- Email: [email protected]
The inner workings of a quorum sensing signal generator
C 0 l\.1: l\.1: E N T Vievvpoint
The inner workings of a quorum sensing signal generator Simon Swift, Gordon S.A.B. Stewart and P. Williams -ACYI homo serine lactones (AHLs) are the signal mol• ecules used by the LuxRI family of quorum sensing regula• tors!. In vitro studies2 with the Agro• bacterium tumefaciens Lux! homo• logue TraI (Ref. 3) have identified the substrates for signal biosynthe• sis that couple components of both amino acid and fatty acid bio• synthesis (Fig. 1). Importantly, this work demonstrates that TraI (and presumably all LuxI homologues) fulfils a catalytic role in AHL bio• synthesis.
N
Quorum sensing
Quorum sensing regulates the ex• pression of ecologically, medically and industrially important bac• terial phenotypes!. The phenom• enon of quorum sensing allows bac• teria to coordinate gene expression with population growth. In micro• biology this is most often described as a cell density-dependent func• tionality. Microorganisms have evolved many different mechanisms of quorum sensing that involve the production, release, sensing and re• sponse to small signalling molecules by individual cells within a bacterial population !,4,5. Control of bioluminescence via the LuxRI quorum-sensing mecha• nism has long been known for Photobacterium (Vibrio) fischeri 6 • In recent years, a variety of charac• teristics under quorum sensing con• trol, which use LuxRI homologues, have been identified in a range of other Gram-negative bacterial. Ex• amples include aspects of virulence of the opportunistic vertebrate pathogen Pseudomonas aeruginosa and the plant pathogen Erwinia carotovora. Although the regulatory details vary considerably, a simple model can be proposed involving an AHL synthase (LuxI homologue) that is responsible for the production of the AHL signal, which accumu-
lates in concert with changes in population size to a threshold where it effects a response through a regu• lator protein (LuxR homologue). A number of phenotypes have been assigned to LuxR-homologue• activated regulons!, including co• ordinated expression of the viru• lence determinants of large bacterial populations of P. aeTuginosa and E. carotovora, which is a feature of their pathogenicity. In A. tume• faciens quorum sensing controls the conjugal transfer of Ti plasmids in the presence of specific nutrients (opines) produced by the host plant. Expression of the tra gene products required for conjugation is induced by TraR (a LuxR homologue) in the presence of the TraI (a LuxI homo• logue)-generated AHL signal mol• ecule, N-(3-oxooctanoyl)-L-homo• serine lactone (OOHL), at the high bacterial cell densities found in the crown gall tumour!,3. Variations in the N-linked acyl moiety of the AHL molecule allow for structurally and functionally distinguishable signals to be syn• thesized by individual LuxI homo• logues 1 • Given the importance and range of characteristics positively regulated by the accumulation of different AHL signals, an under• standing of the biosynthetic mecha• nism is important. The recent work of More et al. 2 provides a significant step towards elucidating the under• lying biochemistry of AHL synthe• sis. Purification of the recombiS. Swift· and G.S.A.B. Stewart are in the Dept of Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington Campus, Leicestershire, UK LE12 5RD; P. Williams is in, and S. Swift is also in, the Dept of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham, UK NG72RD. ·tel; +44115 9516115, fax; +44115 9516162, e-mail: [email protected]
Copyright Q 1996 Elsevier Science Ltd. All rights reserved. 0966 842X196/$1S.00 TRENDS IN MICROBIOLOGY
463
VOL.
4
No. 12
nant hexahistidinyl-TraI (HI)TraI) protein has enabled the synthesis of OOHL from purified substrates2 in vitro. S-adenosylmethlonlne as a substrate
Preliminary experiments with LuxI in crude P. fischeri extracts7 proposed S-adenosylmethionine (AdoMet) as the donor of the homo serine lactone (HSL) moiety; however, other possible substrates have since been considered. For ex• ample, HSL accumulation has been proposed as a general signal for starvation8, allowing the attractive hypothesis of a role for substrate availability in the AHL biosynthetic reaction, with HSL providing the amino acid component. In vitro, HI)TraI catalyses the biosynthesis of OOHL in the pres• ence of an extract of soluble Escher• ichia coli proteins (S28 extract) and AdoMet (Ref. 2). Substitution of AdoMet by homoserine lactone does not lead to OOHL formation, which means that AdoMet must provide the homo serine lactone2 • Consistent with this conclusion, in vivo experiments using E. coli methionine and homo serine auxo• trophs in the presence of cyclo• leucine (an inhibitor of AdoMet synthesis) and bearing a plasmid en• coding the luxI gene have provided additional support for AdoMet, but not HSL, as a LuxI substrate9 • The fatty acyl moiety Is derived from the acyl-acyl carrier protein
HI)TraI catalyses the biosynthesis of OOHL in the presence of S28 extract and AdoMet (Ref. 2). In cell-free extracts of P. fischeri, both coenzyme A (CoA) and the acyl carrier protein (ACP) adduct of 3oxohexanoic acid were suggested to be potential acyl side chain donors in OOHL synthesis7• To identify the origin of the fatty acyl moiety of
PH: S0966·842X(96)30036·X DECEMBER
1996
COMMENT
o 3-oxooctanoyl-ACP
o
,,+~o--Jdenine ~ H .. ~d H2Ntr H:i
C
HO HO AdoMet
H 0
N-acylation and lactonization
__.
HX ~
_.-H,c......s~denine HO
HO MTA
~
N
OOHl
:
0
i
H H
o
Fig. 1. A model outlining the biosynthesis of N-(3-oxooctanoyl)+homoserine lactone (OOHL). The 3-oxooctanoyl group from 3-oxooctanoyl-acyl carrier protein (3-0xooctanoyl-ACP) is acquired by Tral (X may be either S or 0 from, for example, a cysteine or serine/threonine residue), which catalyses the formation of the amide bond between the amino group of 5-adenosylmethionine (AdoMet) and C-I of the fatty acid. This reaction is followed by lactonization, creating OOHL and 5'-methylthioadenosine (MTA). Adapted from More et al. 2 •
N-( 3-oxohexanoy l)-L-homoserine lactone (OHHL), the 528 extract was investigated. Dialysis of the S28 extract abolished any detectable OOHL synthase activity, but this could be restored by the addition of malonyl-CoA (but not acetyl• CoAl and NADPH (or NADH). Experiments in which cerulenin was used as an irreversible inhibi• tor of fatty acid biosynthesis dem• onstrated that ongoing fatty acid metabolism was required for OOHL biosynthesis. Interestingly, the bio• synthesis of a related AHL [N-(3hydroxybutanoyl)-homoserine lac• tone; HBHL] involved in quorum
sensing regulation of bioluminesc• ence in Vibrio harvey; requires ac• tive fatty acid biosynthesis 10. How• ever, it should be remembered that the AHL synthase in this case is not a Lux! homologue and that there is almost certainly at least one alternative mechanism for AHL synthesis 11 ,12. To determine whether the ACP adduct could donate the acyl chain, a quaternary amine derivative of 3oxo-octanoic acid was used to load the active tmol of the phosphopenta• thionine group of ACP. The result• ant 3-oxo-octanoyl-ACP (OOACP) replaced the requirement for the 528
TRENDS IN MICROBIOLOGY
464
VOL.
4 No_ 12
extract or the dialysed S28 extract plus malonyl-CoA and NADPH, al• lowing biosynthesis of OOHL in the presence of Ado Met and HeT ral. Hence, it was concluded that the fatty acid moiety derives from the acyl-ACP and not the correspond• ing acyl-CoA (Ref. 2). Significantly, this suggests that the LuxI homo• logues must interact selectively with the acyl-ACP carrying the specific acyl group for the given AHL. In line with this conclusion, it has been demonstrated that AdoMet and the hexanoyl-ACP, but not hexanoyl• CoA, act as substrates for a mal• tose-binding-protein-LuxI fusion
in vitr0 9,13.
Perspectives
It will be important to confirm that other LuxI homologues use acyl• ACP and AdoMet as substrates and to compare these with the sub• strate preferences of the LuxLM familyl1,ll of AHL synthases. The challenge remains to elucidate t~e mechanism of AHL biosynthesls and to identify the LuxI homologue structure/function relationships. An understanding of the mechanism of signal generation may then allow disruption of signal production and the control of expression for a vari• ety of important gene products. A striking feature of LuxI homo• logues is that when they are cloned into E. col; from the wild-type organisms, they retain acyl moiety specificity without the presence of obvious structural motifs that de• fine subfamilies that make specific AHLs. Elucidation of the structural basis for this selectivity is an impor• tant question for the future, the an• swer to which may have relevance to the study of fatty acid biosynthesis. Finally, the slow rate of the Hr Tra! catalysed reaction using puri• fied substrates in vitro2 suggests that a rate enhancing factor may be miss• ing. It is possible that an NAD moi• ety may fulfil this role, perhaps as an allosteric modifier of the LuxI homologue. Although the require• ment for NADPH in OOHL bio• synthesis2 can be explained by its participation in the elongation re• actions of fatty acid biosynthesis l 4, the substitution of NADH to the same effectiveness in this reaction2 is surprising. It will be interesting,
DECEMBER 1996
C
therefore, to see whether the NAD moiety provides any additional functionality . References 1 Swift, S. et al. (1996) Trends Biochem. Sci. 21,214-219 2 More, M.l. et al. (1996) Science 272, 1655-1658 3 Piper, K.R., Beck von Bodman, S. and Farrand, S.K. (1993) Nature 363, 448-450 4 Kell, D.B., Kaprelyants, A.S. and
Response from Winans and More e are grateful for the opportu• nity to reply to Dr Swift's in• W sightful summary. The finding that
S-adenosylmethionine (AdoMet) provides the homo serine lactone (HSL) moiety of N-(3-oxooctanoyl)• L-homoserine lactone (OOHL) may seem surprising, as AdoMet is best known as a donor of methyl groups. However, AdoMet is also known to donate 3-carboxy-3-aminopropyl groups. For example, it modifies a histidine residue of the translation elongation factor EF2 of eukaryotes and the Archaea 1. The 3-carboxy-3aminopropyl group is then N-meth• ylated three times at the expense of AdoMet. The resulting modified histidine is known as diphthamide and is the target of ADP-ribosyl• ation by diphtheria toxin, by exo• toxin A of Pseudomonas aeruginosa and by an endogenous cytoplas• mic ADP-ribosylase. 3-carboxy3-aminopropyl groups are also donated to diacylglycerol in Rhodo• bacter sphaeroides, creating a phos• phate-free membrane lipid that is synthesized in phosphate-limiting environments 2 • The finding that the fatty acid group of OOHL is derived from acyl-acyl carrier protein (ACP) was less surprising. Although many dif• ferent HSLs have been described, these pheromones differ only in the length and oxidation state of their acyl moieties 3 • All acyl groups have even numbers of carbons and all have keto groups, hydroxyl groups or methylene groups at the 3-carbon position. Significantly, all these substrates are available during the synthesis of fatty acids. This helps
OJ\t1J\t1ENT
Grafen, A. (1995) Trends Eco/. Evo/. 10, 126-129 5 Wirth, R., Muscholl, A. and Wanner, G. (1996) Trends Microbiol. 4, 96-103 6 Meighen, E.A. (1994) Annu. Rev. Genet. 28,117-139 7 Eberhard, A. et al. (1991) Arch. Microbiol. 155, 294-297 8 Huisman, G.W. and Kolter, R. (1994) Science 265, 537-539 9 Hanzelka, B.L. and Greenberg, E.P. (1996) J. Bacterio/. 178,5291-5294 10 Cao, J.G. and Meighan, E.A. (1993)
J. Bacterio/. 175,3856-3862 11 Bassler, B.L., Wright, M. and Silverman, M.R. (1994) Mol. Microbiol. 12,403-412 12 Gilson, L., Kuo, A. and Dunlap, P.V. (1995) J. Bacteriol. 177,6946-6951 13 Schaefer, A.L. et al. Proc. Natl. Acad. Sci. U. S. A. (in press) 14 Cronan, J.E. and Rock, c.o. (1996) in Escherichia coli and Salmonella: Cellular and Molecular Biology (2nd edn) (Neidhardt, F.C., ed.), pp. 612-636, ASM Press
to explain why the expression in Escherichia coli of HSL synthases from diverse bacteria leads to the production of the cognate HSL. As summarized by Swift, we pre• viously reported in vitro synthesis of OOHL using purified Tral (a Luxl homolog), AdoMet and an E. coli crude extract as a source of acyl• ACP. When this extract was dial• yzed, synthesis required malonyl• CoA and either NADPH or NADH. Swift expressed surprise that NADH could substitute for NADPH in this reaction. We also were surprised and can make only two comments. First, when limiting amounts of these reductants were provided, NADPH supported synthesis more effectively than NADH. Second, although it is often stated that NADPH is required both for the 3ketoacyl-ACP reductase and for the enoyl-ACP reductase that act in fatty acid biosynthesis, there is evi• dence that both steps are carried out by several redundant enzymes4• One of the enoyl-ACP reductases uses NADH rather than NADPH. It would seem inescapable that at least one 3-ketoacyl-ACP reductase must also be able to utilize NADH. We agree with Swift that more work needs to be done on the re• action mechanism of auto inducer synthases. A two-step reaction was recently proposed in which an auto• inducer synthase is first acylated and then donates its acyl group to AdoMet (Ref. 5). In support of this model, we found that Tral that is chemically acylated by 3-oxo• octanoylthiocholine iodide (OOTC) can donate this acyl group to AdoMet, resulting in OOHL pro• duction. However, when OOTC was removed by gel filtration rather
than by dialysis, this activity was not detected. One explanation could be that OOTC was not fully re• moved by dialysis and that this com• pound chemically acylated AdoMet, leading to nonenzymatic synthesis of OOHL. The model that Tral is transiently acylated predicts that addition of radiolabelled malonyl• CoA to a dialyzed crude extract would lead to radiolabelling of TraI. However, so far, we have not de• tected radiolabelling of TraI by this procedure. A second prediction of this model is that it should be poss• ible to combine Tral with 3-oxo• octanoyl-ACP (OOACP), separate these proteins and then combine AdoMet with Tral. However, we have not detected OOHL in such experiments. We currently regard a single-step reaction mechanism, in which acyl groups are directly donated to AdoMet, as plausible. Swift points out that TraR (a LuxR homolog) and OOHL acti• vate transcription of the tra regulon of various Ti (tumor-inducing) plas• mids and that opines, which are nu• trient sources released from crown gall tumors, provide an essential en• vironmental queue. We have two comments. First, like TraR and Tral, several quorum-sensing proteins function as part of a larger signal transduction cascade. A particularly interesting example was reported at a recent symposium (L.S. Pierson, D.W. Wood and S.T. Chancey, pers. commun.). Transcription of the phzFABCD operon of Pseudo• monas aureofaciens, which directs the synthesis of phenazine anti• biotics, requires PhzR and PhzI. Ex• pression of the phz operon also re• quires the two-component kinase LemA and the response regulator
Copyright 01996 Elsevier Science Ltd. All rights reserved. 0966 842X1961$15.00 TRENDS IN MICROBIOLOGY
465
VOL. 4
PH: S0966-842X(96)20023-X
No. 12 DECEMBER 1996
The inner workings of a quorum sensing signal generator Simon Swift, Gordon S.A.B. Stewart and P. Williams -ACYI homo serine lactones (AHLs) are the signal mol• ecules used by the LuxRI family of quorum sensing regula• tors!. In vitro studies2 with the Agro• bacterium tumefaciens Lux! homo• logue TraI (Ref. 3) have identified the substrates for signal biosynthe• sis that couple components of both amino acid and fatty acid bio• synthesis (Fig. 1). Importantly, this work demonstrates that TraI (and presumably all LuxI homologues) fulfils a catalytic role in AHL bio• synthesis.
N
Quorum sensing
Quorum sensing regulates the ex• pression of ecologically, medically and industrially important bac• terial phenotypes!. The phenom• enon of quorum sensing allows bac• teria to coordinate gene expression with population growth. In micro• biology this is most often described as a cell density-dependent func• tionality. Microorganisms have evolved many different mechanisms of quorum sensing that involve the production, release, sensing and re• sponse to small signalling molecules by individual cells within a bacterial population !,4,5. Control of bioluminescence via the LuxRI quorum-sensing mecha• nism has long been known for Photobacterium (Vibrio) fischeri 6 • In recent years, a variety of charac• teristics under quorum sensing con• trol, which use LuxRI homologues, have been identified in a range of other Gram-negative bacterial. Ex• amples include aspects of virulence of the opportunistic vertebrate pathogen Pseudomonas aeruginosa and the plant pathogen Erwinia carotovora. Although the regulatory details vary considerably, a simple model can be proposed involving an AHL synthase (LuxI homologue) that is responsible for the production of the AHL signal, which accumu-
lates in concert with changes in population size to a threshold where it effects a response through a regu• lator protein (LuxR homologue). A number of phenotypes have been assigned to LuxR-homologue• activated regulons!, including co• ordinated expression of the viru• lence determinants of large bacterial populations of P. aeTuginosa and E. carotovora, which is a feature of their pathogenicity. In A. tume• faciens quorum sensing controls the conjugal transfer of Ti plasmids in the presence of specific nutrients (opines) produced by the host plant. Expression of the tra gene products required for conjugation is induced by TraR (a LuxR homologue) in the presence of the TraI (a LuxI homo• logue)-generated AHL signal mol• ecule, N-(3-oxooctanoyl)-L-homo• serine lactone (OOHL), at the high bacterial cell densities found in the crown gall tumour!,3. Variations in the N-linked acyl moiety of the AHL molecule allow for structurally and functionally distinguishable signals to be syn• thesized by individual LuxI homo• logues 1 • Given the importance and range of characteristics positively regulated by the accumulation of different AHL signals, an under• standing of the biosynthetic mecha• nism is important. The recent work of More et al. 2 provides a significant step towards elucidating the under• lying biochemistry of AHL synthe• sis. Purification of the recombiS. Swift· and G.S.A.B. Stewart are in the Dept of Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington Campus, Leicestershire, UK LE12 5RD; P. Williams is in, and S. Swift is also in, the Dept of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham, UK NG72RD. ·tel; +44115 9516115, fax; +44115 9516162, e-mail: [email protected]
Copyright Q 1996 Elsevier Science Ltd. All rights reserved. 0966 842X196/$1S.00 TRENDS IN MICROBIOLOGY
463
VOL.
4
No. 12
nant hexahistidinyl-TraI (HI)TraI) protein has enabled the synthesis of OOHL from purified substrates2 in vitro. S-adenosylmethlonlne as a substrate
Preliminary experiments with LuxI in crude P. fischeri extracts7 proposed S-adenosylmethionine (AdoMet) as the donor of the homo serine lactone (HSL) moiety; however, other possible substrates have since been considered. For ex• ample, HSL accumulation has been proposed as a general signal for starvation8, allowing the attractive hypothesis of a role for substrate availability in the AHL biosynthetic reaction, with HSL providing the amino acid component. In vitro, HI)TraI catalyses the biosynthesis of OOHL in the pres• ence of an extract of soluble Escher• ichia coli proteins (S28 extract) and AdoMet (Ref. 2). Substitution of AdoMet by homoserine lactone does not lead to OOHL formation, which means that AdoMet must provide the homo serine lactone2 • Consistent with this conclusion, in vivo experiments using E. coli methionine and homo serine auxo• trophs in the presence of cyclo• leucine (an inhibitor of AdoMet synthesis) and bearing a plasmid en• coding the luxI gene have provided additional support for AdoMet, but not HSL, as a LuxI substrate9 • The fatty acyl moiety Is derived from the acyl-acyl carrier protein
HI)TraI catalyses the biosynthesis of OOHL in the presence of S28 extract and AdoMet (Ref. 2). In cell-free extracts of P. fischeri, both coenzyme A (CoA) and the acyl carrier protein (ACP) adduct of 3oxohexanoic acid were suggested to be potential acyl side chain donors in OOHL synthesis7• To identify the origin of the fatty acyl moiety of
PH: S0966·842X(96)30036·X DECEMBER
1996
COMMENT
o 3-oxooctanoyl-ACP
o
,,+~o--Jdenine ~ H .. ~d H2Ntr H:i
C
HO HO AdoMet
H 0
N-acylation and lactonization
__.
HX ~
_.-H,c......s~denine HO
HO MTA
~
N
OOHl
:
0
i
H H
o
Fig. 1. A model outlining the biosynthesis of N-(3-oxooctanoyl)+homoserine lactone (OOHL). The 3-oxooctanoyl group from 3-oxooctanoyl-acyl carrier protein (3-0xooctanoyl-ACP) is acquired by Tral (X may be either S or 0 from, for example, a cysteine or serine/threonine residue), which catalyses the formation of the amide bond between the amino group of 5-adenosylmethionine (AdoMet) and C-I of the fatty acid. This reaction is followed by lactonization, creating OOHL and 5'-methylthioadenosine (MTA). Adapted from More et al. 2 •
N-( 3-oxohexanoy l)-L-homoserine lactone (OHHL), the 528 extract was investigated. Dialysis of the S28 extract abolished any detectable OOHL synthase activity, but this could be restored by the addition of malonyl-CoA (but not acetyl• CoAl and NADPH (or NADH). Experiments in which cerulenin was used as an irreversible inhibi• tor of fatty acid biosynthesis dem• onstrated that ongoing fatty acid metabolism was required for OOHL biosynthesis. Interestingly, the bio• synthesis of a related AHL [N-(3hydroxybutanoyl)-homoserine lac• tone; HBHL] involved in quorum
sensing regulation of bioluminesc• ence in Vibrio harvey; requires ac• tive fatty acid biosynthesis 10. How• ever, it should be remembered that the AHL synthase in this case is not a Lux! homologue and that there is almost certainly at least one alternative mechanism for AHL synthesis 11 ,12. To determine whether the ACP adduct could donate the acyl chain, a quaternary amine derivative of 3oxo-octanoic acid was used to load the active tmol of the phosphopenta• thionine group of ACP. The result• ant 3-oxo-octanoyl-ACP (OOACP) replaced the requirement for the 528
TRENDS IN MICROBIOLOGY
464
VOL.
4 No_ 12
extract or the dialysed S28 extract plus malonyl-CoA and NADPH, al• lowing biosynthesis of OOHL in the presence of Ado Met and HeT ral. Hence, it was concluded that the fatty acid moiety derives from the acyl-ACP and not the correspond• ing acyl-CoA (Ref. 2). Significantly, this suggests that the LuxI homo• logues must interact selectively with the acyl-ACP carrying the specific acyl group for the given AHL. In line with this conclusion, it has been demonstrated that AdoMet and the hexanoyl-ACP, but not hexanoyl• CoA, act as substrates for a mal• tose-binding-protein-LuxI fusion
in vitr0 9,13.
Perspectives
It will be important to confirm that other LuxI homologues use acyl• ACP and AdoMet as substrates and to compare these with the sub• strate preferences of the LuxLM familyl1,ll of AHL synthases. The challenge remains to elucidate t~e mechanism of AHL biosynthesls and to identify the LuxI homologue structure/function relationships. An understanding of the mechanism of signal generation may then allow disruption of signal production and the control of expression for a vari• ety of important gene products. A striking feature of LuxI homo• logues is that when they are cloned into E. col; from the wild-type organisms, they retain acyl moiety specificity without the presence of obvious structural motifs that de• fine subfamilies that make specific AHLs. Elucidation of the structural basis for this selectivity is an impor• tant question for the future, the an• swer to which may have relevance to the study of fatty acid biosynthesis. Finally, the slow rate of the Hr Tra! catalysed reaction using puri• fied substrates in vitro2 suggests that a rate enhancing factor may be miss• ing. It is possible that an NAD moi• ety may fulfil this role, perhaps as an allosteric modifier of the LuxI homologue. Although the require• ment for NADPH in OOHL bio• synthesis2 can be explained by its participation in the elongation re• actions of fatty acid biosynthesis l 4, the substitution of NADH to the same effectiveness in this reaction2 is surprising. It will be interesting,
DECEMBER 1996
C
therefore, to see whether the NAD moiety provides any additional functionality . References 1 Swift, S. et al. (1996) Trends Biochem. Sci. 21,214-219 2 More, M.l. et al. (1996) Science 272, 1655-1658 3 Piper, K.R., Beck von Bodman, S. and Farrand, S.K. (1993) Nature 363, 448-450 4 Kell, D.B., Kaprelyants, A.S. and
Response from Winans and More e are grateful for the opportu• nity to reply to Dr Swift's in• W sightful summary. The finding that
S-adenosylmethionine (AdoMet) provides the homo serine lactone (HSL) moiety of N-(3-oxooctanoyl)• L-homoserine lactone (OOHL) may seem surprising, as AdoMet is best known as a donor of methyl groups. However, AdoMet is also known to donate 3-carboxy-3-aminopropyl groups. For example, it modifies a histidine residue of the translation elongation factor EF2 of eukaryotes and the Archaea 1. The 3-carboxy-3aminopropyl group is then N-meth• ylated three times at the expense of AdoMet. The resulting modified histidine is known as diphthamide and is the target of ADP-ribosyl• ation by diphtheria toxin, by exo• toxin A of Pseudomonas aeruginosa and by an endogenous cytoplas• mic ADP-ribosylase. 3-carboxy3-aminopropyl groups are also donated to diacylglycerol in Rhodo• bacter sphaeroides, creating a phos• phate-free membrane lipid that is synthesized in phosphate-limiting environments 2 • The finding that the fatty acid group of OOHL is derived from acyl-acyl carrier protein (ACP) was less surprising. Although many dif• ferent HSLs have been described, these pheromones differ only in the length and oxidation state of their acyl moieties 3 • All acyl groups have even numbers of carbons and all have keto groups, hydroxyl groups or methylene groups at the 3-carbon position. Significantly, all these substrates are available during the synthesis of fatty acids. This helps
OJ\t1J\t1ENT
Grafen, A. (1995) Trends Eco/. Evo/. 10, 126-129 5 Wirth, R., Muscholl, A. and Wanner, G. (1996) Trends Microbiol. 4, 96-103 6 Meighen, E.A. (1994) Annu. Rev. Genet. 28,117-139 7 Eberhard, A. et al. (1991) Arch. Microbiol. 155, 294-297 8 Huisman, G.W. and Kolter, R. (1994) Science 265, 537-539 9 Hanzelka, B.L. and Greenberg, E.P. (1996) J. Bacterio/. 178,5291-5294 10 Cao, J.G. and Meighan, E.A. (1993)
J. Bacterio/. 175,3856-3862 11 Bassler, B.L., Wright, M. and Silverman, M.R. (1994) Mol. Microbiol. 12,403-412 12 Gilson, L., Kuo, A. and Dunlap, P.V. (1995) J. Bacteriol. 177,6946-6951 13 Schaefer, A.L. et al. Proc. Natl. Acad. Sci. U. S. A. (in press) 14 Cronan, J.E. and Rock, c.o. (1996) in Escherichia coli and Salmonella: Cellular and Molecular Biology (2nd edn) (Neidhardt, F.C., ed.), pp. 612-636, ASM Press
to explain why the expression in Escherichia coli of HSL synthases from diverse bacteria leads to the production of the cognate HSL. As summarized by Swift, we pre• viously reported in vitro synthesis of OOHL using purified Tral (a Luxl homolog), AdoMet and an E. coli crude extract as a source of acyl• ACP. When this extract was dial• yzed, synthesis required malonyl• CoA and either NADPH or NADH. Swift expressed surprise that NADH could substitute for NADPH in this reaction. We also were surprised and can make only two comments. First, when limiting amounts of these reductants were provided, NADPH supported synthesis more effectively than NADH. Second, although it is often stated that NADPH is required both for the 3ketoacyl-ACP reductase and for the enoyl-ACP reductase that act in fatty acid biosynthesis, there is evi• dence that both steps are carried out by several redundant enzymes4• One of the enoyl-ACP reductases uses NADH rather than NADPH. It would seem inescapable that at least one 3-ketoacyl-ACP reductase must also be able to utilize NADH. We agree with Swift that more work needs to be done on the re• action mechanism of auto inducer synthases. A two-step reaction was recently proposed in which an auto• inducer synthase is first acylated and then donates its acyl group to AdoMet (Ref. 5). In support of this model, we found that Tral that is chemically acylated by 3-oxo• octanoylthiocholine iodide (OOTC) can donate this acyl group to AdoMet, resulting in OOHL pro• duction. However, when OOTC was removed by gel filtration rather
than by dialysis, this activity was not detected. One explanation could be that OOTC was not fully re• moved by dialysis and that this com• pound chemically acylated AdoMet, leading to nonenzymatic synthesis of OOHL. The model that Tral is transiently acylated predicts that addition of radiolabelled malonyl• CoA to a dialyzed crude extract would lead to radiolabelling of TraI. However, so far, we have not de• tected radiolabelling of TraI by this procedure. A second prediction of this model is that it should be poss• ible to combine Tral with 3-oxo• octanoyl-ACP (OOACP), separate these proteins and then combine AdoMet with Tral. However, we have not detected OOHL in such experiments. We currently regard a single-step reaction mechanism, in which acyl groups are directly donated to AdoMet, as plausible. Swift points out that TraR (a LuxR homolog) and OOHL acti• vate transcription of the tra regulon of various Ti (tumor-inducing) plas• mids and that opines, which are nu• trient sources released from crown gall tumors, provide an essential en• vironmental queue. We have two comments. First, like TraR and Tral, several quorum-sensing proteins function as part of a larger signal transduction cascade. A particularly interesting example was reported at a recent symposium (L.S. Pierson, D.W. Wood and S.T. Chancey, pers. commun.). Transcription of the phzFABCD operon of Pseudo• monas aureofaciens, which directs the synthesis of phenazine anti• biotics, requires PhzR and PhzI. Ex• pression of the phz operon also re• quires the two-component kinase LemA and the response regulator
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