alteration in the contents of unsaturated fatty acids in dnaa mutants of escherichia coli

Alteration in the contents of unsaturated fatty acids indnaA mutants of Escherichia coli

Emi Suzuki, 1 Taemi Kondo, 2 Masaki Makise, 2 ShinjiMima,2 Kenji Sakamoto, 2 Tomofusa Tsuchiya 2 andTohru Mizushima 2*1Laboratory for Pharmacokinetics, PreclinicalDevelopment Laboratories, Research and DevelopmentDivision, Nippon Hoechst Marion Roussel, Ltd, 1-3-2,Minamidai, Kawagoe 350-11, Japan.2Faculty of Pharmaceutical Sciences, OkayamaUniversity, 1-1-1, Tsushima-naka, Okayama 700, Japan.

Summary

DnaA protein, the initiator of chromosomal DNA repli-cation in Escherichia coli , has a high affinity for acidicphospholipids containing unsaturated fatty acids.We have examined here the fatty acid compositionof phospholipids in dnaA mutants. A temperature-sensitive dnaA46 mutant showed a lower level ofunsaturation of fatty acids (ratio of unsaturated tosaturated fatty acids) at 42 8C (non-permissive tem-perature) and at 37 8C (semi-permissive temperature),but not at 28 8C (permissive temperature), comparedwith the wild-type strain. Plasmid complementationanalysis revealed that the dnaA46 mutation is respon-sible for the phenotype. Other temperature-sensitivednaA mutants showed similar results. On the otherhand, a cold-sensitive dnaAcos mutant, in which over-initiation of DNA replication occurs at low tempera-ture (28 8C), showed a higher level of unsaturation offatty acids at 28 8C. Based on these observations, wediscuss the role of phospholipids in the regulationof the activity of DnaA protein.

Introduction

The initiation of replication of chromosomal DNA is co-ordinated with cell division. It has been proposed thatDNA replication in bacterial cells is initiated on membranesand that the activities of replication proteins are regulatedby membrane components (the replicon model) (Jacob etal., 1963).

Chromosomal DNA replication in Escherichia coli is initi-ated at a unique sequence, oriC, the origin of chromosomal

DNA replication. DnaA protein, the initiator of DNA replica-tion in E. coli, specifically binds to oriC and causes duplexopening (Hirota et al., 1970; Fuller and Kornberg, 1983;Bramhill and Kornberg, 1988). DnaA protein has a highaffinity for ATP and ADP; the ATP-binding form is activein the oriC replication system in vitro, whereas the ADP-binding form is inactive (Sekimizu et al., 1987). Synthe-sized organic compounds that were designed to block theATP binding to DnaA protein specifically inhibited oriCDNA replication in vitro (Mizushima et al., 1996a). Wehave shown recently that induction of an artificially con-structed mutant DnaA protein, which has decreasedATPase activity, led to overinitiation of DNA replicationin cells, resulting in a dominant lethal phenotype (Mizu-shima et al., 1997a). These results suggest that adeninenucleotide binding to DnaA protein regulates the initiationof chromosomal DNA replication in E. coli cells.

Acidic phospholipids, such as cardiolipin (CL) and phos-phatidylglycerol (PG), decrease the affinity of adeninenucleotide for DnaA protein (Sekimizu and Kornberg,1988; Yung and Kornberg, 1988; Castuma et al., 1993;Mizushima et al., 1996b). Thus, it has been proposedthat phospholipids regulate the activity of DnaA proteinin cells (Sekimizu and Kornberg, 1988). This notion wassupported by recent genetic studies (Xia and Dowhan,1995; Shinpuku et al., 1995; Mizushima et al., 1996c).Phospholipids affect the activity of DnaA protein eitherpositively or negatively in vitro. That is, phospholipidsinhibit the binding of ATP to DnaA protein, resulting inthe inhibition of DNA replication, whereas they activatedthe ADP-binding form of DnaA protein to the ATP-bindingform in the presence of a high concentration of ATP bystimulating the exchange reaction of ADP with ATP (Seki-mizu and Kornberg, 1988). Thus, the role of phospholipidsin the regulation of the activity of DnaA protein in cellsremains to be elucidated.

We reported recently that the amounts of a-ketoacyl(acyl carrier protein) synthase II and long-chain fatty acidtransport protein, which are involved in fatty acid metabol-ism, were increased in temperature-sensitive dnaA mutants(Ohba et al., 1997), suggesting alteration in the fatty acidcomposition in the mutants. We speculated that examina-tion of the fatty acid composition in dnaA mutants wouldprovide useful information for understanding the role ofphospholipids in the regulation of the activity of DnaA pro-tein. Accordingly, in the present study, we have investigated

Molecular Microbiology (1998) 28(1), 95–102

Q 1998 Blackwell Science Ltd

Received 4 September, 1997; revised 7 January, 1998; accepted 9January, 1998. *For correspondence. E-mail [email protected]; Tel. and Fax (86) 251 7958.

m

the influence of mutations in the dnaA gene on the fattyacid composition of E. coli cells. We focused on the unsatu-rated fatty acids, because the interaction of phospholipidswith DnaA protein requires unsaturated fatty acids in thephospholipids (Yung and Kornberg, 1988).

Results

Phospholipid composition of the dnaA46 mutant

We used temperature-sensitive dnaA mutants, includingthe dnaA46 mutant, to examine the influence of mutationsin the dnaA gene on the phospholipid and fatty acid com-positions. We reported previously that these mutantsshowed inhibition of DNA synthesis at 428C but not at378C or 288C, whereas they showed other phenotypes,such as immotility, a higher level of DNA supercoilingand low activity of thymidine transport at 378C but not at288C (Mizushima et al., 1994; 1996d; 1997b). Alterationsin the expression of fatty acid metabolism-related proteinsin the dnaA mutants were also observed at 378C but not at288C (Ohba et al., 1997). In this study, we examined thecompositions of phospholipids and fatty acids in dnaAmutants at 288C, 378C and 428C. Even at 428C, tempera-ture-sensitive dnaA mutants could grow to reach an opticaldensity of 1.0 at 600 nm under the present conditions. Thismay be because the synthesis of macromolecules otherthan DNA continues for some period in these mutants at428C. We observed filamentous cell structures of tempera-ture-sensitive dnaA mutants after incubation at 428C, butnot at 378C or 288C (data not shown).

We examined first the phospholipid compositions of thednaA46 mutant and the wild-type strain. The ratio betweenphosphatidylethanolamine (PE), CL and PG was slightlyaffected by the mutation; the content of PE in the mutantwas higher or lower than that in the wild-type strain at288C or 428C respectively (Table 1). The alteration in thephospholipid composition caused by elevation of the incu-bation temperature was manifested in different waysbetween the mutant and the wild-type strain: the amountof PE increased and the amounts of CL or PG decreasedupon elevation of the incubation temperature in the wild-type strain, whereas the elevation shifted the amounts ofphospholipids in the opposite direction in the dnaA46mutant (Table 1).

Decrease in unsaturated fatty acids in the dnaA46mutant

We compared the composition of fatty acids in the totalphospholipids of the dnaA46 mutant (KS1003) with thatin the wild-type strain (KS1001) (Fig. 1A). We focusedon the unsaturated fatty acids, because the interaction ofDnaA protein with phospholipids depends on the presenceof unsaturated fatty acids in the phospholipids (Yung and

Kornberg, 1988). A striking difference in the content offatty acids between the mutant and the wild-type strainwas observed for C16:1 (palmitoleic acid) and C18:1 (cis-11,cis-vaccenic acid), which are the major unsaturated fattyacids (Fig. 1A). Their amounts in the mutant decreasedmore drastically by elevating the incubation temperaturethan did those in the wild-type strain (Fig. 1A). We deter-mined the level of unsaturation of fatty acids (the ratio ofunsaturated to saturated fatty acids) in the dnaA46 mutantand the wild-type cells. As shown in Fig. 2, the level ofunsaturation of fatty acids in the total phospholipids ofthe mutant was lower than that of the wild-type strain athigher temperatures (378C and 428C) but not at 288C.

Analysis of the fatty acid composition of each phospho-lipid revealed that the influence of the dnaA46 mutation onthe fatty acid composition was different between CL andthe other two phospholipids (PE and PG). In general, thefatty acid composition of CL was not significantly affectedby the dnaA46 mutation and the incubation temperaturewhen compared with the compositions of PE and PG.For example, the amounts of C18:1 (cis-11) in PG and PEof the mutant growing at 428C were much lower, whereasthe amount in CL was much the same as that in the wild-type strain (Fig. 1B–D). As a result, the decrease in thelevel of unsaturation of fatty acids in the dnaA46 mutantat higher temperatures is apparent in PE and PG but notin CL (Fig. 2).

To confirm that the drastic decrease in the level ofunsaturation of fatty acids in KS1003 at higher tempera-tures is caused by the dnaA46 mutation, we carried out acomplementation experiment using the pHB10S plasmidcarrying the wild-type dnaA gene (Shinpuku et al., 1995).When introduced into KS1003, the pHB10S plasmid com-plemented both the temperature-sensitive growth pheno-type (data not shown) and the phenotype of the lower

Q 1998 Blackwell Science Ltd, Molecular Microbiology, 28, 95–102

Table 1. Composition of phospholipids in the dnaA46 mutant and thewild-type strain.

Phospholipid (mol%)

Strain Growth temperature (8C) PE CL PG

KS1001 28 72 11 17(wild type) 37 82 10 9

42 85 7 8

KS1003 28 81 8 10(dnaA46 ) 37 81 9 10

42 76 12 12

Exponentially growing KS1001 (wild type) and KS1003 (dnaA46 )were centrifuged when the optical density at 600 nm reached 0.5.After extraction of the total phospholipids, individual phospholipidswere separated by solid-phase extraction. Their contents were thencalculated from the fatty acid contents measured by the capillaryGC method, as described in Experimental procedures. Averagevalues of triplicates are given, and the deviation was less than 5%of each value.

96 E. Suzuki et al.

level of unsaturation of fatty acids (Table 2). The vector,pBR322, did not affect the level of unsaturation of fattyacids in KS1003 (Table 2). These results indicate that thedecrease in the level of unsaturation of fatty acids inKS1003 was indeed caused by the dnaA46 mutation.

Fatty acid compositions of other temperature-sensitive dnaA mutants

A number of temperature-sensitive dnaA mutants havebeen isolated, and the positions of their mutations havebeen identified (Skarstad and Boye, 1994). We examinedthe fatty acid composition in other temperature-sensitivednaA mutants, and they all showed lower levels of unsa-turation of fatty acids at 378C but not at 288C comparedwith the wild-type strain (Table 3), as in the case of thednaA46 mutant (Table 2). The levels of unsaturationof fatty acids in the dnaA205 (KS1006) and dnaA508(KS1007) mutants, which have mutations in the C- andN-terminal regions of DnaA protein respectively, were


Fig. 1. Fatty acid composition in the dnaA46 mutant. Exponentially growing KS1001 (wild type) and KS1003(dnaA46 ) were centrifuged whenthe optical density at 600 nm reached 0.5. Extraction of the total phospholipids and purification of each phospholipid were performed asdescribed in Experimental procedures. The contents of fatty acids in the total phospholipids (A), PE (B), CL (C) and PG (D) were determinedas described in Experimental procedures. The composition of each fatty acid is shown as the relative value to the total content of fatty acids.Average values of triplicates were given, and the deviation was less than 5% of each value. Fatty acids: C14:0, myristic acid; C14:1,myristoleic acid; C16:0, palmitic acid; C16:1, palmitoleic acid; DC17:0, cis-9-10-methylene-hexadecanoic acids; C18:0, stearic acid; C18:1cis-9,oleic acid; C18:1cis-11, cis-vaccenic acid; C18:2, linoleic acid.

Fig. 2. Effect of growth temperature on the ratio of unsaturatedfatty acids in the dnaA46 mutant and the wild-type strain. The ratioof unsaturated to saturated fatty acids (mol mol¹1) was calculatedusing the data presented in Fig. 1.

Fatty acid composition in dnaA mutants 97

more than half of the wild-type strain’s level (Table 3). Incontrast, the levels of unsaturation of fatty acids in thednaA602 (KS1008) mutant, which has mutations in theATP-binding domain of the protein, was less than a quarterof that in the wild-type strain (Table 3). In all dnaA mutantstested, the decrease in the level of unsaturation of fattyacids was apparent in PE and PG but not in CL (Table3), as in the case of the dnaA46 mutant (Table 2).

Increase in unsaturated fatty acids in the dnaAcosmutant

The results described above suggest that a decrease inthe activity of DnaA protein in cells led to the lower levelof unsaturation of fatty acids. There is another type ofdnaA mutant, a dnaAcos mutant, in which overinitiationof chromosomal DNA replication was observed at low tem-perature (288C), but not at 428C (Kellenberger-Gujer et al.,1978; Katayama and Kornberg, 1994). The replication

activity of DnaA protein seems to be much higher in thismutant at 288C (Kellenberger-Gujer et al., 1978; Katayamaand Kornberg, 1994). For a further examination of the rela-tionship between the activity of DnaA protein and the fattyacid composition, we investigated the fatty acid composi-tions in the dnaAcos mutant (KA441) and the wild-typestrain (KH5402) growing at 288C, 378C and 428C. As inthe case of temperature-sensitive dnaA mutants incu-bated at 428C, the dnaAcos mutant can grow to reachan optical density of 1.0 at 600 nm even at 288C (data notshown). As shown in Fig. 3, the dnaAcos mutation affectedthe fatty acid composition of total phospholipids, especiallywhen the incubation was performed at a low temperature(288C). For example, the amount of C16:1 was higher inthe mutant than in the wild-type cells at 288C. The levelof unsaturation of fatty acids in the total phospholipids ofthe dnaAcos mutant was much higher than that of thewild-type strain at low temperatures (288C or 378C) butnot at 428C (Fig. 4). Analysis of the fatty acid composition


Table 2. Plasmid complementation analysis forthe phenotype of a lower level of unsaturationof fatty acids in the dnaA46 mutant.

Ratio of unsaturated fatty acidsGrowth Phospholipidtemperature

Strain (8C) Total PE CL PG

KS1001 28 1.26 1.37 0.37 2.04(wild type) 37 1.07 1.08 0.58 1.60KS1003 28 1.28 1.37 0.51 1.81(dnaA46 ) 37 0.49 0.49 0.37 0.58KS1003/pHB10S 28 1.31 1.40 0.46 2.08

37 1.05 1.03 0.68 1.49KS1003/pBR322 28 1.40 1.49 0.62 1.95

37 0.41 0.39 0.33 0.59

Exponentially growing KS1001 (wild type), KS1003 (dnaA46 ), KS1003 harbouring pHB10S(the wild-type dnaA gene) and KS1003 harbouring pBR322 (vector) were centrifuged whenthe optical density at 600 nm reached 0.5. After extraction of the total phospholipids, individualphospholipids were separated by solid-phase extraction. The content of fatty acids was deter-mined, and the ratio of unsaturated to saturated fatty acids (mol mol¹1) was calculated asdescribed in Experimental procedures. Average values of triplicates are given, and the devia-tion was less than 5% of each value.

Table 3. Ratio of unsaturated fatty acids invarious dnaA mutants. Ratio of unsaturated fatty acids

Growth Phospholipidtemperature

Strain (8C) Total PE CL PG

KS1001 28 1.26 1.37 0.37 2.04(wild type) 37 1.07 1.08 0.58 1.60KS1006 28 1.39 1.44 0.74 1.89(dnaA205 ) 37 0.73 0.72 0.52 0.98KS1007 28 1.28 1.35 0.55 1.80(dnaA508 ) 37 0.71 0.67 0.33 0.95KS1008 28 1.33 1.39 0.68 1.81(dnaA602 ) 37 0.24 0.20 0.29 0.36

Exponentially growing KS1001 (wild type), KS1006 (dnaA205 ), KS1007 (dnaA508 ) andKS1008 (dnaA602 ) were centrifuged when the optical density at 600 nm reached 0.5. Theratio of unsaturated fatty acids (mol mol¹1) was calculated as described in the footnote toTable 2. Average values of triplicates are given, and the deviation was less than 5% of eachvalue.

98 E. Suzuki et al.

of each phospholipid revealed that the increase in the levelof unsaturation of fatty acids in the dnaAcos mutant at alower temperature is apparent in PE and PG but not inCL (Fig. 4).

Effect of the expression of DnaA protein inoriC-deleted mutant

As for the molecular mechanism behind the decrease inthe amount of unsaturated fatty acids in the tempera-ture-sensitive dnaA mutants, we considered that DnaAprotein is involved in the transcriptional regulation ofgenes that affect the fatty acid metabolism, as DnaA pro-tein regulates the expression of various genes negatively(Skarstad and Boye, 1994). On the other hand, it wasalso possible that the inhibition of DNA replication by thednaA mutations caused the decrease in unsaturated fattyacids. To understand the mechanism, we examined theeffect of the expression of the wild-type DnaA protein inthe KA450 strain. As the KA450 strain has an amber muta-tion in the dnaA gene and its oriC is deleted (Katayama,1994), the strain grows in a manner independent of thefunction of DnaA protein. In other words, the expressionof DnaA protein in KA450 does not affect DNA replication.

Thus, if inhibition of DNA replication caused the decreasein unsaturated fatty acids in the temperature-sensitive dnaAmutants, the expression of the wild-type DnaA proteinin KA450 would not affect the amount of unsaturatedfatty acids. KA450 cells were transformed with pHB10S,


Fig. 3. Fatty acid composition in the dnaAcos mutant. Exponentially growing KH5402 (wild type) and KH441 (dnaAcos) were centrifugedwhen the optical density at 600 nm reached 0.5. The contents of fatty acids in the total phospholipid (A), PE (B), CL (C) and PG (D) weredetermined as described in the legend to Fig. 1. Average values of triplicates were given, and the deviation was less than 5% of each value.

Fig. 4. Effect of growth temperature on the ratio of unsaturation offatty acids in the dnaAcos mutant and the wild-type strain. Theratio of unsaturated to saturated fatty acids (mol mol¹1) wascalculated using the data presented in Fig. 3.


the expression plasmid for the wild-type DnaA protein, orpBR322 (vector), and the compositions of fatty acids in thetotal phospholipids of the transformants were examined.As shown in Fig. 5, the composition of fatty acids in thetotal phospholipids in KA450 was affected by the expres-sion of the wild-type DnaA protein. The level of unsatura-tion of fatty acids in the total phospholipids of KA450/pHB10S and KA450/pBR322 were 0.88 and 0.69, respec-tively, indicating that the expression of DnaA proteinincreases the level of unsaturation of fatty acids even inthe KA450 strain. Thus, the results suggest that DnaA pro-tein affects the level of unsaturation of fatty acids by itsdirect involvement in fatty acid metabolism and not byaltering DNA replication.

Discussion

In this study, we have shown that the level of unsaturationof fatty acids decreased in the temperature-sensitive dnaAmutants grown at higher temperatures. We have also shownthat the level of unsaturation of fatty acids increased in thednaAcos mutant grown at lower temperatures. As the repli-cation activity of DnaA protein in the temperature-sensitivednaA mutants at higher temperatures seems to decreaseand that in the dnaAcos mutant at lower temperaturesseems to increase, we have considered the possibilitythat the level of unsaturation of fatty acids is controlledaccording to the replication activity of DnaA protein.

The interaction of acidic phospholipids with DnaA pro-tein requires the presence of unsaturated fatty acids inthe phospholipids; the interaction of DnaA protein with PGwith unsaturated fatty acids is much stronger than thatwith PG with saturated fatty acids (Yung and Kornberg,1988). Thus, the observations in this study suggest that

the interaction of phospholipids with DnaA protein in thetemperature-sensitive dnaA mutants is weakened by ele-vating the incubation temperature and becomes strongerin the dnaAcos mutant by lowering the incubation tempera-ture. Taken together with the earlier observation that acidicphospholipids inhibited DNA replication in vitro throughinhibition of ATP binding to DnaA protein (Sekimizu etal., 1988), we propose that acidic phospholipids regulatethe activity of DnaA protein in cells negatively and thatcells adapt to a decrease or increase in the activity ofDnaA protein by decreasing or increasing the inhibitoryeffect of phospholipids (i.e. by decreasing or increasingthe content of unsaturated fatty acids).

As for the molecular mechanism behind the alteration inthe amount of unsaturated fatty acids in dnaA mutants, theresults from experiments using KA450 (Fig. 5) suggestthat the alteration is not the result of the inhibition ofDNA replication by the dnaA mutations. Thus, we surmisethat DnaA protein is involved in the transcriptional regula-tion of genes that affect fatty acid metabolism. DnaA pro-tein regulates the expression of various genes negatively,such as the dnaA, mioC, rpoH, guaBA and uvrB genes,through specific binding to DnaA boxes located in the 58

upstream regulatory region or coding region of thesegenes (Skarstad and Boye, 1994). On the other hand, puri-fied DnaA protein alters DNA supercoiling through non-specific binding to DNA (Mizushima et al., 1996e). Wealso found an increase in DNA supercoiling in tempera-ture-sensitive dnaA mutants (Mizushima et al., 1996d). AsDNA supercoiling profoundly affects the transcription of var-ious genes (Higgins et al., 1988; Dorman et al., 1988; 1990;Mizushima et al., 1993; 1997c), we have proposed thatDnaA protein is involved in the regulation of gene expres-sion via alteration in DNA supercoiling (Mizushima et al.,1996d). We consider that DnaA protein is involved in theregulation of genes that affect the fatty acid compositionand that a change in the activity of DnaA protein causedby mutations affects this regulation, resulting in the alter-ation in fatty acid composition. We found recently thata-ketoacyl (acyl carrier protein) synthase II and long-chain fatty acid transport protein, which are involved infatty acid metabolism, were increased in the tempera-ture-sensitive dnaA mutants growing at high temperatures(Ohba et al., 1997). These enzymes seem to be candi-dates for enzymes that are responsible for alteration ofthe fatty acid composition in dnaA mutants.

Experimental procedures

E. coli strains

Temperature-sensitive dnaA mutants [KS1003 (dnaA46 ),KS1006 (dnaA205 ), KS1007 (dnaA508 ), KS1009 (dnaA602 )and its isogenic wild-type strain (KS1001)] were from our labora-tory stocks (Shinpuku et al., 1995). KA450 [DoriC-1071::Tn10,


Fig. 5. Effect of the expression of DnaA protein on fatty acidcomposition in KA450. Exponentially growing KA450/pHB10S (thewild-type dnaA) and KA450/pBR322 (vector) were centrifuged at378C when the optical density at 600 nm reached 0.5. The contentsof fatty acids in the total phospholipid were determined asdescribed in the legend to Fig. 1. Average values of triplicates aregiven, and the deviation was less than 5% of each value.

100 E. Suzuki et al.

rnhA199(Am), dnaA17(Am), trpE9829(Am), tyrA(Am), thr,ilv, thyA] (Katayama, 1994), a cold-sensitive dnaAcos mutant(KA441), and its isogenic parent strain (KH5402) were kindlyprovided by Dr T. Katayama (Kyushu University, Japan)(Katayama and Kornberg, 1994).

Analysis of fatty acid composition

Full-growth suspensions of E. coli cells were diluted 100-foldwith Luria–Bertani (LB) medium and cultured. Exponentiallygrowing E. coli cells in LB medium were harvested by centri-fugation when the optical density at 600 nm reached 0.5. Totallipids were extracted by the method of Bligh and Dyer (1959)and applied to SPE cartridges (NH2-phase and SI-phase) toseparate each phospholipid, using a solid-phase extractionmethod (Suzuki et al., 1993; 1997a,b).

The phospholipids were esterified with 14% trifluoroboronin anhydrous methanol, and their fatty acid contents wereanalysed by gas chromatography. The apparatus used wasa Hewlett Packard 5890A gas chromatograph with a capillarycolumn (0.25 mm internal diameter ×15 m, 0.25 mm, Supel-cowax 10; Supelco) and a hydrogen flame ionization detector.The column was operated at 1708C for 20 min, and the tem-perature was raised by 48C min¹1 to 1908C for 15 min. Heliumwas used as the carrier gas at a flow rate of 27 cm s¹1. Theinjection volume was 2 ml (split ratio 1:50).

Acknowledgements

We are grateful to Drs T. Katayama and K. Sekimizu (KyushuUniversity, Japan) for providing the bacterial strains and com-ments on this manuscript respectively. This work was fundedby grants for scientific research from the Ministry of Educa-tion, Sciences, Sports and Culture, Japan.

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alteration in the contents of unsaturated fatty acids in dnaa mutants of escherichia coli

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