©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inorganic Polyphosphates in the Acquisition of Competence in Escherichia coli(*)

Celina E. Castuma (1), Ruiping Huang (2), Arthur Kornberg (1)(§), Rosetta N. Reusch (2)(¶)

From the (1) Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 and the (2) Department of Microbiology, Michigan State University, East Lansing, Michigan 48824

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A complex of polyhydroxybutyrate (PHB), Ca, 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 PHBCapolyP complex perturbs the conformation of the lipid matrix, making it more permeable to charged molecules and thus allowing the entry of DNA.


INTRODUCTION

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 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).

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 CapolyP. 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.()

The identity and content of PHB in the novel membrane complexes have been established by chemical and immunological assays and by 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).

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.


EXPERIMENTAL PROCEDURES

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 MnCl4HO, 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 PHBCaPolyP Complex

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 25 cm) with 100 mM NaCl as eluent and analyzed by polyacrylamide gel electrophoresis (PAGE) (Clark and Wood, 1987).

The PHBCapolyP 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.

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 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).

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 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%.


RESULTS

Inorganic PolyP in the Membranes of Competent Wild-type and ppkCells-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 ().

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).

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 PHBCapolyP 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 PHBCapolyP 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.


DISCUSSION

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 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.

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 PHBCapolyP 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 PHBCapolyP complex in the cytoplasmic membrane coincided with the appearance of semiregular, protein-free plaques containing shallow particles.

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 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.

The presence of the PHBCapolyP 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

Assays with PPX and PPK were each used to estimate the polyP content (see ``Experimental Procedures''). Values are expressed in terms of phosphate residues.


  
Table: PolyP distribution in the membrane fractions of competent cells from ppk mutant

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).


  
Table: Total cellular content of polyP increases in competent ppk cells

PolyP was extracted and quantitated by assays with PPX and PPK (see ``Experimental Procedures''). Values are expressed in terms of phosphate residues.



FOOTNOTES

*
This work was supported by Grants GM-07581 and GM-33375 from the National Institute of General Medical Sciences and a National Science Foundation CAA Award (to R. N. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests may be addressed. Tel.: 415-723-6167; Fax: 415-723-6783.

To whom correspondence and reprint requests may be addressed. Tel.: 517-355-9307; Fax: 517-353-8957.

The abbreviations used are: PHB, polyhydroxybutyrate; polyP, polyphosphate; PPK, polyphosphate kinase; PPX, exopolyphosphatase; DPH, 1,6-diphenyl-1,3,5-hexatriene; NPN, N-phenyl-1-naphthylamine; MES, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.

R. N. Reusch, unpublished results.


ACKNOWLEDGEMENTS

We thank LeRoy Bertsch for helpful suggestions in preparing the manuscript.


REFERENCES
  1. Ahn, K., and Kornberg, A.(1990) J. Biol. Chem. 265, 11734-11739 [Abstract/Free Full Text]
  2. Akiyama, M., Crooke, E., and Kornberg, A.(1992) J. Biol. Chem. 267, 22556-22561 [Abstract/Free Full Text]
  3. Akiyama, M., Crooke, E., and Kornberg, A.(1993) J. Biol. Chem. 268, 633-639 [Abstract/Free Full Text]
  4. Calvin, W., and Hanawalt, P. C.(1988) J. Bacteriol. 170, 2796-2801 [Medline] [Order article via Infotrieve]
  5. Castuma, C. E., Crooke, E., and Kornberg, A.(1993) J. Biol. Chem. 268, 24665-24668 [Abstract/Free Full Text]
  6. Clark, J. E., and Wood, H. G.(1987) Anal. Biochem. 161, 280-290 [Medline] [Order article via Infotrieve]
  7. Cornibert, J., Marchessault, R. H., Benovit, H., and Neill, G.(1971) Macromolecules 3, 741-746
  8. Crooke, E., Akiyama, M., Rao, N., and Kornberg, A.(1994) J. Biol. Chem. 269, 6290-6295 [Abstract/Free Full Text]
  9. Hanahan, D.(1983) J. Mol. Biol. 166, 557-580 [Medline] [Order article via Infotrieve]
  10. Kornberg, A.(1994) in Phosphate in Microorganisms: Cellular and Molecular Biology (Torriani-Gorini, A., Yagil, E., and Silver, S., eds) pp. 204-208, American Society for Microbiology, Washington, D. C.
  11. Kulaev, I. S., and Vagabov, V. M.(1983) Adv. Microb. Physiol. 24, 83-171 [Medline] [Order article via Infotrieve]
  12. Mandel, M., and Higa, A.(1970) J. Mol. Biol. 53, 159-162 [Medline] [Order article via Infotrieve]
  13. Osborn, M. J., Gander, J. E., Paris, E., and Carson, J.(1972) J. Biol. Chem. 247, 3962-3972 [Abstract/Free Full Text]
  14. Reusch, R. N.(1989) Proc. Soc. Exp. Biol. Med. 191, 377-381 [Abstract]
  15. Reusch, R. N.(1992) FEMS Microbiol. Rev. 103, 119-130
  16. Reusch, R. N., and Sadoff, H. L.(1983) J. Bacteriol. 156, 778-788 [Medline] [Order article via Infotrieve]
  17. Reusch, R. N., and Sadoff, H. L.(1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4176-4180 [Abstract]
  18. Reusch, R. N., Hiske, T. W., and Sadoff, H. L.(1986) J. Bacteriol. 168, 553-562 [Medline] [Order article via Infotrieve]
  19. Reusch, R., Hiske, T., Sadoff, H., Harris, R., and Beveridge, T.(1987) Can. J. Microbiol. 33, 435-444 [Medline] [Order article via Infotrieve]
  20. Reusch, R. N., Sparrow, A. W., and Gardiner, J.(1992) Biochim. Biophys. Acta 1123, 33-40 [Medline] [Order article via Infotrieve]
  21. Seebach, D., Brunner, A., Bürger, H. M., Schneider, J., and Reusch, R. N.(1994) Eur. J. Biochem. 224, 317-328 [Abstract]
  22. Shinitzky, M., and Barenholz, Y.(1978) Biochim. Biophys. Acta 515, 367-394 [Medline] [Order article via Infotrieve]
  23. Sklar, L. A.(1980) Mol. Cell. Biochem. 32, 169-177 [Medline] [Order article via Infotrieve]
  24. Smith, M., Jessee, J., Landers, T., and Jordan, J.(1990) Focus 12, 38-40
  25. Wood, H. G., and Clark, J. E.(1988) Annu. Rev. Biochem. 57, 235-260 [CrossRef][Medline] [Order article via Infotrieve]
  26. Wurst, H., and Kornberg, A.(1994) J. Biol. Chem. 269, 10996-11001 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.