ELSEVIER
FEMS Microbiology
Letters 125 (1995) 219-224
Distribution of the flavohaemoglobin, HMP, between periplasm
and cytoplasm in Escherichia coli
Subhash G. Vasudevan
a9bT
* , Pan
Tang ‘, Nicholas E. Dixon b, Robert K. Poole c7d
a Department of Molecular Sciences, James Cook University, Townsuille, Qld 4811, Australia
’ Centre for Molecular Structure and Function, Research School of Chemistry, Australian National Vniuersity, Canberra, Australia
’ Diuision of Life Sciences, King’s College London, UK
d DiGsion of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University,
Canberra, Australia
Received 22 September
1994; revised 10 November
1994; accepted
11 November
1994
Abstract
of the soluble flavohaemoglobin
(HMP) of Escherichiu coli has been determined. Cells
cloned hmp gene on a multicopy plasmid were fractionated by osmotic shock and lysozyme
subcellular fractions showed the CO-binding haemoprotein to be cytoplasmic. However,
raised to purified HMP revealed approximately 30% of the protein to be periplasmic in the
analysis also revealed substantial levels of periplasmic HMP in a strain expressing only
but none in an hmp mutant. The results are discussed in relation to protein function and the
similar distribution reported for Vitreoscilfa globin.
The subcellular
distribution
over-expressing HMP from the
treatment. Spectral analysis of
Western blotting using antibody
over-expressing
strain. Western
chromosomally encoded protein
Keywords: Haemoglobin;
Globin-like
proteins; Cytoplasm;
Periplasm;
1. Introduction
Globin-like proteins occur in several prokaryotic
and eukaryotic microorganisms.
With the exception
of leghaemoglobin
(a plant-produced protein), which
provides an oxygen-buffered
environment for symbiotic rhizobia [l], and the FIXL oxygen sensor of
Rhizobium meliloti [2], the functions of these proteins are unknown. The Vitreoscilla globin (VGB or
VHB) 131, when over-expressed in Escherichiu coli,
is found in both periplasm and cytoplasm [4], appar-
* Corresponding author. Tel: + 6177 81 4343; Fax: + 61 77 25
1394; E-mail: [email protected]
0378-1097/95/$09.50
0 1995 Federation
SSDlO378-1097(94)00501-X
of European
Microbiological
Oxygen utilisation;
Escherichia coli
ently consistent with the proposed function of VGB
in facilitating oxygen transfer from the medium to
terminal respiratory oxidases [5].
The E. coli globin-like
protein HMP [6] is a
soluble protein with two structural domains: the Nterminal portion shows striking similarity to VGB
and leghaemoglobin,
whilst the C-terminal portion is
related to proteins in the ferredoxin-NADP+
reductase (FNR) family [7]. Purified HMP contains haem
B and FAD [8], and binds and reduces oxygen at the
expense of electrons derived from NADH [9]. The
kinetics of redox change of the chromophores during
oxygen consumption suggest that HMP might act as
an oxygen sensor [9]. To shed light on the possible
role of HMP in facilitating oxygen transfer from the
Societies.
All rights reserved
220
S.G. Vasudecan et al. /EMS
Microbiology Letters I25 (I995J 239-224
growth medium to cytoplasmic sites of oxygen reduction (as proposed for VGB), we have determined
the subcellular localisation of both the HMP apoprotein and the holoenzyme.
which was resuspended in 4 ml of 0.1 M Tris-HCl
buffer, pH 8. Volumes of fractions were recorded
and used to calculate the recoveries of proteins [12].
2.3. Western blotting and detection
2. Materials
and methods
2.1. Bacterial strains, media and growth conditions
E. coli K12 strains RSC516 (AN1459/pBR322)
and RSC521 (AN1459/pPL341)
expressing wildtype and elevated levels of HMP, respectively, have
been described [6]. Strain RB9060 ( AglnB Ahmp)
was kindly provided by Drs. J. Lui and B. Magasanik [lo]. Cells were grown at 37°C in LB medium
at pH 7.0 with ampicillin (100 pg ml-‘)
where
appropriate. Cultures were grown in l-l Erlenmeyer
flasks containing l/5 their volume of medium, and
cells were harvested in the late exponential phase of
growth (c. 16 h) at an A,,, of about 1.5.
2.2. Cell fractionation
The method adopted was a scaled-up modification
of that of Manoil and Beckwith [ll]. Cells centrifuged from 200-ml cultures (10000 g X 8 min,
4°C) were suspended for 5 min in 20 ml of cold
spheroplast solution [ll] containing 0.5 M (not 0.5
mM) sucrose. A sample (‘whole cells’; 5 ml) was
frozen for subsequent analysis. The remainder was
centrifuged (10000 g X 5 min, 4°C); the pellet was
warmed to room temperature and, after 5 min, suspended with shaking in 15 ml of ice-cold water.
After 45 s at 0°C 0.75 ml of 20 mM MgCl, was
added and the osmotically-shocked
cells were centrifuged again to give a supernatant fraction (‘periplasm’). The pellet was resuspended in 10 ml of cold
spheroplast buffer to which was added 1 ml of
lysozyme solution (2 mg ml-‘) and 10 ml of cold
water. Cells were maintained on ice for 5 min and
then centrifuged to give a supematant fraction (‘cytoplasm I’) and a pellet, which was resuspended in
15 ml of 10 mM Tris-HCl (pH 8). After three freeze
(77 K)/thaw
(28°C) cycles, 0.65 ml of 1 M MgCl,
and 0.4 ml of deoxyribonuclease
I (1 mg ml-‘) were
added. The lysed spheroplasts
were centrifuged
(16 000 g X 20 min, 4°C) to give a supematant
fraction (‘cytoplasm
II’) and a membrane
pellet,
HMP was purified essentially
as described [8]
from an overproducing
strain RSC 419 [6]. New
Zealand White rabbits were administered
subcutaneous injections at multiple sites along the spine
with a homogeneous
mixture of 400 pg purified
HMP in Freund’s complete adjuvant. Subsequently
two booster injections (200 pg of HMP in Freund’s
incomplete adjuvant) were administered at two week
intervals. The antibodies were used in Western blots
as described [13] and detected using goat anti-rabbit
IgG-alkaline phosphate conjugate.
2.4. Assays
Malate dehydrogenase activity was determined in
50 mM potassium phosphate (pH 7.6) containing 100
PM NADH and 500 PM oxaloacetate 1141. The rate
of absorbance change at 340 nm was corrected where
necessary (membrane fraction) for oxaloacetate-independent NADH consumption.
fi-Lactamase activity
was determined spectrophotometrically
at 250 nm
[15] in a l-ml reaction volume containing 6 ~1 of
ampicillin solution (100 mg ml-‘). P-Galactosidase
was assayed according to Miller [16]. The HMP
haemoprotein (holoprotein) was quantified from CO
difference spectra at room temperature [8] using a
Cary 219 recording spectrophotometer
at a scan rate
of 2 nm ss’ and a spectral bandwidth of 0.5 nm or
(results not shown) a Johnson Foundation SDB 3
dual-wavelength
scanning spectrophotometer.
Quantification was made from the Soret peak-trough amplitude using (E~~~-E~~~) = 144 mM_’ cm-‘. Protein concentrations were determined by the Markwell
[17] or Lowry methods.
3. Results
3.1. Subcellular distribution
the HMP ffauohaemoglobin
of marker enzymes and
Several cell disruption procedures were tested for
their ability to yield fractions of appropriate volume
S.G. Vasudevan et al. /FEMS
Microbiology
and concentration
for spectral assay of HMP holoprotein. Release of periplasm by chloroform [181 or
an alternative lysozyme-EDTA
procedure [ 151 proved
inferior to the protocol in Materials and methods.
The first gave incomplete release of P-lactamase
from concentrated cell suspensions while the second
yielded a ‘periplasmic fraction’ contaminated
with
P-galactosidase.
Table 1 shows, from a typical experiment, the
subcellular distributions of malate dehydrogenase and
@-lactamase, marker enzymes for the cytoplasm [19]
and periplasm 1111, respectively.
/3-Lactamase was
detectable only in the periplasmic fractions of both
strains. In RSC516 (control), the combined cytoplasmic fractions contained 94% of the recovered malate
dehydrogenase, as anticipated. In RSC521 (HMP+),
75% was found in the cytoplasm, and 19% in the
periplasmic fraction, suggestive of some cytoplasmic
leakage, as observed in [15]. Nevertheless, the distribution of the spectrally-detected
HMP in RSC521
closely followed that of malate dehydrogenase with
86% in the combined cytoplasmic
fractions. The
malate dehydrogenase activity appears to be significantly lower in RSC.521, but too little is known
about the function of HMP to explain this observation. In two further fractionations (not shown) using
the osmotic shock-lysozyme
method, ,f3-galactosidase was used as a cytoplasmic
marker enzyme.
Neither this activity nor spectrally assayed HMP
could be detected in the periplasm or membrane
fractions: all the recovered activity was in the combined cytoplasmic fractions. Again, p-lactamase was
wholly in the periplasmic fraction. The recoveries of
Table 1
Subcellular
fractionation
Fraction
Periplasm
Cytoplasm I
Cytoplasm II
Membranes
of E. coli RSC521 (HMP+)
Recovered
221
Letters 125 (1995) 219-224
A’
436
Cd)
I
4i2
Fig. 1. CO difference absorption spectra at room temperature of
intact cells of E. co& RX521
(HMP+ 1 and derived subcellular
fractions. The spectra shown are of (a) intact cells, (bl periplasmic
fraction, (cl cytoplasmic fraction II and Cd) membranes. The bar
represents a AA of 0.05, except in (a) where it .is 0.1. Protein
concentrations (mg ml-‘) were (a) 4.0, (b10.43, (cl 1.52, and (d)
0.5. Distinctive
features of the spectrum are marked (at the
wavelength in nm).
total protein and HMP, both of which could be
assayed in intact cells as well as in subcellular
fractions, were always 88-99% (protein) and 87117% (HMP), which demonstrate the validity of the
fractionation procedure.
Fig. 1 shows CO difference spectra for RSC521
over-expressing
HMP. The peak at 418-420 nm in
and RSC516 (control):
distributions
of HMP and marker enzvmes
2
protein or activity in fraction (o/o)
a HMP
b Malate dehydrogenase
’ p-lactamase
RX521
RSC516
RSC521
RSC516
RSC521
14.3
8.6
77.1
0
2.1
13.6
80.6
3.6
19
19.3
55.5
6.3
100
0
0
0
100
0
0
0
Cells were osmotically shocked to release periplasmic contents and lysed by lysozyme and freeze-thawing to release cytoplasmic contents
and membranes. For details,
see Materials and methods. Footnotes give actual concentrations
and activities measured in selected fractions.
a Concentrations in whole cells and cytoplasm II were 0.57 and 0.69 nmol mg-‘, respectively. HMP was not reliably quantified in RSC.516.
’ Activities in cytoplasm II were 3.03 (RSC516) and 0.82 (RSC5211 pm01 min-’ mg- ’
’ Activities in periplasmic fraction were 25.5 (RSC516) and 14.5 (RSC.521) AA,,
units mini
mg- ‘.
222
XC. Vasudeuan et al. / FEMS Microbiology Letters 125 (I 995) 219-224
(a) to (cl is due to the CO adduct of HMP and the
trough at 436-438 nm is due to loss of the unligated
Fe” haemoprotein.
The (Y and p features of the
spectra (not shown) were as reported previously
[6,8]. Absorption bands at 416 and 432 nm in spectra
of membranes
(Fig. Id) are characteristic
of cytochrome o, not HMP. Spectra of cells and membranes of RSC516, expressing only chromosomally
encoded HMP, also showed cytochrome o, which
was not detectable in any other fraction. Typically,
90% of the total cytochrome o measured in intact
cells was recovered in the membrane fraction, again
validating the fractionation procedure.
3.2. Localisatian
blotting
of the HMP protein
HMP---,
PF-_)
by Western
While the assays of subcellular fractions showed
that the HMP holoprotein was located in the cytoplasm, they cannot comment on the distribution of
the apoprotein. Immunochemical
detection with antiHMP serum was therefore used to determine the
distribution of total (holo- plus apo-> HMP. Fig. 2
shows Western blots of the SDS-PAGE gels from
the experiment shown in Table 1. Each lane was
loaded with 5 pg of cell protein. The prominent
band detected with HMP antiserum corresponds to a
protein of 44 kDa, as anticipated
[6]. Bands of
similar mobility were observed for all fractions. The
band in the membrane fraction was weak, whilst the
strongest was observed in periplasmic fractions. In
the cytoplasm II fraction, two additional bands corresponding to polypeptides of about 30 and 29 kDa
were apparent and are attributed to proteolytic fragments of HMP. Close examination of the blot membranes also revealed an extremely faint 30 kDa band
for the periplasmic fraction. However, the clearly
different ratio of intensities of the 44 kDa band to
those of the proteolytic fragments in the periplasmic
compared with the cytoplasmic
fractions strongly
suggests that HMP in the periplasmic fractions could
not have arisen from cytoplasmic leakage, in accordance with the marker enzyme distributions. Similar
results were obtained in four Western blots on samples from two separate fractionation
experiments.
We estimate from the intensities of bands that about
30% of total HMP (allowing for the different volumes of the fractions) is in the periplasm in RSC521
18.5
Fig. 2. Western blot analysis of proteins in subcellular fractions
from E. coli RX521
(HMP+). The samples were obtained as
described in Materials and methods. Samples corresponding to 5
pg total protein were suspended in SDS-PAGE loading solution
and heated to 95°C for 3 min followed by separation on 0.1%
SDS-IO% PA gel. The proteins were transferred to a nitrocellulose
membrane by electroblotting,
probed with rabbit antibodies to
HMP and finally identified by secondary goat anti-rabbit antibodies coupled to alkaline phosphatase using BClP/NBT
substrate
solution. The membrane was blocked with 3% gelatine and the
antibody solutions contained 1% gelatine as a carrier protein. The
band positions shown on the right are those of pre-stained molecular mass standards (BioRad, USA). The arrows at the left show
the positions of HMP (44 kDa) and of proteolytic fragments (PF)
of 30 and 29 kDa.
(HMP+). The electrophoretic mobilities of the approx. 44-kDa HMP derived from the cytoplasmic
and periplasmic
fractions (Fig. 2) were identical,
suggesting that HMP is not processed proteolytically
to remove a short N-terminal leader peptide on transport through the cell membrane. In fractions from the
control strain RSC516, the HMP band was always
very faint (three fractionation
experiments),
but it
was again most prominent in the periplasmic fractions (results not shown).
To confirm the specificity of the antiserum, we
prepared and analysed fractions from the Ahmp mutant strain RB9060. No HMP was detectable in
Western blots of any fraction. When pPL341 was
introduced into this mutant strain, expression of HMP
was weaker than in RSC521, but again a substantial
S.G. Vasudevan et al. / FEMS Microbiology
proportion of HMP was in the periplasmic fraction.
Less of the proteolytic fragments were detectable in
the cytoplasmic fractions of RB9060/ pPL341 (results not shown).
4. Discussion
All microbial globin-related
oxygen-binding
proteins appear soluble, with the exception of Rhizobium FIXL. Although FIXL is anchored to the cytoplasmic membrane,
a truncated form (FIXL*) is
soluble and retains activity [2]. The Alcaligenes
flavohaemoglobin
FHP [20] is cytoplasmic,
not
periplasmic, based on spectroscopic assays similar to
those reported in this paper, and the yeast flavohaemoglobin YHB is also cytoplasmic [21].
A more complex picture derives, however, from
studies of expression in E. coli of the Vitreoscilla
ugb gene. Subcellular fractionation and proteinase K
accessibility studies showed VGB in periplasmic and
cytoplasmic fractions 141. The N-terminal domain of
VGB has been suggested to specify export of the
protein to the periplasm, without its being cleaved
during translocation.
Interestingly,
the N-termini of
HMP and VGB show considerable similarity (Fig.
3a): of the first 30 residues, 16 are identical and a
further 4 show conservative differences [6]. Of the
16 residues shown by use of phoA fusions to specify
export of VGB [4], 10 are identical in HMP and a
Letters I25 (I 995) 219-224
223
further 3 are similar. The amino acid substitutions in
HMP do not affect the net charge of zero [4]. KyteDoolittle analysis [4] of the VGB sequence shows
that lysine-11, conserved in HMP, is responsible for
reducing the overall hydrophobicity of the terminus.
The Hopp and Woods hydrophobicity
plot obtained
using the GeneJockey (Biosoft, Cambridge, UK) sequence processor shows the remarkable similarity
between VGB and the N-terminal domain of HMP
(Fig. 3b).
We conclude that synthesis of HMP in E. coli
from a multicopy plasmid results in transport of a
significant
fraction
as the apoprotein
into the
periplasm. The haem-containing
holoprotein is found
only in the cytoplasm, as for VGB expressed in E.
coli [4]. A similar intracellular distribution appears to
occur also for chromosomally-transcribed
hmp,
which raises the question of its physiological significance. Other haemoproteins,
like the cytochromes c
[22], are located in the periplasm of E. coli, and
membrane proteins have been implicated in haem
transport and/or
assembly of these cytochromes
[22,23]. A function in the periplasm for a form of
HMP that lacks haem is unclear. However, we note
that a NADPH-dependent
flavin-containing
reductase in erythrocytes and liver binds haem and may
serve to protect cell proteins from haem inhibition
and the oxidative damage that might be caused by
free haem [24].
Acknowledgements
R.K.P. thanks the Nuffield Foundation for a Research Travel Grant, SERC for Research Grant GR/
H/92265,
and the John Curtin School for a Visiting
Fellowship. S.G.V. thanks James Cook University
for start-up grant IRA 151572. We thank Dr. Wilf
Armarego for his interest in this work and Mr. Greg
White for technical assistance.
/
2
4
I
,
I
I
6
8
18
12
Amino acid number
I
14
I
16
I
18
Fig. 3. (a) Comparison of sequences of the 18 N-terminal aminoacid residues of HMP and VGB. Identical residues are shaded;
similar residues are boxed. (b) Hopp and Woods hydrophobicity
plots of HMP (solid line) and VGB (broken line); a sliding
window of 6 residues was used.
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