1 Laboratory of Microbial Genetics, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea
2 Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology, Seoul 136-791, Korea
3 Department of Microbiology, Hannam University, DaeJeon 300-791, Korea
Correspondence
Yong Keun Park
ykpark{at}korea.ac.kr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence of the cfa gene determined in this study is AF417203.
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INTRODUCTION |
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Chang & Cronan (1999) suggested that the formation of cyclopropane fatty acids (CFAs) in the membrane is a major factor that protects Escherichia coli from acid shock. They also demonstrated that the sensitivity to acid shock is dependent on CFAs themselves because cfa mutant strains became resistant to acid shock by incorporation of CFAs from the growth medium or by introduction of a functional cfa gene on a plasmid.
Since the discovery of lactobacillic acid in 1950, CFAs have been detected in membrane phospholipids of a variety of eubacteria (Goldfine, 1972). These unusual fatty acids are formed in situ by the transfer of a methyl group from S-adenosyl-L-methionine to a double bond of unsaturated fatty acids (UFAs) of a phospholipid molecule (Law, 1971
; Huang et al., 2002
). CFA synthase, which catalyses this reaction, is encoded by the cfa gene (Grogan & Cronan, 1984
, 1986
; Taylor & Cronan, 1976
). CFA synthase binds to the bilayer of the phospholipid substrate and cyclopropanates the phospholipid (Taylor & Cronan, 1979
). This unique membrane modification occurs preferentially in the late exponential and early stationary phase (Cronan, 1968
; Cronan et al., 1979
; Law, 1971
). In E. coli the cfa gene has been shown to have two promoters (Wang & Cronan, 1994
): one promoter has the consensus sequence of a
70-dependent promoter whereas the other is growth phase dependent and is recognized by the alternative sigma factor RpoS (also called
38 and
S). Wang & Cronan (1994)
suggested that the CFA synthesis during the stationary phase is due to the increased transcription of cfa mediated by the RpoS-dependent promoter in E. coli. The
70-dependent promoter is responsible for the low level of CFA synthesis in the exponentially growing cultures. It was also suggested that the growth-phase-dependent synthesis of CFA in E. coli was due to the instability of CFA synthase (Chang et al., 2000
; Wang & Cronan, 1994
). Chang & Cronan (1999)
demonstrated that CFA formation could be considered as a conditional and post-synthetic modification of the bacterial membrane.
It has been reported that Salmonella spp. have CFAs in the cell membrane (Grogan & Cronan, 1997; Lechivalier, 1977
; Saha & Chakraborty, 1992
); however, little is known about CFA in S. typhimurium, except for its presence in the membrane. The aim of this study was to determine the pattern of CFA synthesis, the growth phase dependence of the expression of CFA synthase, and the role of CFA in the response to acid shock during stationary phase in S. typhimurium.
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METHODS |
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Mutation of the CFA synthase gene (cfa).
In order to delete part of the cfa gene (deletion fragment, 870 bp), fusions of DNA fragments were generated by PCR as follows. To clone the upstream part of the cfa gene, the primers KCFAU1 (5'-gagctcTAACATGGTGGGGGAGTT-3') and KCFAU2 (5'-ggatccAACCGAAAATTGTTAGCGT-3') were used; the 5' tails, which contained cleavage sites for SacI and BamHI (underlined) are shown in lower case. The primers KCFAD1 (5'-ggatccTTGCGAAGGTCTGGATGT-3') and KCFAD2 (5'-tctagaGCGCGTAAATACCACCTG-3') were used for amplifying the downstream fragment; the 5' tails, which contained cleavage sites for BamHI and XbaI (underlined) are shown in lower case. The PCR products were cloned into pGEM T-easy vector (Promega) and the cloned vectors were digested with restriction enzymes as described above. Subsequently, the digested upstream and downstream fragments of cfa gene were ligated by T4 ligase (Takara). Using the product of this ligation as a template, a further PCR was carried out with the primers KCFAU1 and KCFAD2; the product was cloned into pGEM T-easy vector (forming pTDCF).
In order to insert a drug-resistance marker (ampicillin-resistance gene, bla) between the upstream and downstream fragments, the ampicillin-resistance gene derived from the pGEM T-easy vector was amplified by the primers amp1 (5'-cccggatccGGCACTTTTCGGGGAAAT-3') and amp2 (5'-cccggatccACTCACGTTAAGGGATTTTG-3'), which carry 5' tails (lower case) and a cleavage site for BamHI (underlined). The PCR product was digested with BamHI and cloned into pTDCF (forming pTAPC). Plasmids pTDCF and pTAPC were digested with SacI and XbaI, and each fragment was cloned into the suicide plasmid pDMS197, which contains the counter-selectable sacB marker. The resulting clones, named pDCF and pAPC, respectively, were transformed into E. coli 7213, and the transformants were conjugated with wild-type S. typhimurium (Edwards et al., 1998
; Fu & Voordouw, 1997
). The cfa deletion mutants and bla insertion mutants were selected in a medium containing 5 % (w/v) sucrose and ampicillin, as described by Edwards et al. (1998)
.
The acid tolerance response (ATR) assay.
The acid-induced stationary-phase ATR was measured as described by Bang et al. (2000). Briefly, cells were grown in 3 ml minimal E medium (pH 8·0; 37 °C; shaking) overnight for 16 h. Then 500 µl sample of each strain was harvested by centrifugation and washed in an equal volume of EG broth at pH 8·0 for the unadapted culture. The samples were reharvested, and the pellets were resuspended in 3 ml EG broth at pH 3·0 for challenge. Aliquots were collected at timed intervals, and viable counts were measured by serial dilution and plating on LB agar. The results are representative of triplicate experiments, with variability observed within 50 % of the reported value.
Cloning of a functional cfa gene and complementation.
To clone the functional cfa gene, the cfa structural gene plus its upstream region was amplified by PCR with the synthetic oligonucleotide primers CCFA-L (5'-cgcggatccGACCACCACCAGCGTAAT-3') and CCFA-R (5'-cccaagcttGTCGACGGCATCAGTTTG-3'), which contain cleavage sites for BamHI and HindIII (underlined) within their 5' tails (lower case). The template for PCR was obtained from the wild-type strain UK-1. The PCR product was digested with BamHI and HindIII and cloned into pACYC184, to produce pACF, containing the functional cfa gene.
After culturing overnight, wild-type, rpoS and cfa mutants of S. typhimurium were inoculated into 50 ml fresh LB medium and incubated at 37 °C with vigorous shaking until the OD600 reached 0·5. Cells were chilled in ice, centrifuged, and the pellets were washed twice with ice-cold glycerol/water, and resuspended in a volume of 15 % glycerol/water equal to that of the pellet. Samples of cells (50 µl) were electroporated with 10 ng pACF by using a Gene Pulser II Electroporation system (Bio-Rad). The transformants were tested for their acid shock sensitivity and fatty acid formation phenotype.
Purification of CFA synthase.
The CFA synthase structural gene cfa was amplified by PCR with the synthetic oligonucleotide primers ECFA-L (5'-ggatccATGAGTTCATCGTGTATAGAA-3') and ECFA-R (5'-aagcttATGCGGGGGAGATGATTA-3'); the 5' tails (lower case) contained cleavage sites for BamHI and HindIII (underlined). The PCR product was digested with BamHI and HindIII and cloned into pGEX-KG vector (forming pECFA). The CFA synthaseGST fusion protein was purified according to the manufacturer's manual (Amersham Pharmacia Biotech). Glutathione-Sepharose 4B resin (Amersham) was added to the solution containing the purified fusion protein, mixed gently for 30 min at room temperature, and the mixture was centrifuged at 500 g for 3 min. After washing the resin four times with 15 ml PBS buffer to remove unbound proteins, CFA synthase was eluted with 20 mM Tris/HCl (pH 8·4) containing 0·04 U thrombin, 50 mM NaCl and 2·5 mM CaCl2 at 22 °C for 22 h. The CFA synthase thus obtained was further purified by preparative SDS-PAGE. The fractions containing CFA synthase were pooled and dialysed against 0·1 M potassium phosphate buffer (pH 7·5).
Antibody preparation.
Using the CFA synthase purified as described above, antiserum was prepared by injecting rabbits with 150 µg of the protein dispersed in complete Freund's adjuvant. Two weeks and four weeks later, the rabbits were immunized again with 50 µg CFA synthase dispersed in incomplete Freund's adjuvant.
Western blotting.
The CFA synthase protein was detected by immunoblot analysis as described previously (Bang et al., 2000), with the following modifications. Western blots were treated with a 1 : 1000 dilution of polyclonal antibody against CFA synthase, followed by incubation with an alkaline-phosphatase-labelled anti-rabbit antibody. Blots were developed by using a chemiluminescent detection kit. To enhance specificity, the antiserum was incubated with proteins from the cfa mutant S. typhimurium strain at 4 °C. The data were obtained from three independent experiments.
Preparation of total fatty acids.
To prepare the total fatty acids from stationary-phase cells, cultures were grown overnight for 16 h. To obtain cells in the exponential growth phase, overnight-cultured cells were inoculated by 1/100 dilution to fresh LB medium and were cultured until the OD600 reached 0·6. Cells thus obtained were centrifuged for 20 min at 5000 r.p.m. The pellet was washed twice with distilled water. The washed pellets were frozen at 20 °C, and then freeze-dried with a vacuum freeze drier.
The total fatty acids extract of cells was prepared as described by Moss & Dees (1975) with the following modifications. The lyophilized cells were saponified by hydrolysis with 10 % (w/v) sodium hydroxide in 50 % (v/v) methanol. The saponified solution was acidified with hydrochloric acid and then methylated with 14 % boron trifluoride in methanol. Fatty acid methyl esters were extracted using n-hexane/diethyl ether (1 : 1, v/v). The organic extract was washed with base by gently mixing 1·2 % sodium hydroxide (w/v) in saturated sodium chloride. The upper phase of the washed extract was analysed by GC/MS using an HP5890 Series II gas chromatograph combined with an HP5970B mass spectrometer (Hewlett Packard).
Analysis of fatty acid methyl esters by GC/MS.
Fatty acid methyl derivatives were analysed by GC/MS at 70 eV in scan mode, using an HP-5MS capillary column (25 mx0·20 mm i.d., with a film thickness of 0·33 µm). The GC injection port temperature was 250 °C and the transfer line temperature was 300 °C. The split ratio was 1 : 10. Helium was used as a carrier gas (flow rate 0·8 ml min1). The operating conditions were as follows: an initial oven temperature of 150 °C was held isothermal for 4 min; a temperature programme of 5 °C min1 continued to a final oven temperature of 300 °C, which was held isothermal for 11 min in order to remove contaminants. The total run time was 45 min. The fatty acids were identified by comparison of retention times and mass fragmentation patterns with authentic standards purchased from Supelco.
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RESULTS |
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Formation of CFAs in S. typhimurium
To investigate the composition of membrane fatty acids during the various growth phases in S. typhimurium, the total fatty acids from exponential- and stationary-phase cells were prepared and analysed by GC/MS. During the exponential phase only low levels of C17 CFAs were detected (Fig. 1a). In stationary-phase cultures the level of C17 CFAs increased and C19 CFAs were also present (Fig. 1b
). However, the content of saturated fatty acids in the membrane increased only slightly and the content of hydroxy fatty acids was constant regardless of the growth phase (Fig. 1c
). The total CFA level began to increase between mid-exponential phase and the stationary phase (OD600 0·60·9) (Fig. 1c
). Fig. 1(c)
also shows a dramatically altered composition of fatty acids: unsaturated fatty acids (UFAs) were nearly quantitatively replaced by their cyclopropane derivatives during stationary phase: methyl cis-9-hexadecanoate (C16 : 19) and methyl cis-11-octadecanoate (C18 : 111) were replaced by methyl cis-11,12-methylenehexadecanoate (C17 : 0
) and methyl cis-9,10-methyleneoctadecanoate (C19 : 0
), respectively.
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We demonstrated that the synthesis of CFA is partly dependent on the RpoS sigma factor (Table 2). Previous data showed that the induction of RpoS in the stationary phase rendered cells resistant to general stresses such as acid shock (Lee et al., 1994
; Small et al., 1994
). Here, we constructed an rpoS cfa double mutant and compared its acid tolerance response with each single mutant. During stationary phase, the double mutant strain completely lacked CFAs in the membrane (data not shown), whereas residual CFAs remained in the rpoS mutant strain (Table 2
). It was expected that strains lacking RpoS as well as CFA would be more sensitive than either single mutant strain. As shown in Fig. 5(a)
, the resistance of the double mutant to acid shock was indeed more impaired than that of the single mutant.
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DISCUSSION |
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In this work, we also showed that in the stationary phase, all C18 UFAs were converted to C19 CFAs in rpoS mutant cells overexpressing CFA synthase (Fig. 2d, Table 2
). In mid-exponential phase, however, all C18 UFAs were not converted to C19 CFAs in rpoS mutant cells overexpressing CFA synthase (Table 2
). The discrepancy could be due to different levels of expression of CFA synthase in the exponential and the stationary phase. Our data, however, showed that the CFA synthase level in an rpoS mutant harbouring pACF was constant regardless of the growth phase (Fig. 4
, lanes 5, 6, 7). The results suggest that the distribution ratio of total CFAs in S. typhimurium may be determined by the specific activity of an enzyme in vivo during the stationary phase. Alternatively, another factor, which is induced by RpoS in stationary phase, may affect the distribution ratio of total CFAs. However, it is certain that the CFA synthase level is essential to the synthesis of CFAs.
In E. coli, the cyclization of fatty acid acyl chains is generally regarded as a means of controlling the penetration of undesirable molecules in order to adapt the cells to adverse conditions (Chang & Cronan, 1999; Grogan & Cronan, 1997
). Among Helicobacter isolates, those identified as gastric colonizers tend to produce a large amount of CFAs. The isolates identified as intestinal colonizers, on the other hand, generally do not produce a large amount of CFAs (Haque et al., 1996
). In addition, Enterococcus faecalis mutants resistant to folic acid antagonists were found to be CFA deficient and more sensitive to acid than the parental strain (Jungkind & Wood, 1974
). Chang & Cronan (1999
) suggested that the protection of cells from acid shock by CFAs might also apply to other bacteria. Here, we considered that protection against acid shock by forming CFAs could also apply to S. typhimurium. It is known that RpoS from S. typhimurium regulates other processes as well as synthesis of CFAs (Lee et al., 1994
; this study). Therefore, it is thought that other physiological processes regulated by RpoS can mask the effects of CFA content on acid shock survival. In this study, however, we showed that in S. typhimurium, an rpoS cfa double mutant was more sensitive to acid shock than either single mutant strain, as is the case for E. coli (Chang & Cronan, 1999
; this study, Fig. 5a
). These results indicate that the residual presence of CFAs in an rpoS mutant allows cells to survive longer against acid shock, because formation of CFAs was not completely blocked in rpoS mutants of E. coli and S. typhimurium (Chang & Cronan, 1999
; this study). Therefore, it is thought that the presence of CFA in the cell membrane is essential to acid tolerance in both organisms.
In this study, we also showed that complete conversion of UFAs to CFAs did not restore the stationary-phase ATR of S. typhimurium fully (Fig. 5b). This incomplete restoration of acid resistance may be due to the deficiency of UFAs in the cell membrane. Chang & Cronan (1999)
showed that incorporation of exogenous C18 UFA into a fabA/cfa mutant strain partially restored acid resistance in E. coli. From these data, we suggest that UFAs as well as CFAs are necessary for full acid resistance of S. typhimurium, although CFAs are a better barrier against acid shock than UFAs.
It has been reported that in S. typhimurium, RpoS was induced by mild acidic shock during the exponential phase (Lee et al., 1994, 1995
). Recently, de Jonge et al. (2003)
showed that under acid-adaptive conditions, both the C17 and the C19 CFA content of S. typhimurium strains increased. It was thus speculated that the acid-induced RpoS in the exponential phase might increase CFA synthase activity in S. typhimurium. Hence, we compared the CFA level in mildly acidic conditions (pH 5·8) and normal conditions (pH 7) during the exponential phase; we observed that the exposure to the acidic condition increased the CFA level (data not shown). It has been reported that the adaptation to mild (pH 5·8) or moderate (pH 4·4) acidic conditions renders cells tolerant to severe acid stress (pH 3) (Lee et al., 1995
). Therefore, it is suggested that the sustained tolerance to acid shock may be partly due to the increase of CFA level by the mild acid shock-induced RpoS during the exponential phase.
DNA base sequence variability in the rpoS genes of various wild-type strains has been reported. The variability correlated with phenotypic differences in the expression of RpoS-controlled genes (Ivanova et al., 1992). Such strain differences may explain the considerable variation in the CFA synthase level observed in several strains of E. coli K-12 expressing various plasmid and phage cfa clones (Grogan & Cronan, 1984
). Wang & Cronan (1994)
showed that E. coli strain FT1 had a lower CFA synthase activity than strain ZK126. They suggested that the difference in the CFA synthase level may be the result of different rpoS alleles, since the expression of another RpoS-regulated gene, poxB, was lower in FT1 than ZK126 (Wang & Cronan, 1994
). Here, we used only one S. typhimurium strain for the analysis of CFA formation. To elucidate the relationship between RpoS and the formation of the CFAs in S. typhimurium more extensively, the analysis of various Salmonella strains is required.
In conclusion, we have demonstrated the formation of CFAs, the physiological role of CFAs, and the expression of CFA synthase in S. typhimurium. We believe that these findings represent the first report of the physiological significance of CFAs in S. typhimurium. To elucidate the mechanism of the increase of CFA content by CFA synthase during the stationary phase in S. typhimurium, further investigation of the transcription of the cfa gene, and the stability and activity of the CFA protein, is required. The identification of other factor(s) regulating the formation of C19 CFA may also be worthwhile.
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ACKNOWLEDGEMENTS |
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Received 20 April 2004;
revised 14 July 2004;
accepted 27 August 2004.
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