Laboratoire de Biologie Molé culaire des Corynébactéries, Institut de Génétique et Microbiologie, UMR C8621 CNRS, Bât. 4091, and Laboratoire des Biomembranes, UMR 8619 CNRS, Bât. 4302, Université Paris XI, 91405 Orsay Cedex, France
Centre de Génétique Moléculaire, CNRS, 91190 Gif sur Yvette, France3
ORSAN SA, 46 rue de Nesle, BP 42, 80190 Mesnil Saint Nicaise, France4
Author for correspondence: Gérard Leblon. Tel: +33 1 69 15 62 81. Fax: +33 1 69 15 57 12. e-mail: leblon{at}igmors.u-psud.fr
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ABSTRACT |
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Keywords: S-layer, freeze fracture, carbon regulation
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INTRODUCTION |
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Very little is known about the regulation of S-layer protein synthesis. Multiple transcription initiation sites have been identified in the operon encoding cell-wall proteins in Bacillus brevis 47 (Adachi et al., 1989 ) and regulation of S-layer protein gene expression has also been observed (Adachi et al., 1991
). The half-life of mRNA has been found to be long: 1015 min in Caulobacter crescentus (Fisher et al., 1988
), 22 min in Aeromonas salmonicida (Chu et al., 1993
), 15 min in Lactobacillus acidophilus (Boot et al., 1996a
) and 14 min in Lactobacillus brevis (Kahala et al. , 1997
). Changes in the S-layer protein have been described in Campylobacter fetus (Garcia et al., 1995
; Dwokin & Blaser 1996
), Lactobacillus acidophilus ATCC 4356 (Boot et al., 1996b
), Bacillus stearothermophilus (Sára & Sleytr, 1994
; Sára et al., 1996
) and Thermus thermophilus HB8 (Olabarría et al. , 1996
). In T. thermophilus HB8, the C- terminal fragment of the S-layer SlpA protein binds to the 5' untranslated leader region of the slpA mRNA, providing evidence for translational auto-regulation in S-layer gene expression (Fernández-Herrero et al., 1997
).
In the amino-acid-producing bacterium Corynebacterium glutamicum , two major proteins, PS1 and PS2, with apparent molecular masses of 67 and 63 kDa (Joliff et al., 1992 ) have been identified in the cell wall. Corynebacterium belongs to the actinomycete subdivision of the Gram-positive bacteria and has a high G+C content (Liebl & Sinskey, 1988
). The gene encoding the PS2 protein (cspB) has been characterized in C. glutamicum (Peyret et al., 1993
). Chami et al. (1995)
showed that if C. glutamicum was grown on solid medium, the surface of cells was totally covered with a highly ordered, hexagonal surface layer, whereas if it was grown in liquid medium, the cell and fracture surfaces were only partially covered by ordered arrays. This partial covering was correlated with there being less PS2 associated with the cell wall. Typically, cells grown on solid medium contained 34 mg PS2 protein (g bacterial dry wt) -1, whereas cells grown in liquid medium to stationary phase contained 16 mg (g dry wt)-1. This suggests that PS2 production depends on the physiological and metabolic state of the cell.
Here we report that the amount of S-layer PS2 protein present depends on the carbon source available in the growth medium. We also show that a low level of PS2 production is associated with partial covering of the cell surface by a crystalline array.
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METHODS |
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pMF2 was derived from pMC1403 (Casadaban et al., 1980 ) as follows. The aphIII gene from Streptococcus faecalis (Trieu-Cuot & Courvalin, 1983
), isolated from pCGL243 (Reyes et al., 1991
) as a XhoI/NotI cassette, and a NotI/XhoI fragment of the icd locus of C. melassecola ATCC 17965 were inserted together into the SalI site of pMC1403 to give pMF2. The icd gene encoding the isocitrate dehydrogenase of C. melassecola was cloned by heterologous complementation of the E. coli mutant EB 106 as described by Eikmanns et al . (1995)
. The chromosomal DNA library used for the isolation of the icd gene of C. melassecola was described by Reyes et al. (1991)
. The E. coliCorynebacterium shuttle vectors pCGL482 and pCGL815 were described by Peyret et al. (1993)
.
Bacterial cells were cultured in MCGC minimal medium as described by Von der Osten et al. (1989) , except that citrate (used as a chelating agent) was replaced by deferoxamine. This medium contained (l-1): 3 g Na2HPO4 , 6 g KH2PO4, 2 g NaCl, 8 g (NH 4)2SO4, 0·4 g MgSO4 . 7 H2O, 40 mg FeSO4 . 7 H2O, 3·9 mg FeCl3, 0·9 mg ZnSO4 . 7 H2O, 0·3 mg CuCl2 . 2 H2O, 3·9 mg MnSO4 . H2O, 0·1 mg (NH4)6Mo7O24 . 4 H2O, 0·3 mg Na2 B4O7 . 10 H2O, 84 mg CaCl2, 4 mg biotin, 20 mg thiamin and 3 mg deferoxamine. The carbon sources were added to a final concentration of 180 mM (30 g l-1 for glucose and 20 g l -1 for lactate) or 90 mM for single or mixed carbon source experiments, respectively. Corynebacterium cells were grown aerobically at 34 °C with shaking (250 r.p.m.). Chloramphenicol (Cm; 15 µg ml-1) or kanamycin (Km; 25 µg ml-1) were added as required.
Construction of the PcspB'lacZ fusion.
The cspB promoter (PcspB) described by Peyret et al. (1993) was isolated as a 554 bp Eco RI/EcoNI fragment and ligated to an EcoNI/Bam HI adaptor constructed with the two synthetic oligonucleotides 5'-TCAAGGAGCCTTCGCCTCTATG-3' and 5'- GATCCATAGAGGCGAAGGCTCCTTG-3'. The resulting EcoRI/Bam HI fragment was inserted between the EcoRI and Bam HI sites of pMF2 to give pCGL1502 (Fig. 1a
), in which the cspB and lacZ genes are fused at the BamHI site. Thus, pCGL1502 carries an in-phase fusion between the first codon of cspB and lacZ. A 7·92 kb PstI fragment from pCGL1502 containing the PcspB'lacZ fusion was inserted into the single PstI site of the E. coliCorynebacterium shuttle vector pCGL482 to give pCGL1504 (Fig. 1b
).
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Extraction of PS2 protein and quantification of PS2 production.
Proteins were extracted from the cell walls of Corynebacterium cultures with 2% (w/v) SDS, as described by Peyret et al . (1993) . A bacterial pellet was obtained from 2 ml cell culture and suspended in 200 µl Tris/HCl buffer (50 mM, pH 6·8) containing 2% SDS. The suspension was heated to 100 °C for 5 min and centrifuged at 16000 g for 3 min. The supernatant containing the proteins associated with the cell wall (mainly PS2) was collected. This treatment does not solubilize the cytoplasmic membrane or result in cell lysis (Bayan et al., 1993
). SDS-PAGE was carried out as described by Laemmli (1970)
, with a 4% acrylamide stacking gel and a 10% acrylamide separating gel. The volume of protein extract used was calculated so as to give samples with equivalent optical density at 570 nm. The samples were denatured by heating at 100 °C for 5 min in the presence of 2% SDS and 1·25% (w/v) ß-mercaptoethanol in 50 mM Tris/HCl pH 6·8. The samples were then subjected to electrophoresis; under these conditions, PS2 protein had an apparent molecular mass of 63 kDa (Joliff et al., 1992
). After electrophoresis, the gels were stained with Coomassie brilliant blue R- 250. PS2 protein was determined by densitometry (ImagQuant) and 1 µg ß-galactosidase was loaded on the gel as a control for quantity. The amount of PS2 protein is expressed in mg protein (g bacterial dry wt)-1. Biomass (Bm) was determined as a function of optical density at 570 nm using an
correlation factor for each Corynebacterium strain: Bm=OD570/
. To determine the
factor, 200 ml bacterial cells were harvested from a culture in stationary phase with a known OD570 value by centrifugation for 20 min at 3500 g. The pellet was dried at 60 °C for 15 h and its weight was determined. This procedure was carried out for three independent cultures. The
factor was calculated for all carbon sources tested.
Freeze-fracture electron microscopy.
A bacterial suspension was placed between a thin copper holder and a thin copper plate and quenched in liquid propane, as described by Gulik- Krzywicki & Costello (1978) and Aggerbeck & Gulik- Krzywicki (1986)
. The frozen sample was fractured at -125 °C in vacuum of about 1·33x10 -5 Pa by removing the upper plate with a liquid-nitrogen- cooled knife in a Balzers 301 freeze-etching unit.
The fractured sample was etched at -105 °C for 35 min and a replica was produced with platinum-carbon or tungsten-tantalum (1·01·5 nm of metal deposited), backed with about 20 nm of carbon. The replica was cleaned by incubation overnight with chromic acid, washed with distilled water and observed in a Philips 410 electron microscope.
Glucose and lactate assays.
For glucose and lactate determinations in culture media, bacterial cells (2 ml) were centrifuged and the supernatant filtered through 0·45 µm pore filters (Millipore). Samples were stored at -20 °C until use. Extracellular glucose and lactate concentrations were determined with a sensitive colorimetric enzyme assay (Sigma Diagnostics glucose procedure no. 315 and Sigma Diagnostics lactate procedure no. 735, respectively). The kits for glucose and lactate assays were purchased from Sigma and used as recommended by the manufacturer.
ß-Galactosidase activity in cytoplasmic extracts.
Bacterial cells (20 ml of stationary phase culture) were collected by centrifugation and resuspended in 2 ml buffer A (100 mM Tris/HCl pH 8, 500 mM KCl, 1 mM MgSO4 . 7H2O, 0·4 mM MnSO4 , 4 mM DTT). The cell suspension was mixed with 1 g glass beads (106 µm diameter; Sigma), and was shaken for two periods of 5 min, each at 1800 vibrations min-1 (Retsch MM 2000). The suspension was centrifuged at 20800 g and the supernatant was used as a cytoplasmic extract. ß- Galactosidase activity was measured as follows. Cytoplasmic extract (270 µl) was mixed with 180 µl of a solution of 4 g ONPG l-1. The absorbance of the mixture at 420 nm (A420) was monitored for 10 min at room temperature. The protein concentration of the extract was also determined by the Lowry method (DC protein assay, Bio-Rad). Specific ß-galactosidase activity is expressed as [A420 min-1 (µg protein)-1]x1000.
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RESULTS |
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Experiments similar to those performed with C. glutamicum ATCC 14752 were then carried out with C. melassecola ATCC 17965 and Corynebacterium sp. 2262. For C. melassecola ATCC 17965, results similar to those observed with C. glutamicum ATCC 14752 were obtained (data not shown). The single carbon source results obtained with Corynebacterium sp. 2262 were similar to those for C. glutamicum ATCC 14752 (data not shown). In cultures with mixed carbon sources, no PS2 was produced in cultures from glucose-grown inocula and there was a decrease in PS2 production in cultures from lactate-grown inocula (results not shown). These observations suggest that the inhibitory effect of glucose consumption was predominant in this strain.
cspB expression level as a function of carbon source
We investigated the regulation of PS2 production by constructing an in-phase fusion between the PcspB promoter and the E. coli lacZ gene. First, pCGL1502 carrying an in-phase fusion between lacZ and the first codon of cspB was constructed in E. coli DH5F' (Fig. 1a
). As pCGL1502 cannot replicate in Corynebacterium, its transfer by electroporation may lead to its integration into the chromosome at either the icd locus or the cspB locus. We transformed the Corynebacterium sp. 2262 strain with pCGL1502 and selected five kanamycin-resistant recombinant strains. The structure of the insertion in these recombinant strains was determined by Southern blotting and a single copy of pCGL1502 was found to have inserted by homologous recombination at the icd locus in each (data not shown). One recombinant strain, Corynebacterium sp. 2262::pCGL1502, was studied further.
Plasmid pCGL1504 was then constructed, containing the PcspBlacZ fusion, and able to replicate in Corynebacterium. This plasmid was constructed in E. coli DH5F' and transferred into E. coli JM110 (Fig. 1b
). pCGL1504 was introduced by electroporation into C. glutamicum ATCC 14752 and Corynebacterium sp. 2262, producing the recombinant strains ATCC 14752(pCGL1504) and 2262(pCGL1504) respectively.
ß-Galactosidase activity was measured after culture on MCGC minimal medium containing either glucose or lactate (Table 1). Activity was 2·4-fold higher in ATCC 14752(pCGL1504), 3·5-fold higher in 2262(pCGL1504) and 7·1-fold higher in Corynebacterium sp. 2262::pCGL1502 grown in the presence of lactate. These ratios are similar to those observed for PS2 production in lactate and suggested that regulation occurs mainly at the level of protein synthesis.
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DISCUSSION |
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The current lack of knowledge about the function of the S-layer in Corynebacterium makes it difficult to relate its regulation by a carbon source to a particular function. The natural carbon source used by Corynebacterium in its biotope is unknown. It is commonly found in the soil (Trautwetter & Blanco, 1988 ) and organic acids may be its principal substrates. The S-layer may therefore be associated with adhesion sites for exoenzymes, surface recognition and cell adhesion to substrates, as has been suggested for the S-layers of several other organisms (Beveridge et al., 1997
).
We showed, using a lacZ fusion, that PS2 production was mainly regulated by changes in cspB gene expression and that secretion was probably not a limiting step in PS2 accumulation at the cell surface. A few examples of the regulation of S-layer formation have been described (Bahl et al., 1997 ), but regulation by carbon source has not previously been reported. In B. brevis 47, one of the five promoters of the operon encoding cell wall proteins is specifically active in the exponential phase of growth (Adachi et al., 1989
). We observed that growth phase had an effect on PS2 production but the promoter sequences of cspB involved have not yet been determined. In T. thermophilus HB8 (Fernández-Herrero et al. , 1997
), there is evidence that slrA encodes a transcriptional repressor of the S-layer gene. It has been suggested that SlrA is also linked with other metabolic pathways, such as those involved in cell wall synthesis.
We observed co-metabolism of both substrates in all three strains, extending the observation by Cocaign (1992) of simultaneous glucose and lactate consumption in C. melassecola ATCC 17965. The inhibitory effect of glucose consumption on PS2 production was much greater in Corynebacterium sp. 2262 than in the other strains. This suggests that the use of metabolic pathways differs between strains. Glucose catabolism by central pathways in C. melassecola ATCC 17965 has been examined using NMR (Rollin et al., 1995
), enzymic (Cocaign-Bousquet et al., 1996
) and mathematical modelling (Pons et al., 1996
) approaches. These studies have shown that the pentose pathway is responsible for almost 50% of glucose catabolism in this strain. One of the key aspects of sugar catabolism in C. melassecola is the manner in which an adequate supply of NADPH is generated to meet anabolic requirements. The pentose phosphate pathway, which involves two NADP-dependent dehydrogenases, is the principal source of NADPH during growth on glucose. The differences in flux distribution for glucose-grown and lactate-grown cells shows clearly that different NADPH-generating reactions operate in lactate-grown cells. During growth on lactate, a modified tricarboxylic acid cycle involving malic enzyme and an unknown enzyme with pyruvate carboxylating activity is thought to operate, accounting for the apparent shortfall of NADPH for anabolic requirements (Cocaign-Bousquet & Lindley, 1995
). Such studies performed to analyse carbon flux in Corynebacterium sp. 2262 support the idea that major differences exist in metabolism of glucose in these two Corynebacterium strains (N. D. Lindley, personal communication).
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ACKNOWLEDGEMENTS |
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Received 7 May 1999;
revised 13 September 1999;
accepted 15 September 1999.