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Signal transfer through the Uhp regulatory system in the signal transduction network ofEscherichia coli

Verhamme, D.Th.

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Citation for published version (APA):Verhamme, D. T. (2002). Signal transfer through the Uhp regulatory system in the signal transduction network ofEscherichia coli.

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Download date: 13 Jul 2020

ChapterChapter 3

Glucose-6-phosphate-dependentt phosphoryl flow

throughh the Uhp two-component regulatory system

Contents s

AbstractAbstract 46 IntroductionIntroduction 46 MaterialsMaterials & Methods 48

Plasmidd construction 48 Purificationn of Hise-UhpA and His6-UhpB' 48 Isolationn of inside-out membrane (ISO) vesicles 49 Inn vitro phosphorylation reactions 49 Immunodetectionn 50

ResultsResults 51 G6P-inducedd autophosphorylation of UhpB 51 Phosphorylationn and dephosphorylation of UhpA by membrane-bound UhpB 53

DiscussionDiscussion 55 AcknowledgementsAcknowledgements 57

Publishedd in Microbiology (2001) 147: 3345-3352. Daniëll T. Verhamme, Jos C. A rents, Pieter W. Postma, Wim Crielaard & Klaas J.. Hel ling werf

ChapterChapter 3

Abstract t

Expressionn of the UhpT sugar-phosphate transporter in Escherichia coli is regulatedd at the transcriptional level via the UhpABC signalling cascade. Sensing of extracellularr glucose-6-phosphate (G6P), by membrane-bound UhpC, modulates a secondd membrane-bound protein, UhpB, resulting in autophosphorylation of a conservedd histidine residue in the cytoplasmic (transmitter) domain of the latter. Subsequently,, this phosphoryl group is transferred to a conserved aspartate residue in thee response-regulator UhpA, which then initiates uhpT transcription, via binding to thee uhpT promoter region.

Thiss study demonstrates the hypothesised transmembrane signal transfer in an invertedd membrane set-up, i.e. in a suspension of UhpBC-enriched membrane vesicles,, UhpB autophosphorylation is stimulated, in the presence of ([y-32P])ATP, uponn intra-vesicular sensing of G6P by UhpC. Subsequently, upon addition of UhpA, veryy rapid and transient UhpA phosphorylation takes place. When P~UhpA is added too G6P-induced UhpBC-enriched membrane vesicles, rapid UhpA dephosphorylation occurs.. So, in the G6P-activated state, UhpB phosphatase activity dominates over kinasee activity, even in the presence of saturating amounts of G6P. This may imply thatt maximal in vivo P~UhpA levels are low and/or that, to keep sufficient P~UhpA accumulatedd to induce uhpT transcription, the uhpT promoter DNA itself is involved inn stabilisation/sequestration of P~UhpA.

Introductio n n

Everyy living cell has to cope with environmental fluctuations in nutrient concentrationn and/or the stringency of 'stress' signals from the environment. Unicellularr organisms such as bacteria have to be especially well equipped to respond appropriatelyy to their (continuously) changing surroundings. To sense, transduce and correctlyy respond to various signals, prokaryotes predominantly rely on the intracellularr covalent modification of sensory and regulatory proteins via transient phosphorylationn of specific histidine and aspartate residues (Stock et al, 1989; Parkinsonn & Kofoid, 1992; Perraud et al, 1999). The two prerequisite components in suchh a system are a sensory histidine-protein kinase (HPK) and its cognate response-regulatorr (RR). Specificity resides in the input domain, triggering autophosphorylation onn a conserved histidine residue in the transmitter domain of the HPK, upon receiving thee proper stimulus. The high-energy phosphoryl group is subsequently transferred to aa conserved aspartate residue in the receiver domain of the RR. The resulting molecularr switch activates an output domain of the RR, which in most cases modulatess transcription of target gene(s) by (enhanced) binding to specific sequences inn their promoter region. The phosphorylated state of RRs is transient, due to intrinsic

46 6

QuantificationQuantification of phosphorylated TCRS-proteins

instabilityy of the phospho-aspartate bond (autophosphatase activity) as well as phosphatasee activity in the HPK transmitter domain or in additional regulatory protein(s).. Jointly, the HPKs and RRs belong to the family of so-called two-componentt regulatory proteins, which can be clustered on the basis of their highly conservedd transmitter and receiver domains (Ronson et al, 1987). Because of their abundancee and multi-regulatory involvement in bacterial signal transduction, the majorr role of His-Asp phosphoryl transfer, based on the two-component paradigm is beyondd doubt. Generally, the more systems a bacterium possesses [from a few in HaemophilusHaemophilus influenzae and Helicobacter pylori (Grebe & Stock, 1999) to around 60 inn Pseudomonas aeruginosa (Rodrigue et al, 2000) and Caulobacter crescentus (Niermann et al, 2001)], the more flexible a lifestyle it can adopt.

Inn Escherichia coli, 29 HPK/RR pairs have been identified (Mizuno, 1997). One off these is the UhpB-UhpA couple (Weston & Kadner, 1988). UhpB is a membrane-boundd sensor kinase, with the notable feature that it contains eight putative transmembranee helices (Island et al., 1992) instead of four for the KdpD sensor (Zimmannn et al., 1995) and two for the other functionally characterised membrane-boundd HPKs in E. coli (Williams & Stewart, 1999). Furthermore, a second intrinsic membranee protein, UhpC, is required to sense the extracellular stimulus for this system,, the presence of glucose 6-phosphate (G6P) in the periplasm. Subsequently, UhpCC activates UhpB, presumably by inducing a conformational change in the latter (Westonn & Kadner, 1988; Island et al, 1992; Island & Kadner, 1993; Wright et al, 2000).. This uncommon form of transmembrane signalling then activates the typical phosphoryl-transferr reactions inherent to a two-component signal transduction pathway.. The cytoplasmic part of UhpB contains the characteristic HPK/transmitter sequencee motifs, i.e. the H-box, as well as the N- and G-box in the ATP-binding catalyticc domain (for a recent comparison see (Kim & Forst, 2001). Autophosphorylationn of UhpB on the conserved histidine (His-313) may occur in a (homo)dimericc structure, with one kinase monomer catalysing phosphorylation of the otherr (i.e. in trans), as has been shown for other HPKs (Ninfa et al, 1993; Dutta et al, 1999).. The RR UhpA, which is composed of a receiver domain that belongs to the Re subfamilyy of receiver domains (Grebe & Stock, 1999) and an output domain belonging too the FixJ/NarL subfamily (Parkinson & Kofoid, 1992), becomes activated by phosphorylationn on Asp-54. Phosphorylated UhpA exhibits enhanced affinity for specificc target sequences in the uhpT promoter region (Merkel et al, 1992; Dahl et al, 1997),, through which it can stimulate UhpT expression. The UhpT transporter enables E.E. coli to take up a broad range of organo-phosphate compounds from its environment, whichh can be used as carbon and energy source (Dietz, 1976). Nevertheless, UhpT expressionn is exclusively induced by extracellular G6P (Winkler, 1970), because of the specificityy of UhpC (Goldenbaum & Farmer, 1980).

Thee uhpABCT operon has been extensively studied for more than 30 years, with regardd to its characteristics of induction, regulation of uhpT transcription by UhpA,

47 7

ChapterChapter 3

UhpBB and UhpC, and specificity and kinetics of transport by UhpT (for review see Kadner,, 1995). Recently, the interaction of the transmitter domain of UhpB with UhpAA has been described in detail (Wright et aL, 2000; Wright & Kadner, 2001). Heree we demonstrate the G6P-induced transmembrane signal transfer through the UhpBCC complex in an in vitro system, which results in UhpB autophosphorylation andd subsequent phosphoryl transfer to UhpA. We have observed that G6P increases thee kinase/phosphatase ratio in the UhpBC complex, thereby generating phosphorylatedd UhpA. Nevertheless, G6P does not seem to suppress the 'default' phosphatase-onn state of UhpB.

Material ss & Methods

PlasmidPlasmid construction DNAA corresponding to the uhpA open reading frame was amplified by PCR with

primerss UHPA1 (5'-GCGGATCCGATGACGATGACAAAATGATCACCGTTGCC CTTATAG-3')) and UHPA2 (5'-GCGCAAGCTTCACCAGCCATCAAACATGC-3').. DNA encoding the cytoplasmic portion of uhpB was amplified by PCR with primerss UHPB1 (5'-GCGGATCCCTGGCTGAACGGTTGCTGG-3') and UHPB2 (5'-GCGCAAGCTTAGACATAGCGTTGAGGTAG-3').. Pwo DNA polymerase (Boehringerr Mannheim) was used, with E. coli MC4100 chromosomal DNA as template.. PCR products were digested with BamHl and Hindlll and cloned into the correspondingg sites of pQE30 (Qiagen), resulting in expression vectors for full-length UhpAA (pQE30uhpA) and a truncated form of UhpB (residues 293-500, pQE30uhpB'). Bothh recombinant proteins (i.e. UhpA and UhpB') are produced with an amino-terminall His6-extension, under control of a phage T5 promoter regulated by IPTG (for furtherr details see Qiagen brochure). The cloned uhpA and uhpB' fragments were confirmedd by DNA-sequence analysis, using an ABI system (sequence facility, Free Universityy Amsterdam).

PurificationPurification ofHisg-UhpA and Hisg-UhpB' His6-taggedd recombinant proteins were isolated from E. coli M15 cells (Qiagen),

transformedd with pREP4 (Qiagen) and either pQE30uhpA or pQE30uhpB', after growthh in Luria-Bertani medium at 37°C and induction with isopropyl-6-D-thiogalactopyranosidee (IPTG; 1 mM). The proteins were purified by nickel chelate affinityy chromatography using a Ni-NTA resin (Qiagen), according to the QIAexpressionistt manual (Qiagen). Purification under non-denaturing conditions resultedd in a highly concentrated, soluble His6-UhpA fraction, whereas overproduced His6-UhpB'' largely ended up in the insoluble fraction, due to the formation of inclusionn bodies. His6-UhpB' was purified from this fraction under denaturing conditionss with 8 M urea. The resulting (dialysed) product was subsequently only

48 8

QuantificationQuantification of phosphorylated TCRS-proteins

usedd to raise antiserum. Final protein preparations were > 95% pure, as analysed on SDS-PAGE.. Protein concentrations were determined using the Bradford assay (Bio-Rad)) with bovine serum albumin as a standard. Proteins were stored in small aliquots att -20°C.

IsolationIsolation of inside-out membrane (ISO) vesicles Forr preparation of UhpBC-enriched- and UhpBC-lacking- (i.e.'empty')

membranee vesicles, E. coli RK1448 transformed with plasmid pRK18 [uhp(AA)(BQ[uhp(AA)(BQ++ (AT)(AT) in pBR322] and strain RK5000 (AuhpABCT) were used, respectively.. Strains and plasmid (Weston & Kadner, 1988) were kindly provided by R.J.. Kadner (University of Virginia, Charlottesville, USA). Cell cultures were aerobicallyy grown in Luria-Bertani medium at 37°C to an OD600 of ~ 3. Cells were harvestedd by centrifugation (20 min at 5,000 x g) at 4°C, washed once with ice-cold bufferr A (100 mM Tris-HCl pH 7.4, 5 raM EDTA, 10% glycerol), and resuspended in 0.011 volume of buffer A with 1 mM PMSF. The cell suspension was passed through a Frenchh pressure cell twice (12,000 psi; 1 psi = 6.9 kPa). Cell debris was subsequently removedd by centrifugation (twice, 20 min at 20,000 x g). The resulting supernatant wass centrifuged at 180,000 x g for 90 min, to collect the membrane fraction. This membranee pellet was washed and resuspended in buffer B (50 mM HEPES pH 7.8, 2 MM KC1, 1 mM EDTA, 10 mM B-mercaptoethanol, 1 mM PMSF, 10% glycerol) and sedimentedd again (45 min at 180,000 x g). The wash-step was repeated with membranee storage buffer (buffer B without 2 M KC1), after which the pellet was resuspendedd in a small volume (usually 400 u.1 per liter of the original culture). This membranee vesicle fraction was frozen in liquid nitrogen and stored at -80°C in small aliquots.. The protein concentration in these samples was estimated using the Bradford assayy (Bio-Rad), with bovine serum albumin as a standard and UhpB enrichment was confirmedd and quantified by Western blotting, using a polyclonal antiserum raised againstt His6-UhpB'.

InIn vitro phosphorylation reactions Too assay autophosphorylation activity in membranes in which UhpBC was either

enrichedd or absent, samples were incubated with [y-32P]ATP (ICN Biomedicals, stock 70000 Ci mmol"1; 1 Ci = 3.7 x 1010 Bq) in a reaction mixture containing 50 mM Tris-HCll pH 8.0, 5 mM MgCl2, 50 mM KC1, 0.5 mM EDTA, 1 mM DTT, 10% glycerol (andd glucose 6-phosphate). Before initiating the reaction with ATP (usually 50 or 100 \iM,\iM, 10-25 Ci mmol1), the vesicles (with an inverted membrane orientation; Futai & Tanaka,, 1975) were made permeable for glucose 6-phosphate (Boehringer Mannheim) byy freezing the reaction mixture in liquid nitrogen, followed by thawing on ice.

Too assay phosphoryl transfer to UhpA, His6-UhpA was added to the autophosphorylationn mixture (now at pH 7.5) after 2-3 min incubation with [y-32P]ATP. .

49 9

ChapterChapter 3

Too assay phosphatase activity (in UhpBC-enriched membranes) towards P-UhpA,, His6-UhpA was first phosphorylated using acetyl phosphate. Acetyl [32P]phosphatee was enzymically synthesised in AcP buffer (25 mM HEPES, 60 mM KAc,, 10 mM MgCl2, pH 7.6) with 50 \id of [y-32P]ATP per unit of E. coli acetate kinasee (Boehringer Mannheim), for 30 min at room temperature. Acetate kinase was removedd from the acetyl- [32P]phosphate containing mixture by filtration through a Centriconn (10 kDa cut-off filter) microconcentrator (Amicon), including extensive filterr rinsing in AcP buffer. His6-UhpA (5-15 |xM) and acetyl [32P]phosphate were mixed,, and incubated for 2.5 hours at 30°C. Similarly, non-radioactive P~UhpA was obtainedd by incubating 5-15 uM His6-UhpA with 10 mM acetyl phosphate and 10 mM MgCl22 in TEDG buffer (50 mM Tris-HCl pH 7.5, 1 mM DTT, 0.5 mM EDTA, 10% glycerol),, at 30°C for 2.5 hours. In both cases, P~UhpA (free of remaining acetyl phosphate,, ATP and/or ADP) was collected by exchanging the buffer via a Bio-Gel P-66 microcolumn equilibrated in TEDG buffer. The actual phosphatase reaction was initiatedd by adding (G6P-containing) membrane vesicles to the P~UhpA product in 50 mMM Tris-HCl pH 7.5, 5 mM MgCl2, 50 mM KC1, 0.5 mM EDTA, 1 mM DTT and 10%% glycerol.

Thee (de)phosphorylation reactions, carried out at room temperature, were quenchedd by adding 3x SDS-loading buffer (containing 30 mM EDTA), after which thee samples were kept on ice. Before loading they were heated for 3 min at 55°C and thenn separated by SDS-PAGE. Gels were first washed in 25 mM phosphate buffer (pH 7.5),, then in demineralised water and subsequently dried under vacuum. Phosphorylatedd proteins were visualised and quantified by autoradiography (Kodak X-Omatt AR film) and/or phosphorimaging (Bio-Rad). The experiments described weree performed at least three independent times with reproducible outcome; typical exampless are shown in the results.

Immunodetection Immunodetection Forr Western blotting, total cellular protein, membrane-protein fractions and

purifiedd His6-UhpB' or His6-UhpA (5-100 ng) were separated by SDS-PAGE and then transferredd to 0.4 im nitrocellulose membranes using a semi-dry blotting system (Bio-Rad).. Membranes were treated with a polyclonal rabbit antiserum against His6-UhpB' (dilutedd 1:5,000) or His6-UhpA (diluted 1:10,000). Antisera were raised in New Zealandd White rabbits (institute facility). Horseradish peroxidase-conjugated goat anti-rabbitt IgG (Bio-Rad) was used as secondary antibody, followed by treatment with SuperSignall chemiluminescent substrate (Pierce). The secondary antibody signal was detectedd and quantified by exposure of the membranes to film (Kodak X-Omat AR), followedd by densitometric analysis.

50 0

QuantificationQuantification of phosphorylated TCRS-proteins

Results s

G6P-inducedG6P-induced autophosphorylation of UhpB Ass activation of the Uhp system in vivo is known to be dependent on the

presencee of extracellular G6P, we initiated experiments to demonstrate the supposed G6P-dependentt UhpB autophosphorylation in an in vitro system. Preliminary experimentss revealed that this would be extremely difficult in membranes derived fromm wild type strains; therefore UhpBC-enriched membranes were used. Membranes fromm a uhpBC deletion strain served as a control. Furthermore, since a French press wass used to generate the membrane fragments, their orientation was predominantly inside-outt (Futai & Tanaka, 1975). Consequently, the presumed binding site of UhpC forr G6P is located on the inside of these membrane vesicles. A major obstacle in this experimentall design is the intra-vesicular localisation of the G6P binding site. We allowedd G6P access to the intra-vesicular compartment in three alternative ways: (i) viaa French pressing the cells in the presence of G6P; (ii) via quickly freezing the vesicless in liquid nitrogen in the presence of G6P and slowly thawing them on ice; and (iii )) via gentle pre-treatment with a low concentration of a non-ionic detergent (0.1% (v/v)) Triton X-100). All three procedures were effective for G6P concentrations above

1 1

membr.. BC

(a) )

P~UhpB B

xii éÊÊ

G6PP +

Fig.Fig. 1. G6P-induced UhpB autophosphorylation. Reactions were initiated by adding 50 uM [y-,2P]ATPP to ISO membranes in phosphorylation buffer pH 8.0. (a) Samples were quenched after 2 min andd separated by SDS-PAGE (11% gels), followed by autoradiography. Lanes: I, UhpB-lacking membranes,, plus 1 mM G6P; 2, UhpB-enriched membranes with no added G6P; UhpB-enriched membranes,, plus 1 mM G6P. XI was an unidentified non-specifically labelled membrane protein whichh was especially competitive with UhpB for ATP. (b) Time-course of autophosphorylation (in arbitraryy units) of membrane-bound UhpB (0.25 uM) in UhpBC-enriched membranes, in the absence (openn symbols) or presence (solid symbols) of 0.5 mM G6P.

22 3

BC++ BC+

10000 0

3 4 5 6 7 8 99 10 T i m f ll / m i n \

51 1

ChapterChapter 3

1000 (xM. As can be clearly seen on the autoradiogram in Fig.la, UhpB (with a calculatedd mass of 48 kDa and migrating at an apparent mass of -50 kDa) was clearly phosphorylatedd in UhpBC-enriched membranes, isolated in the presence of G6P (lane 3).. Without G6P added only slight (constitutive) UhpB autophosphorylation was observedd (lane 2). As expected, this band was completely absent in membranes that lackk UhpBC (lane 1). The additional bands in the gels represent unidentified and non-specificallyy labelled membrane proteins, of which especially XI was highly competitivee with UhpB for the added [y-32P]ATP. The time dependence of autophosphorylationn of membrane-bound UhpB is shown in Fig. lb. P~UhpB formationn was linear during the first minute after addition of ATP and reached a maximumm after 2 min. Subsequently gradual dephosphorylation occurred.

Ass an alternative we also tested right-side-out (RSO) membrane vesicles, preparedd according to the method of Kaback (1971) and optimised for autophosphorylationn assays as described by Jung et al. (2000). These experiments qualitativelyy confirmed the result obtained with ISO-vesicles (data not shown).

Wee then analysed the concentration range of glucose 6-phosphate that stimulates UhpBB autophosphorylation in this in vitro assay, to determine the apparent Km for transmembranee signalling via UhpBC. Experiments using all three above-mentioned methodss to allow G6P access to the intra-vesicular compartment were performed at a rangee of physiological G6P concentrations (0-300 \xM). Inclusion of G6P via the freeze-thaww treatment gave the most reproducible results (as determined via Phosphor-Imagerr pixel quantification of P~UhpB bands) for the initial rate of P~UhpB formation.. A typical example of such an experiment is shown in Fig. 2, where a high ATPP concentration (250 u,M) was used to assure maximal initial rates. The half-maximall rate of P~UhpB formation, measured at 40 uM in the shown experiment, was observedd at 25 ) \xM G6P.

Fig.Fig. 2. Initia l rate of UhpB autophosphorylation ass a function of G6P concentration. G6P was incorporatedd via freeze-thaw treatment. UhpBC-enrichedd membranes (0.15 iM UhpB) in phosphorylationn buffer (pH 8.0) were incubated withh 250 uM ATP and the indicated G6P concen-trations.. The reactions were initiated by the ad-ditionn of [y-32P]ATP and were quenched after 25 s.

00 50 100 150 200 250 300 [G6P]] uM

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52 2

QuantificationQuantification of phosphorylated TCRS-proteins

Too obtain additional kinetic characteristics of membrane-bound UhpB auto-phosphorylation,, the same assay as described above was done, but with varying concentrationss of ATP, and the initial rate (i.e. within 30 s of ATP addition) of P~UhpBB formation for each ATP concentration (now with a saturating concentration off G6P) was determined. A typical result is shown in Fig. 3. From these experiments, thee Km

A p for the initial rate of autophosphorylation of membrane-bound UhpB was estimatedd to be ~ 250 u.M.

Fig.Fig. 3. Initia l rate of UhpB autophosphorylation ass a function of ATP concentration. The reactions,, initiated with the in-dicated [y-32P]ATP concentrationss (10 Ci mmol"1) added to UhpBC-enrichedd membranes (0.5 \iM UhpB) in phosphorylationn buffer (pH 8.0), containing 0.5 mMM G6P, were quenched after 10 s.

00 150 300 450 600 750 [ATP]] \M

PhosphorylationPhosphorylation and dephosphorylation of UhpA by membrane-bound UhpB Too investigate phosphoryl transfer from UhpB to UhpA, membrane-bound UhpB

wass first incubated with [y-32P]ATP to achieve maximum autophosphorylation of the kinasee (Fig. 4a, lane 1). Subsequently, purified His6-UhpA was added. In Fig. 4a this iss shown for a UhpB:UhpA molar ratio of 1:1 (lane 2). The phosphoryl transfer reactionn was quenched after 30 s. Within this time interval 60% of the label detected inn UhpB was released as inorganic phosphate and 25% re-appeared as [32P]~UhpA. Thee underlying time-course of UhpA phosphorylation was further analysed for differentt UhpB:UhpA molar ratios. P~UhpA formation appeared to be rapid and transientt (Fig. 4b), with an optimum ratio (i.e. maximum level of UhpA phosphorylation)) of 1:5 (UhpB:UhpA). This relatively low optimal ratio may be due too a very active phosphatase activity of UhpB towards P~UhpA. By first phosphorylatingg UhpA, using acetyl [32P]phosphate, this could be further addressed. Duee to the enzymatic procedure used to synthesise acetyl [32P]phosphate, this reaction resultedd in phosphorylated UhpA with a high specific activity, but a low chemical concentrationn (~ 0.5 \iM) and an excess of non-phosphorylated UhpA. This may representt the in vivo situation; however, a higher initial P~UhpA concentration was neverthelesss desirable. This was achieved by synthesising non-radioactive P~UhpA separatelyy and combining it with 32P~UhpA just prior to initiating the phosphatase assay.. The experiment displayed in Fig. 5a and 5b shows that upon addition of

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53 3

ChapterChapter 3

UhpBC-enrichedd membranes, P~UhpA was rapidly dephosphorylated, as compared withh the addition of membranes without UhpBC. This rapid dephosphorylation occurredd independently of the presence or absence of intra-vesicular G6P (compare openn and solid symbols) and the absolute concentration of P~UhpA (compare Fig. 5a andd 5b).

(a) ) 11 2 10000 0

P-UhpB B

xii m

P-UhpA A

33 4.5 Timee (min)

Fig.Fig. 4. Phosphoryl transfer from membrane-bound P~UhpB to His6-UhpA. (a) UhpB autophosphorylationn in UhpBC-enriched membranes, containing 1 uM membrane-bound UhpB and initiatedd with 50 uM [y-32P]ATP in phosphorylation buffer pH 7.5, was either quenched after 2 min (lanee 1) or 1 (iM His6-UhpA was added after 2 min and the reaction was quenched after an additional 300 s (lane 2). (b) Time course of (de)phosphorylation of UhpA (open symbols, regular lines) and UhpBB (solid symbols, dotted lines), at increasing UhpA:UhpB ratios. UhpA (final concentration 0, 0.125,, 0.625, 1.25 and 2.5 uM, respectively) was added to the autophosphorylation mixture (0.25 uM membrane-boundd UhpB, initiated with 100 uM [y-32P]ATP) at 2 min and samples were quenched afterr the indicated times. Symbols indicate different UhpA:UhpB ratios: crosses, 0:1; squares 10:1; circless 5:1; triangles 2.5:1 and diamonds 0.5:1.

54 4

QuantificationQuantification of phosphorylated TCRS-proteins

U T I 1 1 1 — *

00 2 4 6 8 10 0 2 4 6 8 10 Timee (min) Time (min)

Fig.Fig. 5. P~UhpA dephosphorylation by UhpBC-enriched membranes. UhpA (10 \iM) was phosphorylatedd using acetyl phosphate, as described in Materials & Methods. Membranes, containing 11 nM (solid/open circles, +/- 1 mM G6P), or no (stars) membrane-bound UhpB, were added to (P~)UhpAA at time 0 and samples were taken at the indicated time-points, (a) < 10% P~UhpA at time 0;; (b) > 60% P~UhpA at time 0.

Discussion n

Althoughh the majority of intracellular phosphoryl transfer pathways based on the two-componentt paradigm use a transmembrane signal transfer step to become activated,, most systems sense an environmental condition that is not easily mimicked inn an in vitro membrane set-up that allows assay of signal-stimulated HPK autophosphorylation.. Fortunately, several bacterial signalling systems with signal inputt and detection confined to the extracellular presence of a single molecular species havee been characterised. In E. coli, direct sensing of a ligand by a sensory HPK (presumably)) occurs in NarX, NarQ (Rabin & Stewart, 1993), PhoQ (Kato et al, 1999),, DcuS (Zientz et al, 1998), TorS (Jourlin et al, 1996), CusS, PcoS (Munson et al,al, 2000) and HydH [ZraS] (Leonhartsberger et al, 2001). Of these, sensitivity tests usingg in vitro autophosphorylation assays have been reported for membrane-bound NarXX (Williams & Stewart, 1997; Lee et al, 1999) and PhoQ (from Salmonella; Castellii et al, 2000; Montagne et al, 2001), as well as for the 'turgor sensor' KdpD despitee the complex nature of the stimulus that activates this system (Jung et al, 2000). .

Thee Uhp system is also activated upon sensing a single extra-cytoplasmic ligand (G6P).. However, the membrane-bound HPK, UhpB, is incapable of showing signal-stimulatedd autophosphorylation on its own (Island & Kadner, 1993). An additional membrane-boundd protein, UhpC, is needed to sense G6P and to elicit

55 5

ChapterChapter 3

autophosphorylationn in the cytoplasmic domain of UhpB. The existence of this proposedd UhpBC signalling complex has been thoroughly tested in vivo by Kadner andd colleagues (Island & Kadner, 1993; Wright et ai, 2000). Here we have investigatedd its phosphorylation characteristics in vitro, using an inverted membrane set-up.. We observed that the range of concentrations at which G6P activates signalling (i.e.(i.e. UhpB autophosphorylation) overlaps with the range of concentrations that in vivo determinee the Uhp output (data not shown; (Shattuck-Eidens & Kadner, 1981). Cooperativee effects downstream in the signal transduction pathway may explain the lowerr overall apparent Ko.5G6P for induction observed in vivo.

Thee three phosphoryl-transfer reactions that occur within the Uhp signal transductionn pathway were addressed here in vitro in a stepwise fashion. The occurrencee of all three activities, viz. autophosphorylation (1), phosphoryl transfer (2) andd phosphatase activity (3), was demonstrated and characterised independently. This representss the first overall in vitro analysis of phosphoryl flow through the Uhp systemm and, indeed, the outcome confirms the hypothesised two-component paradigm. However,, none of the three systems adequately imitates the in vivo situation, where all threee regulatory Uhp proteins are simultaneously present. Under these conditions, sensingg of G6P by UhpC induces autophosphorylation in UhpB and P~UhpB instantaneouslyy serves as a substrate for UhpA. The latter is present in a large molar excesss over UhpB (data not shown).

Heree we have demonstrated that the G6P concentration controls the rate of UhpAA phosphorylation via the rate of UhpB phosphorylation. G6P does not directly affectt the rate of phosphoryl transfer from P~UhpB to UhpA, nor the phosphatase activityy of UhpB towards P~UhpA. In our phosphoryl transfer assay, the default phosphatasee state (Wright et ai, 2000) of UhpB was only overcome for a few minutes afterr addition of ATP, whereas, as demonstrated with the P~UhpA phosphatase assay, thee presence of G6P in UhpBC-enriched vesicles could not switch off UhpB phosphatasee activity towards P~UhpA. This 'single regulation' phenomenon, i.e. the UhpBB phosphatase activity remains constant while G6P turns on the UhpBC kinase activity,, is in agreement with the general model proposed by (Ninfa, 1996). The dependencee of UhpB kinase activity on the ATP concentration implies that in vivo, withh cytoplasmic ATP concentrations in the order of 4-5 mM (Rohwer et ai, 1996), thee UhpBC sensor will be 'kinase on' with respect to UhpA phosphorylation, as long ass extracellular G6P is being sensed.

Forr the EnvZ/OmpR system it has recently been shown that P~OmpR is stabilisedd when bound to its own target promoter DNA (Qin et al., 2001). This followedd a study that demonstrated enhancement of OmpR phosphorylation bound to promoterr DNA (Ames et ai, 1999). In the former study, P~OmpR-DNA sequestration appearedd to inhibit phosphatase activity by EnvZ (via squelching of (P~)OmpR). The possibilityy of squelching/sequestration of UhpA and OmpR by the phosphoryl transfer domainn of UhpB and EnvZ, respectively, has recently been described in detail (Wright

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QuantificationQuantification of phosphorylated TCRS-proteins

etet al, 2000; Wright & Kadner, 2001). However, the 'P~UhpA protecting' role of uhpTuhpT promoter DNA in the presence of UhpB has not yet been addressed. Moreover, thee in vivo relevance of these reported phenomena in wild-type cells remains to be clarified,, taking into account that both systems have in common that their RRs exist in largee molar excess relative to their cognate HPKs (-100:1), and that there are only a limitedd number of RR target promoters on the chromosome. Furthermore, this HPK:RRR molar ratio, as well as the demonstration of HPK-RR squelching inhibition, makess enhanced phosphorylation in a pre-existing HPK-RR complex, as very recently reportedd for FixL-FixJ (Tuckerman et al., 2001), unlikely for the Uhp system, when takingg into account that for the latter system the HPK and RR are present in equimolar concentrationss (Tuckerman et al., 2001).

Finally,, our quantification of the in vivo concentration of UhpA (2-3 \iM; unpublishedd data), in combination with the recently reported affinity of (P~)UhpA for thee promoter region of uhpT (Olekhnovich et al., 1999; Chen & Kadner, 2000), impliess that the uhpT promoter region is already saturated with UhpA even in the absencee of extracellular G6P. However, P~UhpA has a much higher affinity for the uhpTuhpT promoter region (Chen & Kadner, 2000). Therefore, in G6P-induced cells phosphorylationn of a relatively small fraction of the total amount of UhpA may well bee sufficient to induce uhpT transcription.

Acknowledgements s Wee thank R.J. Kadner for making strains and plasmid pRK18 available. R.

Cordfunke,, M. Hendrix and I. Nugteren-Roodzant are gratefully acknowledged for expertt technical assistance. This work was supported by the Netherlands Organisation forr Scientific Research (NWO), through the division for Earth and Life Sciences (Gebiedd ALW).

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