Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Centre National de la Recherche Scientifique, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
Correspondence
Danièle Cavard
cavard{at}ibsm.cnrs-mrs.fr
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ABSTRACT |
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INTRODUCTION |
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The colicin A lysis protein (Cal) is responsible for colicin A release. It is synthesized like every lipoprotein as a precursor form (pCal) which is modified (pCalm) before being processed. The mature form of Cal polymerizes and is found in the outer membrane (Cavard, 1991). Colicin A is found in every cell compartment, except in the inner membrane. In the outer membrane, colicin A is complexed with an outer-membrane protein, either a porin or OMPLA. These hetero-oligomers of colicin A, called colicins Au, are stable in urea and in SDS, and dissociate after boiling in SDS, as do the Cal polymers. In most colicins Au, colicin A interacts through its C-terminal domain with either OmpC or OmpF porins (Cavard, 2002
). Such an interaction has previously been demonstrated in vitro (Dover et al., 2000
). A role for colicins Au in colicin A release has been suggested, but is not obvious since cells devoid of porins and, consequently, of most colicins Au, secrete colicin A. Furthermore, cells that do not express Cal produce colicins Au, although they do not release colicin A (Cavard, 2002
).
Whether colicins Au play a role in colicin A secretion, they must interact with Cal. Release of colicin A was studied in strains differing in expressing Cal. Two steps of colicin A release were described, one of which at least was dependent on Cal. Cal was found in extracellular fractions together with colicins Au. It is suggested that colicins Au and Cal interact and are part of a specific machinery for colicin A secretion.
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METHODS |
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Growth conditions.
Strains were grown at 37 °C with shaking in LuriaBertani (LB) medium with ampicillin (100 µg ml1) as required. To induce colicin synthesis, mitomycin C, which is a mutagenic agent, was added at 300 ng ml1 to a culture whose optical density value at 600 nm had been adjusted to 1. Sodium azide (2 mM) was added when necessary at the time of induction.
Colicin A assays.
These were performed as described previously (Cavard, 1976). A 100 µl aliquot of a dilution in LB medium of the sample to be assayed was added to 1·3 ml of MC4100 cells grown in LB medium to an OD600 value of 0·25. After incubation for 20 min at 37 °C with shaking, 100 µl of an SDS solution at 7·5 mg ml1 was added. The OD600 value of the samples was measured 10 min later. The percentage ratio was determined from the OD ratio of colicin-treated and control samples and was converted to killing units (KUs). The number of KUs obtained was multiplied by the dilution factor of the assayed sample and by 3·25x108, which corresponds to the number of MC4100 cells added to the assayed sample.
Extraction of released and unreleased colicin A.
After mitomycin C addition, the culture in LB medium (50 ml) was incubated for various times, before being centrifuged at 12 500 g. The supernatant was added to 60 % ammonium sulfate (390 g l1). The precipitate was harvested by centrifugation and re-dissolved in 5 ml of 20 mM Tris/HCl (pH 6·8), 10 % (v/v) glycerol (SP). The cell pellet was suspended in 4 ml of 10 mM Tris/HCl (pH 6·8), 500 mM NaCl. After 30 min at room temperature, cells were re-centrifuged and incubated in 3 ml water for 30 min before being centrifuged again. The NaCl and water supernatants were combined and designated the salt extract (SE). The final cell pellet was suspended in 1 ml of 10 mM Tris/HCl (pH 8), 1 mM EDTA, 100 µg lysozyme. After 1 h at room temperature, 2 ml of 10 mM Tris/HCl (pH 8) was added. Cells were incubated for 2 h at 4 °C before centrifugation, then washed once with 1 ml of 10 mM Tris (pH 6·8). Both supernatants were mixed and corresponded to the lytic extract (LE).
Urea and trypsin treatment.
SP, SE and LE were dialysed in 10 mM Tris/HCl (pH 6·8) before centrifugation at 75 000 g (TLA 100.2 rotor; Beckman) for 30 min at 4 °C. The pellets were resuspended in 8 M urea in 10 mM Tris/HCl (pH 6·8), vortexed, incubated at room temperature for 30 min and washed once. The pellets were resuspended in 100 mM Tris/HCl (pH 8) to which 100 µg bovine trypsin ml1 (Sigma) was added. The suspension was incubated at 37 °C for 30 min. Trypsin was then inactivated by the addition of 1 mM PMSF.
Protein analysis.
Proteins were resolved by SDS-PAGE, as described previously (Cavard, 2002). The SDS-polyacrylamide gels contained either 11 or 6 % acrylamide. Urea/SDS-polyacrylamide gel contained 7 % acrylamide and 7 M urea. To detect Cal, analyses were performed in Tricine/SDS gels (Schägger & von Jagow, 1987
), with a continuous running gel of 13·5 % acrylamide. Proteins were transferred onto nitrocellulose filters (200 nm pore size; Schleicher & Schuell). The membranes were incubated first with monoclonal antibody (mAb) 1C11 to colicin A at 1 : 5000, or with polyclonal antibodies (pAbs) to OmpF (Cavard, 2002
) or to Cal (Howard & Lindsay, 1992
) at 1 : 1000, and, then, with phosphatase-conjugated anti-mouse or anti-rabbit antibodies as appropriate. Protein bands were visualized by incubation with Nitro Blue Tetrazolium in the presence of MgCl2 and 5-bromo-4-chloro-3-indolyl phosphate disodium salt.
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RESULTS |
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To obtain unreleased colicin A, the pellet of induced cells was suspended in buffer containing 1 M NaCl and centrifuged to collect the SE, as described by Herschman & Helinski (1967) and Schwartz & Helinski (1971)
. However, the amount of colicin A recovered increased when the NaCl concentration of the buffer was decreased in such a way that it was higher in the absence than in the presence of NaCl (not shown). The centrifuged cells were then incubated in 1/10 volume of 10 mM Tris/HCl (pH 6·8) containing 0·5 M NaCl, centrifuged and washed in water before re-centrifugation. The NaCl and water washes were combined and represented the SE. The cells in the pellet were then treated with EDTA/lysozyme and the resulting sphaeroplasts were lysed at low osmotic pressure. The lysed cells were centrifuged and washed once. The pooled supernatants corresponded to the LE. The final pellets containing mainly DNA and cell envelopes were discarded.
Each pool obtained from induced W3110(pColA9) cells was analysed on SDS-polyacrylamide gels stained with Coomassie brilliant blue (Fig. 1). The amount of colicin A varied significantly in each pool with the time of induction, as expected. At zero time of induction, there was no colicin A in any of the pools. After 90 min induction, there was a significant amount of colicin A in the LE and a low amount in the SE. After 180 min induction, most colicin A was found in the SP extract, low amounts in the SE and traces in the LE. In W3110(pJMM1) cells devoid of Cal, colicin A was only found in the LE at all times after induction. Thus, with the help of Cal, colicin A synthesized in the cell cytoplasm moved first to a cell location where it could be extracted by washing with buffer, before being released into the medium.
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Cal and colicins Au are present in the insoluble fraction of each pool of released and unreleased colicin A
The presence of Cal and colicins Au in the three pools of colicin A was not expected since they are membrane-associated and should not be present in aqueous solutions. To know whether they were associated with either membrane fragments or organelles such as pili or vesicles, the SP, SE and LE pools obtained from W3110 degP(pColA9) cells were dialysed and centrifuged at high speed. The pellets were resuspended in 8 M urea, centrifuged, washed and resuspended in 100 mM Tris/HCl (pH 8) before being treated or not with trypsin and analysed on urea/SDS-polyacrylamide gel in order to detect colicins Au. Colicins Au were indeed best detected in SDS-polyacrylamide gels containing 7 M urea, confirming they are not formed on the SDS-polyacrylamide gels and suggesting they were associated with complexes of high molecular masses. Urea is known to suppress non-specific aggregation of membrane proteins in SDS-polyacrylamide gels (Soulié et al., 1996). On the blot of the gel probed with mAb to colicin A, colicins Au were detected without monomeric colicin A in the unheated samples of the insoluble fractions treated with urea. They dissociated into monomeric colicin A after boiling. Colicins Ao were present in the heated as in the unheated samples with a band with an apparent molecular mass of about 260 kDa. All colicin A species were destroyed by trypsin. On the blot probed with pAbs to OmpF, colicins Au were detected in the unheated samples, but not in the trypsin-treated ones. In the heated samples, OmpC and OmpF were detected in both trypsin-treated and untreated fractions. The same results were obtained from each pool of colicin A (shown for SE in Fig. 3
a). Thus, colicins Au could be isolated from monomeric colicin A by centrifugation, but not from colicins Ao. The colicin part of colicins Au was sensitive to trypsin, but not their porin part, indicating that colicin A was associated with porins integrated into the outer membrane.
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Thus, both Cal and colicins Au were found extracellularly in the insoluble fractions of each pool of released and unreleased colicin A.
Steps of colicin A release are impaired in cells with an unacylated Cal precursor
Whether one or both steps of colicin A release were dependent on Cal, they should be modified in cells with a non-functional Cal. The above experiments were repeated in cells carrying a plasmid with a mutated cal gene. Amino acid substitutions of the cysteine residue present at the N-terminal end of mature Cal inhibit Cal acylation and processing and make the cal gene product unstable (Cavard et al., 1987). Plasmids pAL16 and pAK31, which contain, respectively, the C1T and C1P substitutions in the cal gene product, were introduced into W3110 degP cells since a Cal form has been shown to be a substrate of the DegP protease (Cavard et al., 1989
). After induction, the C1T (see below) and C1P (not shown) Cal precursors were expressed and remained stable, indicating that the DegP protease hydrolysed the unacylated precursor of Cal (pCal).
The production and release of colicin A were analysed after induction of W3110 degP cells carrying pColA9 or pAL16 (Fig. 4a) or pJMM1 (not shown). In W3110 degP(pColA9) cells, the distribution of colicin A in the three pools SP, SE and LE was similar to that obtained in W3110(pColA9) cells, but the amount of colicin A released was greater (83 % of the KUs were found in the medium) due to the increased Cal stability, as reported previously (Cavard et al., 1989
). In contrast, in W3110 degP(pAL16) cells, the colicin A content of each pool was drastically modified, as 2x1011 KUs were found in the medium (8 % of the total KUs), 6x1011 KUs in the SE (26 % of the KUs) and 1·5x1012 in the LE (65 % of the KUs). Colicin A release was inhibited as expected, but some colicin might be extracted in the SE in contrast to what was observed in the absence of Cal, as the harvest of colicin A in W3110 degP(pJMM1) cells was similar to that observed in W3110(pJMM1) cells. Both steps of colicin A release were impaired, the second step more drastically than the first, indicating that pCal was not as efficient as Cal.
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Loss of OmpF in the absence of Cal in degP cells
Colicins Au were detected similarly in the unheated samples of W3110 degP cells carrying pColA9 or pAL16 or pJMM1 when analysed on a blot probed with mAb to colicin A. However, on a blot probed with pAbs to OmpF, they were not detected in the unheated samples of the W3110 degP(pJMM1) cells, while they were in the cells carrying either pColA9 or pAL16 (Fig. 5). In the heated samples, both OmpC and OmpF were present in the W3110 degP cells carrying either pColA9 or pAL16, while only OmpC was detected in the cells harbouring pJMM1 cells. W3110 degP(pJMM1) cells behave like an ompF mutant in which colicins Au are detected by mAb to colicin A, but not by pAbs to OmpF (Cavard, 2002
). W3110 degP cells lost OmpF after transformation with pJMM1, even when not induced for colicin A production (Fig. 5b
), suggesting that the colicin AOmpF complex made during spontaneous colicin A production was toxic in the absence of Cal. Either wild-type Cal or non-functional C1T Cal protected the cells, suggesting an interaction between Cal and/or pCal with the colicin AOmpF complex.
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The export of colicins Au to the outer membrane is Sec-independent
The formation of colicins Au has been shown to be a rapid process as monitored by radiolabelling of their heat-labile forms. Kinetics indicated that they rapidly reached the outer membrane (Cavard, 2002). To determine whether colicins Au were using the Sec secretion pathway as porins do (Danese & Silhavy, 1998
), induction of colicin A was performed in temperature-sensitive (ts) sec mutants. Isogenic strains HJM114, CJ105 secA ts and CJ107 secY ts carrying pColA9 were grown at 30 °C and induced for colicin A production at 42 °C. In these conditions, the export of Sec-exported proteins such as
-lactamase was inhibited as described previously (Cavard, 1992
). After 3 h induction, samples of the cultures were run on an urea/SDS-polyacrylamide gel (Fig. 6
a). On the blot probed with mAb to colicin A, colicins Au were similarly observed in the unheated samples of wild-type and sec ts mutant cells. Monomeric colicin A and colicins Ao were detected regardless of whether the cells had been boiled in sample buffer before loading. On the blot probed with pAbs to OmpF, colicins Au were detected in the unheated samples of each strain tested, whereas the two porins OmpC and OmpF were observed in the heated samples. Thus, colicins Au seemed to require neither SecA nor SecY to exhibit heat-modifiability.
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DISCUSSION |
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Many lines of evidence for interactions between Cal and colicins Au are reported. First, Cal and colicins Au were both found in the pools of released and unreleased colicin A, demonstrating they were loosely associated with the outer membrane and might become extracellular. Both Cal and colicins Au co-localized after centrifugation of the pools and were insoluble in 8 M urea, suggesting they were associated with the same organelle. Second, colicins Au produced either in the presence of pCal or in the absence of Cal were unstable and some of them dissociated into colicin A monomers. Third, in the absence of Cal and/or pCal, degP cells lost OmpF, suggesting that colicins Au made of colicin A bound to OmpF were toxic and interacted with Cal and/or pCal which detoxified them.
The extracellular release of colicins Au might be due to their interaction with Cal since it did not occur in the absence of Cal. Porins, which are part of colicins Au complexes, are integral outer-membrane proteins and, consequently, refractory to removal into aqueous solutions. In contrast, Cal binds to the membrane by its diglyceride moiety as do all lipoproteins and this hydrophobic association might be removed (Jakubowski et al., 2003; Sagulenko et al., 2001
). Both Cal and colicins Au might be present in a structure that would constitute either a barrier or a conduit for colicin A. Cal would allow association of the structure with the outer membrane. The Cal sequence is homologous to that of VirB7, the lipoprotein of the type IV secretion system of A. tumefaciens (Fig. 7
), as pointed out previously (Shirasu et al., 1990
). VirB7 forms homodimers and heteromultimeric complexes at the outer face of the outer membrane (Anderson et al., 1996
; Spudich et al., 1996
; Baron et al., 1997
; Jakubowski et al., 2003
; Sagulenko et al., 2001
). In the same way, homodimers of Cal and/or pCal were observed and Cal seemed to interact with hetero-oligomeric complexes such as colicins Au. Preliminary results indicated that expression of the virB7 gene was as deleterious for cells as that of the cal gene, but did not complement the cal gene deletion as observed by transformation of W3110(pJMM1) cells with pPC978 (not shown).
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Until now, colicin A secretion was thought to be caused by the loss of integrity of the bacterial envelope since a drop in the absorbance of the cultures, called quasi-lysis, occurs at the time of colicin A release (Pugsley & Schwartz, 1984; Baty et al., 1987
). Quasi-lysis is provoked by outer membrane phospholipase A (OMPLA) activation following the induction of the colicin lysis protein (Dekker et al., 1999
). However, it is not known whether the cells lysed during release are the induced cells or the uninduced ones in which either the uptake or the action of mitomycin C does not take place. Numerous uninduced cells that do not contain colicin have been observed by electron microscopy in colicin A-induced cultures (Cavard et al., 1984
) and by immunofluorescence microscopy in colicin K-induced cultures (Mulec et al., 2003
). Lysis of the uninduced cells would explain the presence of cytoplasmic and periplasmic proteins in the medium together with colicin A, but colicin A would be released by a specific machinery present in induced cells.
Colicins Au appeared to be formed by binding of colicin A to mature porins already incorporated in the outer membrane. Their assembly did not need the Sec translocase in contrast to that of porins. Porins are produced as precursors that are processed after Sec translocation by the leader peptidase before integration into the outer membrane. Colicin A lacks a signal peptide and is thus exported by an unknown pathway first to the periplasm where it has been observed in cells expressing Cal (Cavard, 2002), suggesting an interaction between Cal and colicin A for crossing the inner membrane. From the periplasm, colicin A would associate with integral outer-membrane proteins such as OmpC and OmpF, OMPLA and maybe LamB and OmpT, to form the heterogeneous group of colicins Au. Colicins Au are produced in an ompT null mutant (Cavard, 2002
), but proteolysis occurred during colicins Au purification which was inhibited by benzamidine (not shown), an OmpT-specific inhibitor (White et al., 1995
), suggesting the presence of OmpT, or homologues, in some colicins Au complexes.
The two gene products of the colicin A operon might be involved in the biogenesis of a machinery for colicin A transport. By making complexes with membrane proteins, both colicin A and Cal might be able to assemble a machinery as efficient as the one encoded by the 10 to 14 genes required for other protein secretion processes described in Gram-negative bacteria (Pugsley, 1993).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 March 2004;
revised 13 May 2004;
accepted 15 June 2004.
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