Protein ProQ influences osmotic activation of compatible solute transporter ProP in Escherichia coli K-12.
ABSTRACT ProP is an osmoregulatory compatible solute transporter in Escherichia coli K-12. Mutation proQ220::Tn5 decreased the rate constant for and the extent of ProP activation by an osmotic upshift but did not alter proP transcription or the ProP protein level. Allele proQ220::Tn5 was isolated, and the proQ sequence was determined. Locus proQ is upstream from prc (tsp) at 41.2 centisomes on the genetic map. The proQ220::Tn5 and prc phenotypes were different, however. Gene proQ is predicted to encode a 232-amino-acid, basic, hydrophilic protein (molecular mass, 25,876 Da; calculated isoelectric point, 9.66; 32% D, E, R, or K; 54.5% polar amino acids). The insertion of PCR-amplified proQ into vector pBAD24 produced a plasmid containing the wild-type proQ open reading frame, the expression of which yielded a soluble protein with an apparent molecular mass of 30 kDa. Antibodies raised against the overexpressed ProQ protein detected cross-reactive material in proQ+ bacteria but not in proQ220::Tn5 bacteria. ProQ may be a structural element that influences the osmotic activation of ProP at a posttranslational level.
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ABSTRACT: ProQ is a cytoplasmic protein with RNA chaperone activities that reside in FinO- and Hfq-like domains. Lesions at proQ decrease the level of osmoregulatory glycine betaine transporter ProP. Lesions at proQ eliminated ProQ and Prc, the periplasmic protease encoded by downstream gene prc. They dramatically slowed the growth of Escherichia coli populations, and altered the morphologies of E. coli cells in high salinity medium. ProQ and Prc deficiencies were associated with different phenotypes. ProQ-deficient bacteria were elongated unless glycine betaine was provided. High salinity cultures of Prc-deficient bacteria included spherical cells with an enlarged periplasm and an eccentric nucleoid. The nucleoid-containing compartment was bounded by the cytoplasmic membrane and peptidoglycan. This phenotype was not evident in bacteria cultivated at low or moderate salinity, or associated with murein lipoprotein (Lpp) deficiency, and it differed from those elicited by MreB inhibitor A-22 or FtsI inhibitor Aztreonam at low or high salinity. It was suppressed by deletion of spr, which encodes one of three murein hydrolases that are redundantly essential for enlargement of the murein sacculus. Prc-deficiency may alter bacterial morphology by impairing control of Spr activity at high salinity. ProQ- and Prc-deficiencies lowered the ProP activity of bacteria cultivated at moderate salinity approximately 70% and 30%, respectively, but did not affect other osmoregulatory functions. The effects of ProQ- and Prc- deficiencies on ProP activity are indirect, reflecting their roles in the maintenance of cell structure.Journal of bacteriology 01/2014; · 2.69 Impact Factor
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ABSTRACT: In this study, culture conditions, including dissolved oxygen (DO) content, presence of osmoprotectants, residual glucose concentration, and ammonium sulfate-feeding strategies, were investigated for decreasing the inhibition effects of acetic acid, ammonium, and osmotic stress on L-lysine fermentation by Escherichia coli. The results revealed that higher DO content and lower residual glucose concentration could decrease acetic acid accumulation, betaine supplementation could enhance osmotic stress tolerance, and variable speed ammonium sulfate-feeding strategy could decrease ammonium inhibition. Thus, with 25 % DO content, 0-5.0 g/L of residual glucose concentration, and 1.5 g/L of betaine supplementation, 134.9 g/L of L-lysine was obtained after 72 h of culture, with L-lysine yield and productivity of 45.4 % and 1.9 g/(L · h), respectively.Applied biochemistry and biotechnology 02/2014; 172(8). · 1.94 Impact Factor
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ABSTRACT: Bacteria respond to elevated osmolality by the accumulation of a range of low molecular weight molecules, known as compatible solutes (owing to their compatibility with the cells' normal physiology at high internal concentrations). The neonatal pathogen Cronobacter sakazakii is uniquely osmotolerant, surviving in powdered infant formula (PIF) which typically has a water activity (aw) of 0.2 - inhospitable to most micro-organisms. Mortality rates of up to 80% in infected infants have been recorded making C. sakazakii a serious cause for concern. In silico analysis of the C. sakazakii BAA-894 genome revealed seven copies of the osmolyte uptake system ProP. Herein, we test the physiological role of each of these homologues following heterologous expression against an osmosensitive Escherichia coli host.Gut Pathogens 01/2014; 6:15. · 2.07 Impact Factor
JOURNAL OF BACTERIOLOGY,
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Mar. 1999, p. 1537–1543Vol. 181, No. 5
Protein ProQ Influences Osmotic Activation of Compatible
Solute Transporter ProP in Escherichia coli K-12
H. JO ¨RG KUNTE,† REBECCA A. CRANE, DOREEN E. CULHAM,
DEBORAH RICHMOND,‡ AND JANET M. WOOD*
Department of Microbiology, University of Guelph, Guelph,
Ontario, Canada N1G 2W1
Received 9 September 1998/Accepted 9 December 1998
ProP is an osmoregulatory compatible solute transporter in Escherichia coli K-12. Mutation proQ220::Tn5
decreased the rate constant for and the extent of ProP activation by an osmotic upshift but did not alter proP
transcription or the ProP protein level. Allele proQ220::Tn5 was isolated, and the proQ sequence was deter-
mined. Locus proQ is upstream from prc (tsp) at 41.2 centisomes on the genetic map. The proQ220::Tn5 and prc
phenotypes were different, however. Gene proQ is predicted to encode a 232-amino-acid, basic, hydrophilic
protein (molecular mass, 25,876 Da; calculated isoelectric point, 9.66; 32% D, E, R, or K; 54.5% polar amino
acids). The insertion of PCR-amplified proQ into vector pBAD24 produced a plasmid containing the wild-type
proQ open reading frame, the expression of which yielded a soluble protein with an apparent molecular mass
of 30 kDa. Antibodies raised against the overexpressed ProQ protein detected cross-reactive material in proQ?
bacteria but not in proQ220::Tn5 bacteria. ProQ may be a structural element that influences the osmotic
activation of ProP at a posttranslational level.
Water flows across biological membranes in response to
osmotic pressure (osmolality) gradients. Turgor pressure de-
velops if cell walls resist osmotically induced water influx.
Osmoregulatory mechanisms adjust cytoplasmic osmolality
by modulating the synthesis, catabolism, uptake, or efflux of
appropriate solutes in response to osmolality changes. Com-
patible solutes are organic solutes, accumulated by bacteria
exposed to hypertonic environments, which do not impair cellular
functions. Physiologists reason that in the absence of osmoreg-
ulatory mechanisms, cytoplasmic osmolality would follow en-
vironmental osmolality, causing unacceptable fluctuations in
cytoplasmic composition, cell volume, and/or turgor pressure
(3, 4, 42).
ProP is an osmoregulatory transporter which mediates the
active accumulation of diverse compatible solutes, including
proline, glycine betaine (N-trimethyl glycine), stachydrine (N-
dimethyl proline) (13, 21), pipecolic acid (8), ectoine (1,4,5,6-
tetrahydro-2-methyl-4-pyrimidine carboxylic acid) (16), and
taurine (23). Gene proP (located at 93 centisomes) is expressed
from ?70- and ?S-dependent promoters. Transcription of proP
is modulated by medium osmolality, carbon source, and cul-
ture growth phase (24, 43–45). ProP is activated by an osmotic
upshift in whole bacteria (11), cytoplasmic membrane vesicles
(26), and proteoliposomes (30). An H?-compatible solute sym-
porter and member of the major facilitator superfamily, the
500-amino-acid ProP protein differs from sequence homo-
logues not implicated in osmoregulation by possessing a 46-
amino-acid carboxyl-terminal extension that is capable of
forming a homodimeric ?-helical coiled coil of limited stability
in vitro (5, 38a).
Mutations pro-219 and pro-220::Tn5 were selected as in-
creasing the resistance of Escherichia coli K-12 derivative RM2
[?(putPA)101] to toxic proline analogue 3,4-dehydroproline.
Mutation proQ220::Tn5 defined a new gene located, by trans-
duction, at 40.4 min on the chromosomal linkage map (27).
Whereas no ProP activity could be detected when proQ220::
Tn5 bacteria were cultivated in a medium of low osmolality, a
partial restoration of ProP activity (41%) was observed when
they were cultivated in a hypertonic medium (0.3 M NaCl
). The mutation did not alter the transcription of a chro-
mosomal proP::lacZ operon fusion (9) in response to increased
medium osmolality, however (27). This report shows that mu-
tation proQ220::Tn5 impairs the osmotic activation of ProP by
acting at a posttranslational level, demonstrates the expression
of ProQ by wild-type bacteria, and reveals the predicted se-
quence of protein ProQ.
MATERIALS AND METHODS
Bacterial strains, plasmids, molecular biological techniques, and growth con-
ditions. The strains and plasmids used for this study are listed in Table 1.
Construction of a prc deletion strain was carried out through a P1-mediated
transduction of E. coli RM2 from strain KS1000, yielding strain WG703. Trans-
ductants were selected on Luria-Bertani (LB) agar containing kanamycin at a
concentration of 50 ?g/ml.
Bacteria were grown aerobically in LB medium (25) or MOPS (morpho-
linepropanesulfonic acid) minimal medium (28) at 37°C. If necessary, antibiotics
were added to the medium at the following concentrations: ampicillin, 100 ?g
ml?1; kanamycin, 50 ?g ml?1; chloramphenicol, 40 ?g ml?1. The hypotonic
medium used to test the prc phenotype (1/2L medium, a salt-free, half-strength
LB medium ) contained Bacto Tryptone (5 g/liter) and Bacto yeast extract
(2.5 g/liter). Routine manipulation of DNA, the construction of recombinant
plasmids, the isolation of chromosomal DNA, electrophoresis of DNA, and
transformation were all carried out by standard techniques described by Sam-
brook et al. (31). DNA sequencing, based on the method of Sanger et al. (32),
was carried out by GenAlyTiC (University of Guelph) or Mobix (Hamilton,
Ontario, Canada). Unless otherwise stated, genetic nomenclature and the num-
bering of DNA sequences are based on release M52 of the E. coli MG1655
genome (accession no. U00096).
To characterize mutation proP219 of E. coli WG170, DNA templates were
synthesized by PCR amplification with synthetic oligonucleotide primers based
on the known sequence of the E. coli K-12 proP locus (accession no. M83089),
and their sequences were determined with the same primers. The overlapping
fragments extended from 282 bp upstream through 79 bp downstream of the
proP open reading frame (ORF), and the full sequence of one DNA strand was
* Corresponding author. Mailing address: Department of Micro-
biology, University of Guelph, Guelph, ON N1G 2W1, Canada.
Phone: (519) 824-4120, ext. 3866. Fax: (519) 837-1802. E-mail: jwood
† Present address: Institut fu ¨r Mikrobiologie und Biotechnologie,
Universita ¨t Bonn, Bonn D53115, Germany.
‡ Present address: 331 Breezewood Crescent, Waterloo, ON N2L
determined. PCR and sequencing were repeated to confirm the single observed
change, from G to A at nucleotide (nt) 1226 of the proP ORF (position 4329305
of the E. coli genome), which would truncate the protein at A408 (at the end of
putative transmembrane helix 11).
To position the Mud1 (lac Ap) insertion of E. coli GJ183 (9) in relation to the
proP promoters, 10 overlapping DNA segments, including proP and flanking
sequences, were PCR amplified. All reactions yielded DNA products of the
expected sizes when E. coli K-12 DNA was used as a template, but two of these
products were missing when E. coli GJ183 DNA was used as a template. Based
on the positions of the corresponding primer sequences, insertion proP227::
Mud1 (lac Ap) interrupted the proP ORF between nt 4329205 and 4329356, in
or after the codon for S375.
Allele proQ220::Tn5 was isolated by selecting bacteriophage Mu dII4042-
derived recombinant plasmids (10, 40), isolated from E. coli WG174 (27), that
conferred kanamycin resistance on strain RM2 Mu cts. Plasmid transductants
were selected on LB medium supplemented with chloramphenicol and kanamy-
cin. Restriction endonuclease analysis of five such plasmids revealed physical
maps which aligned with one another and with the 40.4-min region of the E. coli
genome (17) to which proQ had been mapped (27). These plasmids (or their de-
rivatives) served as templates for proQ sequencing (both DNA strands) with a
primer based on the IS50 regions of Tn5 and others predicted by the emerging
sequence. The deduced sequence in the region of the Tn5 insertion was con-
firmed by PCR amplification and sequencing of the corresponding 609-bp DNA
fragment from E. coli K-12; it also corresponds to the extended yebJ sequence
cited in release M52 of the E. coli MG1655 genome (accession no. AE000277).
In allele proQ220::Tn5, the transposon had been inserted after nt A314 of the
proQ ORF (position 1913173 of the E. coli genome), interrupting the codon for
To effect proQ overexpression, the proQ ORF of E. coli K-12 was amplified as
described previously (2) with primers 5?proQ (5?-GGC TCC ATG GAA AAT
CAA CCT AAG TTG-3?) and 3?proQ (5?-GGA TAA GCT TTC AGA ACA
CCA GGT GTT-3?), the former designed to create an NcoI site at the proQ
initiation codon. The amplified fragment and pQE60 (Qiagen, Santa Clarita,
Calif.) or pBAD24 (12) vector DNAs were cleaved with restriction endonucle-
ases NcoI and HindIII, and the desired DNA fragments were purified, mixed,
Preparation, solubilization, and analysis of cells and subcellular fractions. To
analyze ProQ expression in cells on a small scale, a 1-ml overnight culture was
centrifuged in a Microfuge for 1 min. Cells were resuspended and boiled in 50 ?l
of sample buffer (15.625 mM Tris-HCl [pH 6.8], 2% [vol/vol] glycerol, 0.5%
[wt/vol] sodium dodecyl sulfate [SDS], 0.05% [wt/vol] bromphenol blue, 1.25%
[vol/vol] mercaptoethanol) following a modification of the method of Sambrook
et al. (31). ProP expression was analyzed as described above, except that the
boiling step was replaced by a 30-min incubation at 37°C. Cells were sheared by
repeated passage through a 26-gauge syringe and centrifuged for 5 min in a
Microfuge. The supernatant was analyzed by SDS-polyacrylamide gel electro-
phoresis (PAGE). For larger-scale preparations, cells cultured in LB medium
were harvested by centrifugation (Sorvall GS3 rotor; 5,000 rpm for 20 min at
4°C), washed twice with saline (0.85% [wt/vol] NaCl), and resuspended in a 1/300
volume of potassium phosphate buffer (0.1 M; pH 7.1). Washed cells were passed
three times through a French pressure cell at a pressure of 1.6 ? 108Pa. The
lysate was centrifuged at a low speed (Sorvall SS34 rotor; 10,000 rpm for 20 min
at 4°C) to remove cellular debris and inclusion bodies. Soluble and particulate
fractions were obtained by ultracentrifugation of the resulting supernatant (Beck-
man 45 Ti rotor; 36,000 rpm [145,000 ? g] for 2 h at 4°C). All fractions were
stored at ?70°C after resuspension of the pellets in the same buffer. Appropri-
ately diluted samples of these fractions were dissolved in sample buffer as de-
scribed above. SDS-PAGE analysis of ProQ was performed with gels comprised
of 12% (wt/vol) acrylamide and 2.6% bis-acrylamide according to the method of
Laemmli (19) with a MiniProtean II cell (Bio-Rad, Mississauga, Ontario, Can-
ada). SDS-PAGE of proteins to resolve ProP was performed with 4 to 15%
polyacrylamide gradient Tris-HCl gels (Bio-Rad).
Western blots were carried out according to the method of Towbin et al. (38).
Proteins were electrotransferred to a nitrocellulose membrane (Bio-Rad) at 4°C
with a constant current of 60 mA in a solution of 15.6 mM Tris, 120 mM glycine,
20% (vol/vol) methanol, and 0.02% (wt/vol) SDS. Membranes were blocked by
incubation in phosphate-buffered saline (PBS) (15) containing 5% (wt/vol) skim
milk powder for 18 h at 4°C, washed three times with PBS-Tween (PBS supple-
mented with 0.1% [vol/vol] Tween 20), incubated with either purified anti-ProQ
or purified anti-ProP in PBS for 1 h at room temperature, washed three times
with PBS-Tween, and incubated with horseradish peroxidase- or alkaline phos-
phatase-conjugated mouse anti-rabbit immunoglobulin G in PBS. Blots were
visualized with the ECL kit (peroxidase; Amersham Life Science) or the BCIP/
NBT reagent system (alkaline phosphatase; Sigma, St. Louis, Mo.) according to
the manufacturers’ instructions. Chemiluminescence was detected by exposing
Kodak XAR5 film to the blot for 2 to 5 min.
Affinity purification of anti-ProP antibodies. Anti-ProP antibodies were raised
against the partially purified ProP protein, the antibodies were adsorbed with an
extract of a proP mutant E. coli strain, and ProP(His)6(the ProP protein with six
additional, carboxyl-terminal histidine residues) was purified by nickel chelate
affinity chromatography as described by Racher et al. (30). Purified ProP(His)6
(2.5 mg) was coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech)
according to the manufacturer’s instructions. To bind the anti-ProP antibodies to
the active resin, 0.5 ml of adsorbed serum was incubated with the resulting resin
for 18 h at 4°C on a rotating platform. Bound antibody was eluted from the
column by washing it with 0.1 M glycine-HCl buffer, pH 2.5. The eluate was
immediately neutralized with 1 M Na2CO3. Fractions containing the affinity-
purified antibodies were pooled and stored at ?40°C.
FIG. 1. ProQ is a hydrophilic protein with sequence similarities to E. coli FinO and other structural elements. The full-length sequence alignment of ProQ, FinO,
HI1669, and HI1670 was created by the manual joining of local alignments identified by BlastP (1). ?, conservative substitution.
TABLE 1. E. coli K-12 derivatives and plasmids
Genotype or description
U169 recA1 endA1 hsdR17 (rK
F??(argF-lac)U169 rpsL150 relA1
araD139 flbB5301 deoC1 ptsF25
proP227::Mud1 (lac Ap)
X90 ?prc3::kan eda-51::Tn10
F?lacZ trp rpsL thi ?(putPA)101
F?his pyrD ?(lon-100) rpsL(pREP4)
?) supE44 ??thi-1 gyrA relA1
E. coli ORF proQ inserted into ex-
pression vector pQE60 (encodes
E. coli ORF proQ inserted into ex-
pression vector pBAD24 (encodes
1538KUNTE ET AL.J. BACTERIOL.
Anti-ProQ antibody preparation. The protein overexpressed by E. coli
SG13009(pJK1) was purified as follows. The fraction recovered by the first low-
speed centrifugation of a French press lysate (described above) was washed twice
as described by Neugebauer (29). The insoluble residue was dissolved by boiling
it for 15 min in Tris-HCl buffer (pH 8.0)–5% (wt/vol) SDS–40 mM dithiothreitol
and resolved by SDS-PAGE as described above. The gel was stained with 0.3 M
CuCl2, the gel slice containing the overexpressed protein was excised, and the
staining was reversed by washing it with 0.25 M EDTA and 0.25 M Tris, pH 9.0
(15). A Bio-Rad electroeluter (model 422) was used to recover the protein from
the gel slice by electroelution for 4 h at a constant current of 10 mA in a solution
of 25 mM Tris, 192 mM glycine, and 0.1% (wt/vol) SDS. Five milliliters of
preimmune serum was taken from each of two New Zealand White female
rabbits, before each rabbit was injected intramuscularly with the protein purified
from strain SG13009(pJK1). Further immunization and serum collection were
conducted as described previously (15), and the antibodies were purified as fol-
lows. Cells from a 1-liter overnight culture of E. coli SG13009(pQE60) were
harvested, resuspended in 15 ml of Na MOPS buffer (0.5 mM; pH 7), and dis-
rupted by four serial passages through a French pressure cell at 15,000 lb/in2. The
cell lysate was coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia
Biotech) as specified by the manufacturer, and the resulting affinity matrix was
used to remove contaminating antibodies from the anti-ProQ serum. Fractions
containing anti-ProQ antibodies were pooled and stored at ?40°C.
Transport measurements. The radial streak test (27) was used to estimate the
ProP activities of E. coli RM2, WG174, and WG703 (as proline analogue sen-
sitivities). Cultures of E. coli RM2 and WG174 were prepared and transport
measured by filtration assay essentially as described previously (26). MOPS
minimal medium (28) was inoculated (0.5% [vol/vol]) with cells from an over-
night culture in LB medium. MOPS medium contained NH4Cl (9.5 mM) as a
nitrogen source, glycerol (5 mg ml?1) as a carbon source, L-tryptophan (245
?M), and thiamine hydrochloride (1 ?g ml?1). Upon reaching stationary phase,
cells were subcultured in the same medium to achieve an optical density at 600
nm (OD600) of 0.5. After growth to an OD of 1, cells were harvested by centrif-
ugation and washed three times in unsupplemented MOPS medium (MOPS
medium lacking phosphate, NH4Cl, and organic nutrients). The optical density
of the cells was adjusted to an OD600of 15.0, and amino acid uptake was
measured in an assay mixture consisting of unsupplemented MOPS medium
containing K2HPO4(2.64 mM), glucose (2 mg ml?1), and NaCl as indicated.
Uptake was initiated by the addition of the substrate L-proline (to a concentra-
tion of 200 ?M; 10 Ci mol?1[370 kBq ?mol?1]), following a 3.0-min preincu-
bation of the cells in the assay mixture. The preincubation time was adjusted for
analysis of the kinetics of activation, as indicated. The assay mixture was sampled
after 20, 40, and 60 s. All assays were done in triplicate, and all experiments were
done at least twice. Each set of replicate assays was used to determine the rate
of amino acid uptake over the 20- through 60-s interval. The rates are cited as
means ? standard deviations.
Protein assays. Protein concentrations were determined by a bicinchoninic
acid assay (34) with a kit obtained from Pierce (Rockford, Ill.), with dilutions of
bovine serum albumin as the standard.
Nucleotide sequence accession number. The nucleotide sequence of proQ was
submitted to GenBank and assigned accession no. L48409.
Isolation and sequencing of proQ. Allele proQ220::Tn5 was
isolated by selecting bacteriophage Mu dII4042-derived recom-
binant plasmids (10, 40) isolated from E. coli WG174 (27), and
the proQ sequence was determined (extending from nt 1913558
through 1912860 of the E. coli genome) (see Materials and Meth-
ods). The proQ ORF was predicted to encode a 232-amino-acid,
basic, hydrophilic protein (molecular mass, 25,876 Da; calcu-
lated isoelectric point, 9.66; 32% D, E, R, or K; 54.5% polar
amino acids) with no obvious N-terminal secretion signal se-
quence. The Tn5 insertion interrupted the sequence at codon
Database analysis indicated few protein sequences similar to
ProQ. Haemophilus influenzae Rd contains two adjacent ORFs
(HI1669 and HI1670, one base out of frame) with strong sim-
ilarities to the N- and C-terminal sequences of ProQ, respec-
tively (Fig. 1). The ProQ sequence is also weakly related to
those encoded in orfR5 (a gene of unknown function within the
conjugal transfer region of Agrobacterium tumefaciens octo-
pine-type Ti plasmids) and in finO of E. coli. FinO is believed
to reduce the expression of genes required for the conjugative
transfer of F and related plasmids by associating with antisense
RNA FinP and its target, the traJ transcript (39).
ProQ regulates ProP posttranslationally. The recently pub-
lished E. coli genome sequence facilitated the placement of the
proQ ORF in relation to its neighbors (Fig. 2). Gene proQ
(identical to the extended ORF yebJ; see GenBank accession
no. AE000277) occurs at 41.2 centisomes in the segment of the
E. coli chromosome flanked by loci cspC and holE (Table 2).
ORF b1832, proQ, prc, and htpX constitute a block of genes
known (or predicted) to be transcribed counterclockwise (in
contrast to the flanking loci). E. coli strains defective in prc fail
to grow at 42°C on solid hypotonic medium (1/2L medium; see
Materials and Methods) and have morphologically elongated
cells when grown at 42°C in the corresponding liquid medium
(14, 33). The previous observation that insertion proQ220::Tn5
impairs ProP activity was attributed to its disruption of locus
proQ (27, 36). Locus prc is downstream from proQ, and a pu-
tative prc promoter exists within the proQ ORF (downstream
from the proQ220::Tn5 insertion [nt 1913173] at nt 1913074 to
1913048 ). It was therefore important to rule out the pos-
sibility that the proQ insertion exerted its effects on ProP by
disrupting prc expression, either through polar effects within an
operon including both proQ and prc or by directly disrupting
transcription from a prc promoter located within proQ.
The phenotypes of proQ and prc mutants were therefore
compared. E. coli WG703 (RM2 ?prc3::kan) showed the ther-
mosensitivity and morphology characteristic of prc mutants
FIG. 2. Position and orientation of proQ on the E. coli genome. Arrows indicate positions and orientations of ORFs (see the text). Known and suggested functions
of some of these loci are listed in Table 2.
TABLE 2. Proteins encoded by genes adjacent to proQ
Gene or ORFPosition (nt)FunctionReference(s)
cspC (msmB)1905250–1905459Cold shock protein; suppressor of chromosomal partitioning defects (mukB);
eukaryotic DNA-binding protein homologue
Similar to the IclR family of transcriptional regulators
Heat shock protein; unknown function
Protease responsible for C-terminal cleavage of PBP3 (FtsI)
Similar to GTPase-activating, human proliferating-cell nucleolar protein p120 (GAP)
Theta subunit of DNA polymerase III
VOL. 181, 1999ProQ INFLUENCES OSMOTIC ACTIVATION OF ProP IN E. COLI1539
during growth on 1/2L medium at 42°C, but it retained proline
analogue sensitivity (indicative of ProP activity) identical to that
of E. coli RM2 (proQ?) and not E. coli WG174 (proQ220::Tn5).
In contrast, E. coli RM2 and WG174 grew on solid 1/2L medium
and did not produce elongated cells during cultivation on the
corresponding liquid medium, both at 42°C. Thus the proQ and
prc mutant phenotypes were different, and the effects of the
proQ220::Tn5 insertion on ProP were not exerted through prc.
By analyzing the impact of proQ220::Tn5 on ?-galactosidase
activity in bacteria bearing operon fusion proP227::Mud1 (lac
Ap), Milner and Wood (27) showed that the mutation did not
alter proP transcription. At the time of these experiments the
proP promoters were not defined, so the position of the fusion
in relation to the proP promoters was unclear. Subsequent ex-
periments revealed that proP is transcribed from two promot-
ers with transcription start sites located 182 bp (P1) and 95 bp
(P2) upstream from proP (24). In order to ensure that the
Mud1 (lac Ap) insertion was not between the two promoters,
its approximate location was determined as outlined in Mate-
ed the proP ORF in or after the codon for S375. This ob-
servation reinforced the conclusion that the proQ220::Tn5
mutation does not alter the transcription of proP.
The fact that ProQ shared some similarity to FinO, a known
translational regulator, stressed the importance of examining
the effect of the proQ220::Tn5 mutation on the level of ProP.
However, mutation proQ220::Tn5 did not influence the level of
ProP detected by Western blot analysis in whole cells culti-
vated in media of elevated osmolarities (data not shown) or in
membrane vesicles prepared from cells grown under those con-
ditions (Fig. 3). Thus, neither transcription nor translation of
proP appears to be altered by mutation proQ220::Tn5.
ProQ is expressed as a soluble protein in wild-type E. coli.
The proQ ORF was amplified with DNA from E. coli K-12
(proQ?) as a template and inserted in vector pQE60, yielding
plasmid pJK1. This system was designed to amplify the expres-
sion of the wild-type proQ gene by placing it under the control
of an IPTG (isopropyl-?-D-thiogalactopyranoside)-inducible
tein with an apparent molecular mass of 30 kDa was present in
cells of E. coli SG13009(pJK1) which were induced with IPTG
but was not in those which were not (data not shown). This
protein was contained in a fraction harvested by low-speed
centrifugation from a French press lysate of these bacteria, sug-
gesting that it was present as inclusion bodies. It was enriched
by washing it with EDTA, deoxycholate, and lysozyme (29),
further resolved from contaminants by SDS-PAGE, and eluted
from the gel for antibody production. DNA sequence analysis
revealed that the proQ locus in this plasmid contained muta-
tion C64T, resulting in the predicted protein modification S22P.
To avoid the formation of inclusion bodies and correct the
cited mutation, the proQ ORF was again amplified, inserted in
vector pBAD24 (12) to yield plasmid pDC77, and expressed in
strain DH5?(pDC77). Sequence analysis revealed that plasmid
pDC77 encoded wild-type ProQ, as expected. Plasmids pJK1
and pDC77 both encoded proteins with apparent molecular
masses of 30 kDa, which could be detected by Western blotting
with antibodies prepared as described above. The protein ex-
pressed from plasmid pDC77 was not concentrated in the pellet
obtained by low-speed centrifugation of a French press lysate. It
was most abundant in the supernatant obtained after subsequent
ultracentrifugation (Fig. 4) and was therefore a soluble protein.
The expression of the putative ProQ protein in E. coli RM2
carrying proQ?was analyzed by SDS-PAGE and Western blot-
ting. A protein with an apparent molecular mass of 30 kDa was
detected in the soluble fraction from strain RM2 but not in
that from strain WG174 carrying proQ220::Tn5 (Fig. 5), sug-
gesting that the 30-kDa protein is the proQ gene product.
Mutation proQ220::Tn5 impairs activation of ProP. The
rates of proline uptake via ProP in proQ?and proQ220::Tn5
FIG. 3. Mutation proQ220::Tn5 does not alter the level of ProP. Membrane
vesicles were prepared from E. coli cells grown in NaCl (0.3 M)-supplemented
MOPS minimal medium. Membrane proteins (20 ?g) were separated by SDS-
PAGE, and Western blots were prepared with purified anti-ProP antibodies as
described in Materials and Methods. Purified ProP(His)6(1.3 ?g) served as a
control. The numbers to the left indicate the positions of molecular size markers
FIG. 4. Expression of proQ with vector pBAD24 yields a soluble protein with a molecular mass of 30 kDa. Shown are a Western blot, visualized with the BCIP/NBT
reagent system, and an SDS-PAGE analysis of the soluble (S) and particulate (P) fractions derived from E. coli DH5?(pDC77) with (I) or without (U) induction of
protein expression by arabinose (2 mg/ml). These are compared with the 30-kDa protein overexpressed by E. coli SG13009(pJK1) (Q). The numbers indicate the
positions of molecular size markers (in kilodaltons).
1540 KUNTE ET AL.J. BACTERIOL.
bacteria were measured as a function of the NaCl concentra-
tion employed to impose an osmotic upshift (Fig. 6). (The
proline uptake observed under these conditions is attributable
to transporter ProP, since it is absent from bacteria further
defective in locus proP .) As previously determined, the
optimal upshift for activation of ProP in wild-type bacteria was
imposed with approximately 0.12 M NaCl. ProP activity was
increased sevenfold to a maximum of 22 nmol/min/mg of pro-
tein. Activation of ProP in the proQ220::Tn5 strain occurred
over a similar range of NaCl concentrations but was very lim-
ited (only a threefold increase to a maximum of 2.5 nmol/min/
mg of protein). The kinetics of ProP activation after an osmotic
upshift imposed with 0.12 M NaCl were determined for proQ?
and proQ220::Tn5 bacteria (Fig. 7). The data were fitted to a
model describing an exponential increase in ProP activity, post-
shift (26; Fig. 7). The level of ProP activity approached by the
proQ mutant bacteria (4.8 ? 0.2 nmol/min/mg of protein) was
fivefold lower than that approached by the wild-type strain
(22.7 ? 0.3 nmol/min/mg of protein). The rate constant (k) for
activation of ProP was reduced 2.6-fold in the proQ mutant
(from 0.75 ? 0.04 to 0.29 ? 0.04 s?1). Thus, insertion
proQ220::Tn5 reduced both the rate and the extent of ProP
activation by an osmotic upshift.
The ProQ protein expressed from plasmid pDC77 was ca-
pable of complementing the proQ220::Tn5 defect. The proline
uptake activities of bacteria cultivated in MOPS minimal me-
dium and subjected to an osmotic upshift (0.12 M NaCl) were
14 ? 4 nmol/min/mg of protein for E. coli RM2 carrying proP?
proQ?, 3 ? 1 nmol/min/mg of protein for E. coli WG174
(pBAD24) carrying proP?proQ220::Tn5, and 12 ? 2 nmol/min/
mg of protein for E. coli WG174(pDC77). The levels of the
ProQ protein expressed by E. coli RM2 and WG174(pDC77)
were comparable under these conditions, and no ProQ protein
was detected in E. coli WG174(pBAD24) (Western blotting data
not shown). Thus, plasmid-based expression of proQ restored
ProP activity to wild-type levels in bacteria harboring a chro-
mosomal proQ mutation.
Cells from diverse organisms can accumulate similar arrays
of organic compounds, all known to be compatible with and/or
to stabilize protein structure, when challenged by hypertonic
FIG. 5. The ProQ protein is expressed by E. coli. A Western blot, visualized
with the ECL reagent system, is shown. Soluble fractions derived from E. coli
RM2 and WG174 and the 30-kDa protein overexpressed by E. coli SG13009
(pJK1) (Q) were probed with antibodies raised against the latter protein. The
arrow marks the 30-kDa immunoreactive protein that is present in E. coli RM2
carrying proQ?but not in E. coli WG174 carrying proQ220::Tn5.
FIG. 6. Mutation proQ220::Tn5 impairs ProP activation. E. coli RM2 carrying proQ?(white circles) and WG174 carrying proQ220::Tn5 (black circles) were
cultivated in MOPS medium, and proline uptake rates were measured as described in Materials and Methods. Supplementary NaCl was added to the transport assay
mixtures at the indicated levels. Error bars indicate standard deviations.
VOL. 181, 1999ProQ INFLUENCES OSMOTIC ACTIVATION OF ProP IN E. COLI1541
environments (35). The machinery of compatible solute accu-
mulation has been described for some organisms (e.g., E. coli
[3, 4, 42]), but its regulation is not well understood. Although
proteins Fis and CAP are involved, no trans-acting transcrip-
tional regulatory element specific to locus proP has been im-
plicated in the impressive modulation of its transcription by
osmotic stress. Transporter ProP is activated, in the absence of
protein synthesis, when whole bacteria (11), cytoplasmic mem-
brane vesicles (26), or proteoliposomes incorporating purified
ProP (30) are subjected to an osmotic upshift with a mem-
brane-impermeant osmolyte. Our research is designed to
elucidate the mechanisms by which ProP senses osmolality
changes and mounts its osmoregulatory response. Since ProP
activity is impaired by insertion proQ220::Tn5 (27, 36), we are
exploring the structure and function of proQ as well as its
relationship to ProP.
In this study we establish that the effects of the insertion on
ProP are due to the altered expression of locus proQ and not
to polar effects on downstream locus prc (see the text and Fig.
2 and 5). The previous conclusion that the Tn5 insertion in
proQ does not influence proP transcription was confirmed.
Database analysis identified two proteins with weak sequence
similarities to ProQ. Within this group of homologues, the re-
lationship that appeared most interesting was the weak simi-
larity with translational regulator FinO. This raised the possi-
bility that ProQ could be acting at a translational level to alter
the levels of ProP protein. However, this study has shown that
the level of ProP protein present in either whole cells (data not
shown) or membrane vesicles is not altered by the Tn5 inser-
tion in locus proQ (Fig. 3).
This study has further shown that the rate and extent of ProP
activation are significantly reduced in a proQ220::Tn5 strain of
E. coli. These reports are significant in documenting the only
trans-acting factor which is known to influence the osmotic
activation of ProP. Gene proQ is predicted to encode a 232-
amino-acid protein that is both basic and hydrophilic in nature.
SDS-PAGE and Western blot analysis indicate that the over-
expressed ProQ protein is soluble, as predicted (Fig. 4). The
subcellular location of the protein in wild-type bacteria re-
mains to be determined, however.
ProP activity is observed in cytoplasmic membrane vesicles
(26) and proteoliposomes prepared with purified ProP (30).
There are some significant differences between the ProP activ-
ities of these vesicle systems and those of whole cells, however.
The hyperosmotic shift which gives maximal ProP activity in
cells (0.2 osM) is lower than that required in membrane vesi-
cles (0.8 osM) (22). As well, ProP is active in whole cells even
without an osmotic shift, whereas ProP activities in proteo-
liposomes and membrane vesicles absolutely require a hyper-
osmotic shift (22, 30). Given these contrasting features of ProP
activities in cells and vesicle systems, we now believe that ProQ
is a structural element which influences the osmotic activation
of ProP at a posttranslational level.
We are grateful to Luz-Maria Guzman for plasmid pBAD24 and to
Gabor Magyar, Terry Beveridge, and Jeff McLean for access to equip-
ment and assistance with experiments.
We also thank the Deutscher Akademischer Austauschdienst
(DAAD) for a postdoctoral fellowship awarded to H.J.K., Karlheinz
Altendorf and his colleagues (Arbeitsgruppe Mikrobiologie, Uni-
versita ¨t Osnabru ¨ck) and the DAAD for sabbatical leave support to
J.M.W., and the Natural Sciences and Engineering Research Council
of Canada for a research grant awarded to J.M.W.
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FIG. 7. Mutation proQ220::Tn5 reduces the rate and extent of ProP activation. E. coli RM2 carrying proQ?(white circles) and WG174 carrying proQ220::Tn5 (black
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