmic fraction ot EF038 grown in MMA operon {Faatz efa/., 1988; Dattananda and Gowrishankar, with 0,3 M NaCi (6); inner membrane fractton of EF038 grown in MMA 1989; Gowrishankar, 1989). The product of prolV did not with 0.3 M NaCt (7): lanes 8 and 9 as lanes 7 and 8 in (A). The solid triangle marks the position of the ProV protein; hybrid proteins are react with the anti-GBBP serum {compare Fig. 2A, lane 7 indicated by arrowheads. with Fig. 2B, lane 1). The ProW protein migrates close to D. Autoradiograph ot lanes 8 and 9 from the Western blot shown in (C). the position of GBBP in our gel system (Fig. 2B). The symbols are as in (B).

Plasmid pOS7 carries the reguiatory sequences required for osmotic control of proU expression grown at high osmolarity or from the proW^ strain EF038 grown at low osmolarity, whereas a reacting band was Expression of the O{proi7-/acZ)hyb2 protein fusion detected in extracts of the latter strain when grown at high carried by plasmid pOS7 (Fig. 1) is osmotically controlled osmolarity. This band corresponds to a protein with an and results in the synthesis of a large amount of a 150kD apparent molecular weight of 44 kD (Fig. 2C, lanes 3 to 5). hybrid protein, which led to the suggestion that the When cells of strain EF038. grown at high osmolarity, were sequences required in cis for the osmoresponsive ex- fractionated the 44 kD protein was detected in both the pression of proU are present on pOS7 {May ef ai, 1986). cytoplasmic and inner membrane fractions (Fig. 2C, lanes However, Ramirez ef ai {1989) reported that the ex- 6 and 7). This protein was aiso found in minicells carrying pression of the hybrid gene (monitored by p-galactosi- either the proU"" plasmid pOS49 {see Fig. 1) or the proV^ dase assays) carried on a multicopy plasmid increases proW* plasmid pOSI 3 {Fig. 2C, lanes 8 and 9). The newly strongly in cultures grown at moderate osmolarity and synthesized pOS49- and pOS13-encoded proteins in the drops sharply when the osmolarity of the growth medium 1524 G.May etal.

A when the pOS7-encoded proU-lacZ fusion was recombined into a X specialized transducing phage and mM NaCI 0 50 TOO 200 300 integrated as a single copy at attB, the osmotically controlled expression of this fusion was identical to that of the original fusion present in the proU operon (data not shown). We therefore conclude that pOS7 carries the regulatory sequences required in cis for the correct osmotic regulation of proU.

Determination of the proU transcriptional start point A computer-aided search for DNA sequences with hom- ology to the of E. coti promoters recognized by the RNA polymerase-670 holoenzyme (Harley and Reynolds, 1987) revealed several possible promoter sequences in the region 5' to the translational 1 - initiation site of prol/at the position 688bp (Fig. 4). The 6- sequence with the highest homology score (54%) accord- ing to the algorithm of Mulligan et at. (1984) is located between position 593 and 620bp. Within this region, a possible -35 and -10 region, separated by 16bp, canbe recognized (Fig. 4). To determine whether this is the proU 3 - promoter, we mapped the transcriptional start site by the o 2 SI nuclease protection assay as described by Aldea et at. £ 1 (1988). As the template for mRNA synthesis, we used plasmid pJL19, a derivative of pUC18 into which a DNA fragment (position 3 to 833 bp) from the proU operon had 50 100 200 300 been cioned. This DNA segment carries the complete NaCI concentrotion [mM) proU regulatory region and the beginning of the proV structural gene. Total RNA was isolated from cultures of Fig. 3. Osmotically controlled synthesis and enzymatic activity of the ProV-p-galactosidase hybrid protein. Strain MC4100(pOS7) was grown the A(proL/)600 strain EF027(pJL19), hybridized against ovemight in glucose MMA with the indicated NaCI concentrations. a radiolabelled, single-stranded proU DNA probe, and A. The cultures were used to prepare total cellular extracts to visualize digested with SI nuclease. Only one set of DNA frag- the hybrid protein (indicated by an arrow) on a 7% polyacrylamide gel. Only the upper part of the Coomassie-Biue-stained gel is shown. ments, differing in size by one base, was protected by the B. Specific p-galactosidase activity was determined in cells from the proU mRNA (Fig. 5). This positions the transcriptional same cultures. initiation site(s) between position 622 and 627 bp (Fig. 5), but the limited resolution of our SI mapping experiment is increased further. This is in contrast to the steady precludes pin-pointing the exact transcriptional start point increase in gene expression when the same fusion is within this region. The number and position of the pro- present as a singie copy in the £ co//chromosome (Barron tected fragments were the same when the mRNA was etai, 1986; May etai, 1986)orwhen the copy number of isolated from cells grown at low- or high osmolarity, the plasmid is lowered (Ramirez et ai, 1989). The data demonstrating that no alternative promoter is used when shown in Fig. 3 demonstrate that the aberrant pattern of the expression of proU is induced. As expected from the p-galactosidase activity found in cells carrying pOS7 is an known regulatory pattern of proL/expression, more proU artefact. The amount of hybrid protein synthesized is not mRNA is synthesized when the cells are grown at high correlated with the measured p-galactosidase activity osmolarity (Fig. 5; compare lanes 1 and 2). Synthesis of the when larger amounts of the hybrid protein are made. This ProU system is reduced when the high-osmolarity growth indicates that most of the overproduced hybrid protein is medium is supplemented with 1 mM glycine betaine present in the cell in an enzymatically inactive form. (Barron etai, 1986; May efa/., 1986), and this is reflected Synthesis of the hybrid protein follows the osmotically by the amount of proU mRNA made under these con- modulated pattern of gene expression extensively docu- ditions (Fig. 5; compare lanes 2 and 3). Taken together, the mented for gene fusions to the proU locus (Gowrishankar, results of these experiments provide good evidence that 1985; Cairney etai, 1985b; Dunlap and Csonka. 1985; the -35 and -10 regions indicated in Fig. 4 define the in Barron et ai. 1986; Gutierrez et al., 1987). Furthermore, vivo proU promoter. Characterization of the proU promoter of Esoherichia coli 1525

I 50 GATATCTCTGGGACAACGTGAAGAGTTGAAGAGTTTCGCCTTCGATTTGTTGCTGGAACTCTACG 100 ACAACGAGTTGCAATACACCGATGAGCTGTACGCCGAAACCCCGTGGGCTGACGATGTGAAAGCG 150 TTTCTCTGTTACAACGCCAATAAGGCTTTGATGAATCTGGGCTACGAACCGTTATTTCCCGCAGA 200 250 AATGGCGGAXGTGAATCCGGCAATCCTCGCCGCGCTTTCGCCGAATGCCGATGASAATCACGATT 300 TCTTTTCCGGTTCAGGCTCCTCTTATGTGATGGGGAAAGCGGTTGAAACAGAAGATGAAGACTGG 350 AATTTCTGAGGGTGTTATTTTCAAAAATATCACTACCCGCAGCAGGGAAATAATTCCCGCCAAAT 400 450 AGCTTTTTATCACGCAAATAATTTGTGGTGATCTACACTGATACTCTGTTGCATTATTCGCCTGA 500 AACCACAATATTCAGGCGTTTTTTCGCTATCTTTGACAAAAAATATCAACTTTCTCGATTTGCTC 550 TCAGCCCTTATATCACGGGAAATTCCGGCGATTTGCTCGCATCAATATTCATGCCACATTTGCCA -35 600 -10 650 TCAGGGGTTGCCTCAGATTCTCAGTATGTTAGGGTAGAAAAAAGTGACTATTTCCATTGGGTAAT Fig. 4. Nudeotide sequence of the proU regulatory region. The -10 and -35 regions of the proU promoter are underlined, as well as the 688 putative ribosome binding site (r.b.s.) for proV. ATATCGACATAGACAAATAAAGGAATCTTTCTATTGCATG... The alteration in the -10 region caused by the i^-^B. Met. . . proU601 mutation is indicated by an arrow. 1

Characterization of a as-acting proU regutafory measuring the uptake of glycine betaine in cells grown in mutation glucose MMA with 75 mM NaCI. The control strains EF047 Strain BRE2074 carries a mutation, proU601. that in- iproPproU) and EF038(proPproL/^) showed no (EF047), creases the expression of the (proU-/acZ)hyb2 fusion or only weak (EF038), uptake of radiolabelled substrate both at high- and low osmolarity relative to the parental under these conditions (Fig. 6). Strain GM238, whioh strain GM37 (Table 1). This mutation is closely linked to. showed wild-type regulation of the proU-lacZ fusion, but separable from, the lac fusion present in strain displayed a much higher rate of glycine betaine uptake BRE2074 when the hybrid gene is transduced with phage than the proU^ strain EF038 (Fig. 6). In contrast, in strain PI into strain MC4100 (Higgins et ai. 1988a). To test the GM239, which showed increased expression of the proU- effect of the proU601 mutation on the expression of the /acZ fusion, the rate of uptake was similar to that of the proU operon, we isolated from strain BRE2074 a wild-type strain, EF038 (Fig. 6). From the data shown in specialized X transducing phage that carried the entire Table 1 and Fig. 6 it is clear that the proU601 mutation is proU-tacZ fusion and the proU601 mutation (see the c/s-acting and affects the proU* operon as weil as the Experimentat procedures). This phage, XpGM22, was hybrid gene used to identify the mutation. then used to isolate lysogens in the proP proU^ strain, To characterize this mutation further, we cloned it EF038. Integration of XpGM22 into the chromosome can together with the *(proty-/acZ)hyb2 fusion from \pGM22 only occur by homologous recombination between the into the tacZ fusion vector, pMLB524 (see the Experi- proUsequences present in the chromosome of EF038 and mentat procedures) and obtained plasmid pOSI 01. When on the \ phage, and this recombination yields a proW^/ grown at low osmolarity, strain MC4100(pOS101) ex- ^{proU-tacZ)hyb2 merodiploid. Depending on the point of pressed the fusion more strongly than strain MC4100 recombination, the proU601 mutation will either be (pOS7), which carries the same (J>(proi-/-/acZ)hyb2 fusion crossed into the proU^ locus or will remain linked to the but not the proU601 mutation (Table 1); thus pOSIOI praL/-/acZfusion. Two classes of \pGM22 lysogens with confers the ProU601 phenotype. At high osmolarity, the different patterns of osmoregulated expression of the tac (i-galactosidase activities of the plasmid-carrying strains fusion were found: one class, represented by strain (Table 1) were not reliable indicators of expression of the GM238, showed a pattern of expression of the fusion (^(p^oL/-/acZ)hyb2 fusion (see Fig. 3). These growth con- similar to that of the wild-type strain, GM37; the second ditions led to much larger amounts of the hybrid protein in class, represented by strain GM239, showed the regula- total cellular extracts of MC4100(pOS101) than in those of tory pattern of the proU601 strain, BRE2074 (Table 1). The MC4100(pOS7), as determined by SDS-PAGE (data not effect of the proU601 mutation on the expression of the shown). Restriction anaiysis revealed no structural differ- proU operon in strains GM238 and GM239 was tested by ences between the mutated (I>(p/'oL/-/acZ)hyb2 fusion 1526 G A/fayetal.

3' ACGT123i present on pOSIOI and the wild-type fusion carried by pOS7. The proUeOI mutation presumably affects trans- cription or translation of the protj operon. To localize the

tf-T proU601 alteration, we cloned twoXmnl-XmnI fragments (position 33 to 376 bp, and position 377 to 785 bp) carrying the 5' end of the proV gene and the proU promoter into vector M13mp19 and determined the DNA sequences of -10 these regions. The mutant differed from the wild-type sequence only at position 619bp, where a G-C base pair was changed to an A-T base pair {Fig. 4). This point mutation is located in the -10 region of the promoter we • 1 proposed for proU, and it increases the homology of this sequence to the consensus sequence of E. coti promoters (Harley and Reynolds, 1987). The presence of pOSI 01 is apparently deleterious to the cell, since derivatives of MC4100(pOS101) were fre- quently found that had acquired secondary mutations on the plasmid and expressed the proU-lacZ fusion at a strongly reduced level both at low- and high osmolarity. An example of such a strain is MC4100(pOS102) (Table 1). Fig, 5. Determination of the prot/transcriptional start point by SI Restriction analysis of pOS102 revealed no difference in nuclease mapping. Total RNA was extracted from the HproU)600 strain EF027(pJLi9) (lanes 1, 2, 3) and EF027(pUC18) (lane 4). Strains were the structure between this plasmid and its parental grown in glucose MMA (lane 1), glucose MMA with 464 mM sucrose plasmid, pOSIOI (data not shown). (lanes 2 and 4), and glucose MMA with 464mM sucrose and 1 mM glycine betaine (lane 3). The M13mp1S recombinant phage used as a template for the synthesis of the radiolabelled proU DNA probe was sequenced according to Sanger etal. (1977) and the sequencing Discussion reactions (A, C, G, TI were electrophoresed on the same gel. The sizes of the DNA fragments protected from SI digestion can be determined In E. coti and S. typhimurium. the efficient uptake of the directly by comparing them with the sequencing ladder {Aldea et al.. osmoprotectant glycine betaine is mediated by the high- 1988). A partial sequence of the coding strand, including the -10 promoter region, is shown on the left; the 3' ends ol the protected affinity binding-protein-dependent transport system en- fragments are indicated by solid triangles. coded by the proU locus (Cairney efa/., 1985b; May efa/., 1986; Higgins ef at., 1987a). Our DNA sequence analysis of part of this operon confirms the findings of Gowri- shankar {1989) regarding the genetic organization of protJ.

Table 1. The effect of the praU60/ mutation on the osmoregulated expression of the 'PiproU- Specific |l-galactosidase lacZ)hyb2 fusion. activity of cells grown rn

Strain Description" MMA MMA + NaCI

Chromosomal fusions GM37 (proU-/acZ)hyb2 0.03 4.94 BRE2074 (pfOU-/acZ)hyb2 proU601 0.37 6.04 GM238 proueo r/<^(p^ot;-/acZlhyb2 0.05 5.52 GM239 proW /<\>lproU-lacZ)hyb2 proueoi 0.36 8.23 Plasmid-encoded fusions MC4100 pMLB524 0 0 MC4100 pOS7 1.56 15,12 MC4100 pOSlOl 5,?7 11.41 MC4100 pOS102 (1 ;'s 0.35

The strains were grown in giucose MMA without or with 0.3 M NaCI (for chromosomal fusions), of with 0.1 M NaCI (for plasmid-enceded fusions). The jl-galactosidase activities shown are the mean values of two independent experiments and are expressed as (XTIOI min ' mg protetn '. a. Strains GM37 and BRE2074 carry no functional proL/operon since the integration of the Xp/acMuiS phage used to isolate the proU-lacZ protein fusion destroys the integrity of this operon (May et al, 1986). The merodiploid strains GM238 and GM239 are derivatives of EF038 iproPproU') in which the xpGM22 transducing phage was integrated by homologous recombination at the proW locus. The ptasmids pOS7, pOSlOl and pOS102 carry the 4'(proL/-/acZ}hyb2 fusion. Characterization of the proU promoter of Escherichia coli 1527

the proU promoter is located 67 bp upstream of the transcriptional initiation codon for proV. The -35 (TTGCCT) and -10 (TAGGGT} sequences, which are o separated by 16bp, show homologies to the consensus sequences of E coti promoters recognized by the RNA- c 1,0- polymerase-fi7o holoenzyme (Harley and Reynolds, 1987; Mulligan ef at., 1984). However, the Pribnow box is unusual since it contains three consecutive G-C base pairs. Among the 263 promoter sequences compiled by Harley and Reynolds (1987) there is no -10 region with such a string of G-C base pairs. At low osmolarity. proU is only weakly expressed, but high osmolarity leads to a strong increase in proU transcription. It seems likely that the unusual DNA sequence of the proU -10 region contributes to the low basal level of expression, possibly 60 90 because it requires a higher energy for strand separation time [sec] and open complex formation. Consistent with this view, we found that the proU601 mutation, which increases Fig. 6. Influence of the proU601 mutation on glycine betaine uptake. The protJ expression but does not affect its osmotic regula- initial rate of glycine betaine uptake was measured in cells from exponentially growing cultures in glucose MMA with 75mM NaCI. The tion, is a G-C to A-T transition in the -10 (TAGGAT) region assay medium contained glycine betaine at a final substrate of the promoter. This mutation increases the homology of concentration of 7jiM and 300mM NaCI to ensure the optimal functioning of the ProU system (Faatz et al., 1988), Strains assayed for the proU -10 region to the consensus Pribnow box glycine betaine uptake were: EF047(prDPpraU) (A); EF038(proPpraC/') (TATAAJ). raising the homology score for the proU pro- (•); GM239(p^oPproL/•/'^(proL/-/acZ)hyb2 proUBOl) (O) and GM238 moter region from 54% to 59% (Mulligan ef ai, 1984). It should also facilitate strand separation because the free energy required for the transition from double helix to Our earlier suggestion (Faatz et ai, 1988) that the first single strands is increased (SG =+1.3 kcal mol ^) for the gene. proV, of this operon encodes GBBP is no longer mutated -10 region. This is consistent with the findings of tenable. The immunological data we present here show Margalit et at. (1988), who have shown that 80% of 'up' that the proV gene product is a 44kD protein partly mutations in the -10 region are correlated with increased associated with the cytoplasmic membrane. Furthermore, free energy. ProV shows strong homologies (Gowrishankar, 1989) to One important result of our work is the finding that the the presumed energy-coupling components of other bind- transcriptional start site(s) of proU is not shifted when ing-protein-dependent transport systems, which are typi- expression of the operon is induced by high medium cally also associated with the cytoplasmic membrane osmolarity. This demonstrates that no alternative pro- (Ames. 1986; Higgins efa/., 1988b; Gallagher efa/., 1989), moter is activated under inducing conditions. For these These data suggest that ProV serves the energy-coupling experiments, we used a multicopy plasmid carrying the function in the ProU transport system. In contrast to the proU regulatory region. We found that the osmotically results presented by Barron etat. (1987), our experiments modulated expression of the (proU-/acZ)hyb2 protein using antisera raised against GBBP and the

Table 2. Bacteria, bacteriophages and plasmids. Strain Description^ Reference/ongin

Strains derived from £ co//K12 MC4100 F S{argF-lac)Ut69, araD139. VSL150, Casadaban (1976) deoCI, relAI. ptsF25. flbB5501, rbsR TGI Mlac-pro). supE. thi. hsd5/f traD36, Carter ef a/, (1985) proA'B- lacPlacZ^M^5 HB290 minB. rpsL. mgl G. Hazelbauer through K. Heller GM37 MC4100 *(proL/-/acZ)hyb2. (Xp/acMu15) May ef a/. (1986) GM238 EF038 proU601/>l'{proU-lacZ)Uyb2 (XpGM22) This study GM239 EF038 proU' /•.t'lproU-lacZ)hy\:)2, proU601 This study (XpGM22) BRE2074 GM37 proU601 Higgins efaf. (1988a) eF027 MC4100A(pfoL/)600 This study EF038 MC4WQMpiitPA)tO1. proPf May ef a/. (1986) EF047 MC4100 \[putPA)tO-i, proPI. ^^proU)600 May ef a/, (1986) EF086 HB290(proU-/acZ)hyb2,(Xp/acMij15) This study Bacteriophage XpGM22 Lac' specialized transducing phage This study carrying the 'I'(pfoL/-/acZ)hyb2 and the proU601 mutation

Plasmids PMLB524 Cloning vector for facZ fusions; titi* Silhavy sfaf. (1984) pUC18 Ctoning vector, bla* Norrander af al. (1983) pOS7 (praL/-tecZ)hyb2; bta^ May era/. (1986) pHG329 Expression vector with lacPO. bla' Stewart ef a/. (1986) pOS13 *(pTOU-/acZ)hyb11; b/a' Faatz ef a/. (1988) pOS25 , proW derivativeofpBR322; b/a *^ Faatz ef a/. (1988) pOS40 Derivative of pHG329 carrying the This study 3' part of the proU operon under lacPO control; bla' pOS49 proW deletion derivative of pOS25 This study pOSIOI iproU-lacZ)hyb2 proU60t: bla' This study pOS102 pOSIOI proU602: bla' This study

a. The symbol 4" indicates the presence of a (acZfusion, and !he abbreviation hyb indicates that the gene fusion encodes a hybrid protein. The AptacMu15 prophage in strains GM37, BRE2074, and EF086 carries the kanamycin resistance gene kan. This gene is also present on phage \pGM22, which is denved from the prophage in strain BRE2074.

Experimental procedures , bacteriophage (Silhavy ef al., 1984). A LacZ' specialized trans- ducing phage, XpGM22, carrying the (pfoU-/acZ)hyb2; Ap/acMul5) and subsequent selection for plates spread with 0.1 ml of a lOmg ml ' solution of 5-bromo-4- Km** transductants: this resulted in strain EF086. chioro-3-indolyl-(i-D-galactoside (KG) in dimethylformamide were used to distinguish between LacZ and LacZ' phenotypes. Kanamycin (Km) and ampicillin (Ap) were added to media at 30 fig ml ^ and 50ji.g ml \ respectively. Transport assays

Genetic procedures and construction of bacteriat strains Glycine betaine uptake was measured as described by May etai. (1986) using methyl-['"CJ-glycine betaine (7,1 mCi mmol '. Standard techniques were used tor the purification and growth of Amersham). Characterization ofthe proU promoter o^ Escherichia coli 1529

Methods used with nucleic acids and construction of pOS7 resulted in strong overproduction of the ProV-p-galacto- piasmids sidase hybrid protein. This hybrid protein sedimented in large amounts with the membrane fraction when French-pressed cells Isolation of plasmid and phage \ DNA and routine manipulations were centrifuged for 30 min in a Sorvali SS34 rotor at 15000 r.p.m. of nucleic acids were all as described (Maniatis e( al., 1982; The pellet was solubilized using a 2% SDS solution, the proteins Silhavy ef al., 1984). DNA sequencing was performed using a were electrophoretically separated by SDS-PAGE. and the hybrid modification of the dideoxy nucleotide chain-termination method protein was electroeluted from the gel. The protein-containing (Sanger etal., 1977; Biggin etat., 1983). solution was concentrated with Aquacide and dialysed against Plasmid pOSIOI was constructed by cloning an £coRI-EcoRI water containing 0.2% SDS. The purity of the protein was restriction fragment from \pGM22 into the IacZ fusion vector. assessed by SDS-PAGE. pMLB524 (Silhavy ef a/., 1984), as described by May ef a/. (1986). The structure of one of the resulting LacZ' plasmids, pOSI 01, was characterized by restriction analysis. Plasmid pOS40 (Fig. 1) Preparation of GBBP and ProV-(i-galactosidase antisera was constructed by cloning a Sal\-Pvu\\ fragment from the proU* and immunological detection of the antigens plasmid pOS25 (Faatz ef a/., 1988; Fig. 1) into the Sa/I and Smal sites in the polylinker region of pHG329 (Stewart ef al.. 1986), Rabbits were given injections of GBBP or ProV-p-galactosidase allowing the expression of the 3' end of the proUoperon under the proteins (Img and 0.5mg of protein, respectively) in 1ml of control of the lac promoter. To reduce the size of the chromo- BBS-buffer (140mM NaCI, 166mM Tris-borat, pH 8.0) and 1 ml of somal material present on pOS25. DNA 3' to the profoperon was complete Freund's adjuvant. After 14 days, the animals were removed by Sa/31 digestion (Silhavy ef a/.. 1984) and DNA 5'to given booster injections of the same doses of protein suspended the proL/regulatory region was removed by digestion with EcoRV in BBS buffer; 14 days after this, the blood of the animals was and religation. One of the proW^ plasmids obtained is pOS49 collected. For the immunological detection ofthe antigens. E. coti (Fig. 1). proteins were separated by SDS-PAGE and electrophoretically transferred (Towbin etal., 1979) to a sheet of nitrocellulose (pore size. 0.45 Jim; Schleicher & Schuell). The bound proteins were then probed with a crude rabbit antiserum. The antigen-antibody Mapping of the proU transcription initiation site complexes formed were visualized with a second goat anti-rabbit immunoglobulin G peroxidase-coupled antibody using 3-3'- The transcriptional start point of the proU operon was detemnined dimethoxybenzidine (Sigma) as a substrate. according to the protocol of Aldea et ai (1988). We constructed a derivative of M13mp18 with an 839bptcoRI-Sa/l insert carrying the proL/regulatory sequences and the 5' end of proV{F\Q. 1). The proU fragment is inserted into the Ml 3 phage such that the Radiotabelling of proteins in minicells. fractionation of universal priming site within the lac-a region is adjacent to the cellular proteins, and SDS-PAGE Sa/I end of the cloned fragment. This EcoRI-Sa/l fragment was cloned in the same way into the polylinker region of pU018, Minicells were isolated from strain EF086 carrying various plas- resulting in plasmid pJLI 9. RNA was extracted from cells carrying mids. Radiolabeiling of the plasmid-encoded proteins with pJL19 or the control plasmid pUC18, grown under different P^S]-methionine (1000 mCi mmol '; Amersham) in the presence conditions. A single-stranded, radiolabelled proU DNA probe was of 0.3 M NaCI was performed as described by Faatz ef at. (1988). prepared by primer extension using the universal lac primer and The proteins were separated by SDS-PAGE (Laemmli. 1970), and the recombinant M13mp18 phage, described above, as the the radiolabelled polypeptides were visualized by autoradiogra- template. The RNA was hybridized to this probe, DNA-RNA phy using Kodak X-omatic 100 film. Periplasmic proteins were hybrids were digested with SI nuclease, and the sizes of the isolated using the cotd osmotic-shock procedure (Neu and protected fragments were determined on a sequencing gel. Heppel. 1965; May efa/., 1986). and proteins from the inner and outer membrane of E. coti were separated by sucrose gradient density centrifugation, as described by Hengge et al. (1983). Purification of GBBP and the ProV-p-galactosidase hybrid protein (i-gatactosldase assays

Periplasmic proteins were isolated from strain EF047(pOS40) Specific (J-galactosidase activity, expressed as micromoles of grown in glucose MMA using the cold osmotic-shock procedure substrate cleaved per min per mg of protein, was assayed as of Neu and Heppel (1965). As the first step in the purification of described by May et at. (1986). GBBP. we employed ion-exchange chromatography on a DEAE- Sephacel column as described by Barron ef al. (1987). Fractions containing GBBB were pooled, and the proteins were concen- Acknowledgements trated with Aquacide (Serva) and diatysed against a large volume of 16mM Tris-HCI (pH 8.3). The proteins were then loaded onto an We thank K. Heller and K. Bauer for generously providing bacterial FPLC ion-exchange column (Mono-Q; Pharmacia), and the strains and plasmids. and A Middendori for expert technical bound polypeptides were eluted using a linear NaCI gradient assistance. We are grateful to R. Vdgele and A. ReidI for their help (0-250mM NaCI in 16mM Tris-HCI, pH 8.3). This yielded 99% with the computer analysis. We appreciate the help of V. Koogle in pure and functional GBBP, as judged by SDS-PAGE and glycine preparing the manuscript. We thank W. Boos, in whose laboratory betaine-binding assays (May et al., 1986). The osmotically this work was carried out, for helpful discussions. Financial induced expression of the *{praL/-/acZ)hyb2 fusion carried by support for this work was provided by a grant from the Deutsche 1530 G.May etal.

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