Unité Microbiologie et Génétique Composante INSA, UMR CNRS-INSA-UCB 5577, INSA Bat 406, 20 Av Einstein, 69621 Villeurbanne, France1
Author for correspondence: Guy Condemine. Tel: +33 472 43 80 88. Fax: +33 472 43 87 14. e-mail: condemin{at}insa.insa-lyon.fr
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ABSTRACT |
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Keywords: general secretory pathway, protein secretion, proteinprotein interaction, secretin
Abbreviations: CMC, carboxymethylcellulose; GSP, general secretory pathway; PGA, polygalacturonate
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INTRODUCTION |
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Besides this conserved core of 12 proteins, some GSP proteins are found in only a few bacteria. GspN has been found in Erw. carotovora but not in Erw. chrysanthemi (Lindeberg & Collmer, 1992 ). GspA, a protein with putative ATPase activity, has only been detected in Escherichia coli and A. hydrophila (exeA) (Francetic & Pugsley, 1996
; Jahagirdar & Howard, 1994
). GspS has only been identified in K. oxytoca, in the enterohaemorrhagic Esc. coli O157:H7 strain, in Erw. chrysanthemi and Erw. carotovora (PulS, EtpO and OutS) (dEnfert & Pugsley, 1989
; Schmidt et al., 1997
; Condemine et al., 1992
; Lindeberg et al., 1996
). These GspS proteins are outer-membrane lipoproteins that stabilize the secretins PulD and OutD and help their insertion in the outer membrane (Hardie et al., 1996
; Shevchik & Condemine, 1998
). The interaction requires a 62 aa domain present at the C-terminal extremity of PulD and OutD (Daefler et al., 1997
; Shevchik & Condemine, 1998
). This domain is also present in the Erw. carotovora OutD protein but not in other secretins. Thus, GspS probably does not exist in other bacteria. GspB has been described in three bacteria: K. oxytoca, Erw. chrysanthemi and A. hydrophila (dEnfert & Pugsley, 1989
; Condemine et al., 1992
; Jahagirdar & Howard, 1994
). It probably also exists in Erw. carotovora (Lindeberg et al., 1996
). In the first two bacteria, the gene gspB is clustered with gspS, whereas in A. hydrophila exeB forms an operon with exeA. ExeB forms a complex with the ATPase ExeA (Schoenhofen et al., 1998
) and is required for efficient secretion of aerolysin in this bacterium. By contrast, a K. oxytoca pulB mutant has no phenotype. In Erw. chrysanthemi, the absence of OutB leads to an intermediate phenotype, with secretion of pectate lyase reduced by 30%. In this study, we analysed the cellular localization of OutB and showed that a mutation in outB can be suppressed by overproduction of OutD. Our results suggest that OutB interacts with OutD.
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METHODS |
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Construction of OutBBlaM protein fusions.
The outB gene was cloned between the EcoRI and KpnI sites of plasmid pJBSKpn, a derivative of plasmid pJBS633 (Broome-Smith et al., 1990 ) that contains an additional KpnI site upstream of the blaM gene. The resulting construct was digested with KpnI and HindIII that cleaves the plasmid downstream of outB to produce exonuclease III-insensitive and sensitive sites, respectively. Deletions in outB were generated with the Exonuclease III deletion kit (USB). After ligation and transformation, the Kanr clones obtained were first replicated on GL plates containing Amp (50 µg ml-1) to identify those producing in-frame OutBBlaM fusion proteins. Four Ampr clones were retained. For these, the MIC of ampicillin required to prevent colony formation by a single cell was determined and nucleotide sequencing was performed to characterize the outBblaM junction.
OutB purification.
The periplasmic soluble form of OutB (PelBspOutB) was overproduced in the NM522/pABSC3 strain. Cells were grown in 100 ml LB containing chloramphenicol. At an OD600 of 0·5, IPTG was added to 1 mM and, after 2 h additional growth, the cells were harvested by centrifugation for 5 min at 5000 g and frozen at -80 °C. The overproduced protein was extracted from cells by three cycles of freeze-thawing (Johnson & Hecht, 1994 ). The supernatant was loaded onto a 15% (w/v) preparative SDS-PAGE. The band containing PelBspOutB was cut out and the protein was extracted by three washes with 10 mM Tris/HCl pH 8·0, 0·1% SDS. The protein was concentrated and injected into a rabbit for antibody production by Valbex (Villeurbanne).
Protein labelling.
Overexpression and exclusive labelling of plasmid-encoded proteins was carried out using the T7 promoter/T7 polymerase system of Tabor & Richardson (1985) .
Gel electrophoresis and immunoblotting.
SDS-PAGE was usually performed according to Laemmli (1970) . Proteins were transferred onto nitrocellulose in a semi-dry apparatus and the membrane was incubated with antibodies and developed with the ECL detection kit (Amersham), as described previously (Shevchik et al., 1996
). The primary antibodies used were anti-OutB diluted 1:5000, anti-BlaM diluted 1:5000, anti-OutD diluted 1:3000 and anti-EGZ diluted 1:3000.
Isolation and analysis of cell fractions.
Exponentially growing cells (OD600 0·81·0) were used for cell fractionation. Cell-membrane fractionation was performed by sucrose gradient centrifugation, as previously described (Shevchik et al., 1996 ). NADH oxidase was assayed as described by Osborn et al. (1972)
. Crude membrane fractions were isolated by centrifugation (200000 g for 2 h) after French press disintegration of cells and resolubilized in 50 mM Tris/HCl pH 8·0.
Cross-linking experiments.
Bacteria were grown in LB medium to an OD600 of 1·0. An aliquot of 1 ml of culture was centrifuged, the bacteria were washed in 10 mM potassium phosphate buffer pH 6·8 and resuspended in the same volume of the buffer. The cells were incubated with 1% formaldehyde at room temperature for 30 min, washed in phosphate buffer, and resuspended in SDS sample buffer for analysis by SDS-PAGE and immunoblotting.
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RESULTS |
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Topological analysis of OutB
Alignment of the sequence of OutB with those of PulB and ExeB shows that the three proteins present a low homology throughout their length with some more highly conserved regions in their C-terminal part (Fig. 1). Their hydropathy profiles, analysed by the method of Kyte & Doolittle (1982)
, are also very similar. In their N-terminal parts, they contain, after a short hydrophilic segment, a highly hydrophobic region of about 20 residues which does not present the features of a signal sequence but which could anchor the protein in the cytoplasmic membrane (Fig. 2
). To elucidate the OutB topology, we constructed in-frame fusions between OutB and the topology probe ß-lactamase. Four in-frame OutBBlaM fusions were obtained with the fusion at residues 5, 6, 41 and 128 of OutB. The first two gave a MIC<2 µg ml-1 to isolated Esc. coli colonies on GL Amp plates whereas the last two gave an MIC of 15 µg ml-1. This suggests that OutB possesses a short N-terminal cytoplasmic extremity, a transmembrane segment and a large C-terminal periplasmic domain. The same MICs were obtained when the constructs were introduced into Erw. chrysanthemi strain A350, in an outS mutant (A1903), or in a polar outC mutant (A1919) that lacks all the other Out proteins, confirming that the topology of the OutBBlaM fusions is the same in Erw. chrysanthemi as in Esc. coli and shows that no other Out protein is able, by interacting with the fusions, to modify the MIC produced.
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DISCUSSION |
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However, OutB presents some unusual properties. When the membranes were separated by sucrose gradient fractionation, OutB was not found in the cytoplasmic-membrane fractions but in higher density fractions that contain outer-membrane proteins. This interaction is strong enough to survive the fractionation procedures. These results may indicate that OutB is, in vivo, anchored in the inner membrane but that its large periplasmic domain allows for an interaction of its C-terminal part with the outer membrane. This interaction would not require an additional Out protein since the separation of OutB with the outer-membrane fractions is not modified by the presence or absence of other Out proteins. Such an association with the outer membrane of a protein predicted to be anchored in the inner membrane has been described for the pseudopilin PulG (Pugsley & Possot, 1993 ), PulC (Possot et al., 1999
) and for ExeB (Howard et al., 1996
). The repeated observation of the presence of ExeB in the outer-membrane fractions has been considered as a contamination (Howard et al., 1996
) but this could be a characteristic of the members of the GspB family. The presence of some GSP components, such as PulC and PulG, in both membrane fractions is not totally surprising since it seems normal that some of the GSP proteins will link the inner-membrane and outer-membrane components of the machinery. The energy-transducing protein TonB, which is involved in the transport of molecules across the outer membrane, has also been found associated with both membranes (Letain & Postle, 1997
). Some properties of ExeB (sequence similarity, topology, a proline-rich segment, a high pI) are similar to those of TonB. Although they are rather rich in proline (9%), there is no proline domain in OutB or PulB (Fig. 1
), their theoretical pIs are 7·4 and 7·1, respectively, and they have no obvious homology with TonB. Thus, the homology between ExeB and TonB may not reflect a general feature of members of the GspB family.
An interesting point of this study is that an outB mutation can be suppressed by the overexpression of outD either from the chromosomal copy or from a plasmid. This observation could explain the discrepancy observed between the phenotypes of exeB, outB and pulB mutants. The effect of the outB mutation on pectate lyase secretion was previously tested (Condemine et al., 1992 ). However, to induce pectate lyase synthesis, galacturonate or polygalacturonate had to be added to the culture medium. Their presence also induced the outCO operon, increasing the level of OutD synthesis. In these conditions, secretion of pectate lyase was only slightly reduced. When an outB mutant was tested for EGZ secretion, which does not necessitate the addition of an inducer, the outB mutant appeared to be secretion deficient. Aerolysin secretion can be tested in A. hydrophila without addition of an inducer, leaving the expression of the exeCN operon at its basal level (Jiang & Howard, 1991
). The exeB mutant appears to be deficient for secretion. On the other hand, the K. oxytoca pulB mutant did not appear to be affected for pullulanase secretion. The phenotype of the pulB mutant was tested in the presence of maltose, which induced both pullulanase and PulD synthesis. It is probable that if pullulanase synthesis could be made independent from maltose induction, a pulB mutant would be secretion deficient.
The suppression of the outB phenotype by overexpression of outD led us to look for evidence of interactions between the two proteins. In Erw. chrysanthemi, OutB was stabilized by OutD. Coexpression of OutB and OutD in the same strain increased the quantity of OutD detectable in the bacteria. We have previously used this type of experiment to show the interactions between OutD and OutS, and between OutD and the secreted proteins (Shevchik et al., 1997 ). However, the OutD stabilization obtained with OutB was less efficient than that observed with OutS or PelB, suggesting that the interaction might be weaker. This lesser effect prevented us from testing the protection by OutB of truncated derivatives of OutD: most of them are unstable and could not be stabilized by OutB to a level allowing for their detection. Protection of OutD by PelBspOutB shows that the anchoring of OutB in the inner membrane is not required for the OutBOutD interaction.
What might the role of OutB be? It has been proposed that ExeB could function with the putative ATPase ExeA to transduce metabolic energy to the opening of the secretion pore (Schoenhofen et al., 1998 ). However, the presence of OutB and PulB in two strains in which no ExeA homologue has been found and the presence of GspA in Esc. coli that does not contain a GspB make this hypothesis doubtful in the case of OutB. The results presented here suggest that OutB is necessary for the proper functioning of the Out machinery, probably by interaction with OutD. The presence of outB and pulB next to outS and pulS could indicate that the two proteins may collaborate to pilot OutD and PulD to the outer membrane. Elucidation of the role of OutB requires further investigation.
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ACKNOWLEDGEMENTS |
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Received 18 October 1999;
accepted 22 November 1999.