From the
A complex of polyhydroxybutyrate (PHB), Ca
Despite the universal use of bacterial transformation procedures
(Hanahan, 1983), little is known about the molecular basis of
competence, the state in which cells become capable of taking up
exogenous DNA. Although the protocols for inducing competence differ
for each species, the procedures include a metabolic shift-down without
carbon limitation in the presence of Mg
Coincident with the appearance of this
complex was a sharp, new thermotropic transition at about 56 °C,
indicative of a profound modification of the lipid bilayer
organization. A strong correlation was noted between the peak intensity
and the degree of competence (Reusch and Sadoff, 1983; Reusch et
al., 1986). In a proposed structure for the complex, the methyl
and methylene groups of a helical PHB face the membrane lipids, thereby
creating an inner framework helix of Ca
The identity and
content of PHB in the novel membrane complexes have been established by
chemical and immunological assays and by
With regard to
polyP, the lack of sensitive methods comparable to those available for
determinations of PHB left the presence and amount of polyP in the
complexes of competent cells relatively uncharacterized. Added to this
uncertainty was the finding that mutants of E. coli which
lacked the capacity to make the predominant, long-chain form of polyP
remained competent for DNA transformation. To determine levels of
polyP, we have isolated enzymes which can be used as reagents for the
sensitive and accurate assay of the polymer. One of these is polyP
kinase (Ahn and Kornberg, 1990; Akiyama et al., 1992) which
measures polyP by converting it to ATP in the presence of ADP; the
others are exopolyphosphatases from E. coli (Akiyama et
al., 1993) and yeast (Wurst and Kornberg, 1994) which hydrolyze
polyP quantitatively to orthophosphate. Both assays, applied to a wide
range of bacterial, fungal, and animal sources of polyP, have yielded
values of polyP that agree within narrow limits.
In the present
study, we have applied these enzymatic methods to confirm the presence
of polyP in the membrane complexes of competent cells, to establish its
stoichiometry with PHB, and, by electrophoretic analysis, to disclose a
novel polyP of 60 to 70 residues which appears in both wild-type and
mutant cells. We have used a variety of fluorescent lipid probes to
demonstrate the development of extensive, rigid domains closely
correlated with competence.
The PHB
Membrane fractions were prepared
essentially as described by Osborn et al.(1972). Cells were
lysed in a French pressure cell, and the membrane fraction was
collected by centrifugation at 100,000
Total polyP from competent
and noncompetent cells was extracted as described by Clark and
Wood(1987). The procedure includes five steps: precipitation with 2%
trichloroacetic acid; treatment with DNase, RNase, and proteinase K;
extraction with phenol-chloroform; precipitation with 100 mM
BaCl
Inorganic PolyP in the Membranes of Competent Wild-type and
ppk
The stoichiometric ratio of PHB to polyP
was assayed in the HPLC-purified complex. For the wild-type (JM101),
the fraction contained PHB and polyP in the stoichiometry of 49 to 25
pmol per mg of protein (average of 2 measurements, based on monomer
molecular weights of 86 for PHB and 80 for polyP).
Inorganic polyphosphate (polyP), a polymer of many tens or
hundreds of phosphate residues, has likely been extant since prebiotic
time. Found in every microbe, plant, and animal, polyP has the
potential for a multitude of functions (Kulaev and Vagabov, 1983; Wood
and Clark, 1988; Kornberg, 1994): generation of high energy bonds (as
in ADP and ATP), phosphorylation of sugars, a reservoir of
orthophosphate, and chelation of divalent metal ions. In its
resemblance to polyanions like RNA and DNA, polyP may also exert
regulatory functions, among them the complex cellular adjustments to
stresses and deprivation (Crooke et al., 1994). Still another
role, proposed by Reusch and Sadoff(1988), is to participate as part of
a membrane complex with Ca
The chloroform extraction procedure separates membrane-bound polyP,
solubilized in a complex with PHB, from cytoplasmic polyP. PolyP levels
in the membranes of wild-type E. coli ( Fig. 1and
) are only about a one-twentieth of those found in the
cytosol (Crooke et al., 1994). For PHB on the other hand,
there is no interference from a nonmembranous, chloroform-soluble form
inasmuch as E. coli lacks the cytosolic granules of PHB
present in many other bacterial species.
Reusch et
al.(1986) had found that 78% of the extractable PHB from competent
cells was localized in the inner (cytoplasmic) membrane fraction.
Although the membrane envelopes of competent cells failed to separate
into distinct cytoplasmic and outer membrane fractions in the presence
of divalent cations (), the results clearly indicate that
the chloroform-soluble polyP is associated with the particulate
fraction, likely in the cytoplasmic membrane.
The chain length of
polyP, more accurately determined by PAGE, appears to be 60 to 70
residues, rather than 130 as determined by size-exclusion
chromatography with polyethylene glycols as standards. The molar ratio
of PHB to polyP for the complex purified by gel permeation
chromatography appears to be 2:1 as previously reported (Reusch and
Sadoff, 1988).
A clue to the mechanism whereby the
PHB
Fluidity, as an operational
term, includes the contributions of both the rate and range of the
molecular motions (Shinitzky and Barenholz, 1978). The conformation of
the membrane complex can be envisaged as an inner framework of polyP
bridged to Ca
The presence of the PHB
Assays with PPX and PPK were each used to estimate
the polyP content (see ``Experimental Procedures''). Values
are expressed in terms of phosphate residues.
Membranes were
separated by the Osborn procedure; NADH oxidase activity and polyP
content were measured as described under ``Experimental
Procedures.'' TB denotes transformation buffer (Hanahan, 1983).
PolyP was
extracted and quantitated by assays with PPX and PPK (see
``Experimental Procedures''). Values are expressed in terms
of phosphate residues.
We thank LeRoy Bertsch for helpful suggestions in
preparing the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
,
and inorganic polyphosphate (polyP) was proposed as the membrane
component responsible for competence for DNA entry in Escherichia
coli (Reusch, R. N., and Sadoff, H. L.(1988) Proc. Natl. Acad.
Sci. U. S. A. 85, 4176-4180). While chemical and
immunological assays and
H NMR have unequivocally
established the identity and content of PHB in the complex, comparable
methods were not available for polyP. With specific enzyme assays
developed for polyP, we have identified, in chloroform extracts of
competent cell membranes, a novel form of polyP of about 60 to 70
residues in a stoichiometric ratio of PHB to polyP of 2:1. In E.
coli mutants, incapable of synthesizing the predominant,
thousand-long polyP chains, appearance of this short polyP and its
inclusion in membranes can account for their capacity to develop
competence and indicates an auxiliary pathway for polyP synthesis. A
variety of fluorescent lipid probes demonstrate the appearance of
extensive rigid domains in membranes of competent cells. We propose
that the PHB
Ca
polyP complex perturbs the
conformation of the lipid matrix, making it more permeable to charged
molecules and thus allowing the entry of DNA.
and
Ca
(Mandel and Higa, 1970). The low temperature and
divalent cations are generally assumed to alter the structure of the
lipid matrix to make it more permeable to the highly polar DNA. In
exploring the basis for competence in Escherichia coli (Reusch
et al., 1986), as well as in more readily transformable
bacterial species (e.g. Azotobacter, Bacillus, and
Hemophilus), Reusch and Sadoff(1983) discovered the uniform
presence of a membrane complex of polyhydroxybutyrate
(PHB),
(
)
Ca
, and inorganic
polyphosphate (polyP).
polyP.
The Ca
ions link the two polymers by means of ionic
bonds with the phosphoryl oxygens of the polyP and by ion-dipole bonds
with the ester carbonyl oxygens of the PHB. Although the validity of
this structure and its capacity to form a passage for DNA entry has not
been established, the complexes do form Ca
channels
in planar lipid bilayers.
(
)
H NMR (Reusch and
Sadoff, 1988; Reusch et al., 1992; Reusch, 1992 and Seebach
et al., 1994). The phase transitions of PHB in liposomes are
weak, broad, and occur well below 45 °C. Inasmuch as the novel
phase transition in competent cells disappears readily and irreversibly
when the cells are suspended in an aqueous medium and even more
completely when exposed to chelating buffers, it could be surmised that
the amphiphilic PHB was associated with water-soluble components and
divalent cations. These moieties were in fact identified as polyP and
Ca
(Reusch and Sadoff, 1988).
Competent Cells
Cells collected in log phase
were made competent essentially by the Hanahan procedure(1983) as
described previously (Reusch and Sadoff, 1988). Strains used were JM101
for wild-type E. coli and CA10 for the
ppk mutant (Crooke et al., 1994).
The pUC 18 plasmid, bearing the ampicillin resistance gene, was added
to competent cells in a volume of 20 µl or less. After 20 min on
ice, cells were heat-shocked in a 42 °C bath for 90 s, chilled to 0
°C, and SOC medium (Hanahan, 1983) was added. After incubation at
37 °C for 30-60 min, cells were plated with or without the
addition of ampicillin, and the efficiency of transformation was
calculated. When cells were labeled with
[
P]phosphate, the specific activity was at or
below 10 µCi/ml culture.
Fluorescence Measurements
Three fluorescent probes
(Molecular Probes, Inc.) were used: 1,6-diphenyl-1,3,5-hexatriene
(DPH), trans-parinaric (octadecatetraenoic) acid, and
N-phenyl-1-naphthylamine (NPN) (Sklar, 1980; Shinitzky and
Barenholz, 1978). Cells were labeled as described previously (Castuma
et al., 1993; Reusch and Sadoff, 1983) after being exposed for
40 min to the transformation buffer (TB) (100 mM KCl, 45
mM MnCl4H
O, 10 mM
CaCl
, and 10 mM MES (pH 6.2, Sigma) (Hanahan,
1983). The lipid/probe molar ratio was 300:1. With DPH, the most widely
used fluorescent probe to monitor membrane fluidity (Shinitzky and
Barenholz, 1978), irradiation with polarized light imposes a
preferential direction of alignment of its electrical vectors; the more
fluid the membrane, the lesser the alignment. Thus, fluorescence
anisotropy is directly proportional to rigidity. The
trans-parinaric acid probe, located closer to the polar head
region than DPH, detects heterogeneous rigid areas within an otherwise
fluid membrane (Sklar, 1980). Fluorescence was recorded in an SLM 8000
spectrofluorimeter; measurements were performed as described previously
(Castuma et al., 1993). The sample was excited with vertically
polarized light; vertical (I
) and
horizontal (I
) emission intensities were
recorded. For DPH and parinaric acids, results are expressed, after
appropriate blank and instrumental corrections (G), as
fluorescence anisotropy (r): r =
((I
/I
G) -
1)/((I
/I
G) + 2).
Extraction of the
PHB
The
complex was extracted from competent cells as described previously
(Reusch and Sadoff, 1988). Competent cells were washed sequentially
with cold methanol and acetone, and the dry residue was extracted with
dry chloroform overnight at 4 °C in a desiccator. The chloroform
layer was filtered and concentrated by evaporation. PolyP was purified
from the aqueous extract of the filtered chloroform solution by
chromatography on a Showdex aqueous size-exclusion column (B-804/S; 8
mm Ca
PolyP Complex
25 cm) with 100 mM NaCl as eluent and analyzed by
polyacrylamide gel electrophoresis (PAGE) (Clark and Wood, 1987).
Ca
polyP complex was purified as
such from the original chloroform extract on a Showdex HPLC nonaqueous
size-exclusion column (K-803; 8 mm
25 cm). Fractions were
assayed for PHB (Reusch, 1989) and polyP (Akiyama et al.,
1992). A fraction in the molecular weight range of 13,000 to 21,000
contained both polymers.
g. Inner and
outer membranes were separated in a linear sucrose gradient (25 to 63%)
by centrifugation for 18 to 20 h at 45,000
g. NADH
oxidase activity was used as an enzyme marker for the cytoplasmic
membrane (Osborn et al., 1972).
at pH 4.5; and dissociation of the precipitate with
Chelex.
Assay of PolyP
PolyP was assayed as a donor in the
conversion of ADP to ATP by polyP kinase (PPK) (Akiyama et
al., 1992). The reaction mixture (10 µl) contained 50
mM HEPES-KOH (pH 7.2), 40 mM
(NH)
SO
, 4 mM
MgCl
, 12 µM [
C]ADP (0.1
µCi/mmol), and 2000 units of PPK (3
10
units/mg). The reaction at 37 °C for 45 min was terminated by
chilling to 0 °C and adding carrier ADP and ATP (to 15 mM
each). Aliquots were spotted on a polyethyleneimine-cellulose F thin
layer chromatography plate; polyP, ADP, and ATP were resolved using 0.4
M LiCl, 1 M HCOOH. The radioactivity that remained at
the origin and that which migrated with ADP, ATP, and the solvent front
was determined for each by liquid scintillation counting. With ADP in
excess, PPK catalyzed the nearly complete conversion of polyP to ATP;
polyP values assayed by hydrolysis to P
by E. coli exopolyphosphatase (PPX) (Akiyama et al., 1993) agreed
within 10%.
Cells-The aqueous extract of the
chloroform extract of competent and noncompetent cells was analyzed by
PAGE (Fig. 1). Aside from the contaminant near the origin that
appeared in all samples, competent cells from wild-type E. coli showed a band which was not susceptible to the action of DNase or
RNase but was removed by treatment with PPK and ADP and by E. coli exopolyphosphatase (Fig. 1A). Remarkably, the same
polyP band was present in competent ppk
cells unable to synthesize long-chain polyP
(Fig. 1B). The efficiency of transformation of the
mutant (6
10
transformants/µg of DNA) was only
slightly less than for the wild-type (9
10
). These
values correlate with the levels of polyP in membrane extracts detected
by the enzymatic assays (); the polyP content of the
ppk
mutant was slightly less than that of
the wild-type. In noncompetent cells, wild-type or mutant, the levels
of polyP in membrane fractions were below the limit of detection
().
Figure 1:
Competent cells
from wild-type and ppk mutant contain polyP.
Log-phase cells were labeled with [
P]P
(see ``Experimental Procedures''). A, aqueous
extracts of chloroform extracts of wild-type cells were treated with
RNase (1 µg/ml), DNase (1 µg/ml), PPK, and PPX; samples were
subjected to PAGE, dried, and visualized in a PhosphorImager scanner
(Molecular Dynamics) (see ``Experimental Procedures'').
B, aqueous extracts of chloroform extracts of wild-type and
ppk
mutant cells were analyzed by
PAGE.
The membrane localization of polyP present in
competent cells was examined by the classic Osborn method, in which the
whole membrane fraction is subjected to separation of the inner and
outer membranes in the presence of EDTA to avoid the fusogenic effect
of divalent cations (Osborn et al., 1972). The separation was
performed in the ppk mutant with and without
the transformation buffer (TB) (). As anticipated, the
EDTA and dilutions employed in the Osborn method dissociated the
complex of polyP with PHB and Ca
. Without addition of
TB, a small proportion of the polyP originally present (as in
) was recovered in the total membrane fraction, and none
after the second step which included EDTA. The NADH oxidase marker
indicates that separation of inner and outer membranes was achieved.
With the addition of TB, almost 30% of the polyP originally present was
recovered in the membrane fractions. The enzyme marker also reflected
the poorer separation of the inner and outer membranes in the presence
of TB. Yet most of the polyP remained associated with the inner
membrane ().
Membranes of Competent Cells Possess Rigid Domains
The
increase in quantum yield of the lipid probe, NPN, with a change in
membrane viscosity generates an abrupt rise in its relative
fluorescence intensity. Such a novel and sharp fluorescence peak at 56
°C, observed in cells that develop competence (Reusch et
al., 1986), was seen also with the ppk mutant, but to a somewhat lesser extent (Fig. 2); this
fluorescent peak was absent in noncompetent cells.
Figure 2:
Fluorescence spectra of competent cells
labeled with N-phenyl-1-naphthylamine (NPN). Fluorescence
measurements were made on cells labeled with NPN (label/lipid molar
ratio of 1:300) (see ``Experimental
Procedures'').
With DPH as a
probe, there is an abrupt loss of fluorescence anisotropy as the cell
membranes undergo a transition from the rigid (gel) to the fluid
(liquid crystalline) phase at about 25 °C (Fig. 3) (Castuma
et al., 1993). In competent cells, there was a marked
persistence of rigidity, likely due to restricted movement of the fatty
acyl chains in contact with the
PHBCa
polyP complex; a second thermotropic
transition at 56 °C was indicative of the total disorganization of
the complex as observed with NPN (Fig. 2). The competent
ppk
mutant also displayed this biphasic
behavior, although the persistent rigidity was less pronounced than in
competent wild-type cells.
Figure 3:
Rigid domains in membranes of cells probed
with DPH. Fluorescence measurements were made on cells labeled with DPH
(label/lipid molar ratio of 1:300) (see ``Experimental
Procedures'').
With trans-parinaric acid as a
probe, noncompetent cells exhibited phase transitions at 25 °C and
near 35 °C, presumably due to lipid-protein interactions
(Fig. 4). In competent cells, the second thermotropic transition
did not occur until near 56 °C, a reflection of the rigidity (32%
of the membrane matrix for wild-type cells and 20% for
ppk cells) imposed by the
PHB
Ca
polyP complex.
Figure 4:
Rigid domains in membranes of cells probed
with trans-parinaric acid. Fluorescence measurements were made
on cells labeled with trans-parinaric acid (label/lipid molar
ratio of 1:300) (see ``Experimental
Procedures'').
Competence Depends on Synthesis of PolyP
An
indication that the polyP in membranes of competent cells arises from
the de novo synthesis of the polymer rather than a
redistribution of cytosolic polyP was found in measurements of the
total content of polyP in competent and noncompetent cells
(I). For the ppk mutant, the
amount of polyP increased by about 50% when cells were rendered
competent, an increase that roughly matched the polyP content in
membranes of competent cells. Furthermore, the polyP in competent cells
as determined by PAGE (data not shown) is of a novel size, about 60
residues, compared to the much longer chains in the cytosol of
noncompetent cells.
and polyhydroxybutyrate
(PHB) in the transport of DNA through cell membranes to transform
bacteria. By means of specific enzymes, the data obtained in this study
have validated a role for polyP in making E. coli competent
for transformation and have added some insights into the mechanism.
Ca
polyP complex achieves competence
for E. coli is found in the physical changes observed in the
membranes of competent cells. As observed by freeze-fracture electron
microscopy (Reusch et al., 1987), incorporation of the
PHB
Ca
polyP complex in the cytoplasmic
membrane coincided with the appearance of semiregular, protein-free
plaques containing shallow particles.
within a surrounding cylinder of the
otherwise shapeless PHB helix (Cornibert et al., 1971). Such
structures would severely decrease the fluidity of the lipid bilayer by
impairing the movements of the phospholipid acyl chains in direct
contact with PHB and creating a preferential orientation for aligning
these chains. These interactions could, in turn, induce rearrangements
of the polar head groups that might produce defects facilitating the
penetration of DNA through the lipid matrix, as suggested earlier
(Reusch and Sadoff, 1988). In the case of electroporation (Calvin and
Hanawalt, 1988; Smith et al., 1990), an electric current
modifies the lipid bilayer by producing ``holes'' for DNA
entry.
Ca
polyP
complex and the alterations in membrane structure that accompany
competence were also observed, although less marked, in the
ppk
mutant which lacks the enzyme that
catalyzes the synthesis of the long-chain polyP (Crooke et
al., 1994). However, the mutant still contains a membrane-bound
form of polyP of about 60 to 70 residues. The development of competence
appears to be associated with a new synthesis of the short polyP as
indicated by a 50% increase in the total cellular polyP content
(I). The nature of the synthesis of this species of polyP,
presumably by a novel pathway, remains to be explored.
Table:
Identification of polyP in the membranes of
competent cells
Table:
PolyP distribution in the membrane fractions of
competent cells from ppk mutant
Table:
Total cellular content of polyP
increases in competent ppk cells
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.