Expression and purification of four different rhizobial acyl carrier proteins The GenBank accession numbers for the nucleotide sequences reported in this paper are AF159244 and AF159243 for the acpP-containing regions of Sinorhizobium meliloti 1021 and of Rhizobium leguminosarum LPR5045, respectively.

López‐Lara, Isabel M.; Geiger, Otto

doi:10.1099/00221287-146-4-839

Cited by 23 publications

(15 citation statements)

References 41 publications

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“…Clearly SinI is quite promiscuous in its ability to interact with the proper acyl carrier protein (ACP). ACPs are involved not only in the synthesis of fatty acyl chains but also in their transfer during phospholipid, lipid A, ␣-hemolysin (23,24), and nodulation (Nod) factor biosynthesis (10,30). In E. coli, all of these functions seem to be realized by a single and absolutely essential ACP, which is produced constitutively from the acpP gene (24,40).…”

Section: Discussionmentioning

confidence: 99%

Characterization of theSinorhizobium meliloti sinR/sinILocus and the Production of NovelN-Acyl Homoserine Lactones

et al. 2002

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Sinorhizobium meliloti is a soil bacterium which can establish a nitrogen-fixing symbiosis with the legume Medicago sativa. Recent work has identified a pair of genes, sinR and sinI, which represent a potential quorum-sensing system and are responsible for the production of N-acyl homoserine lactones (AHLs) in two S. meliloti strains, Rm1021 and Rm41. In this work, we characterize the sinRI locus and show that these genes are responsible for the synthesis of several long-chain AHLs ranging from 12 to 18 carbons in length. Four of these, 3-oxotetradecanoyl HL, 3-oxohexadecenoyl HL, hexadecenoyl HL, and octadecanoyl HL, have novel structures. This is the first report of AHLs having acyl chains longer than 14 carbons. We show that a disruption in sinI eliminates these AHLs and that a sinR disruption results in only basal levels of the AHLs. Moreover, the same sinI and sinR mutations also lead to a decrease in the number of pink nodules during nodulation assays, as well as a slight delay in the appearance of pink nodules, indicating a role for quorum sensing in symbiosis. We also show that sinI and sinR mutants are still capable of producing several shortchain AHLs, one of which was identified as octanoyl HL. We believe that these short-chain AHLs are evidence of a second quorum-sensing system in Rm1021, which we refer to here as the mel system, for "S. meliloti."Quorum sensing is a widespread phenomenon among gramnegative bacteria (for recent reviews see references 9, 16, and 33). This form of cell density-dependent gene regulation is mediated by sensing the concentrations of low-molecularweight compounds called autoinducers, which are produced by the bacteria. A few organisms such as Photobacterium fischeri and Pseudomonas aeruginosa serve as model systems for quorum sensing. P. fischeri, in which autoinduction was first identified, has a relatively simple quorum-sensing system. An autoinducer synthase, LuxI, produces the N-(3-oxohexanoyl)-Lhomoserine lactone (oxo-C 6 -HL), which is recognized by the LuxR regulator (11,22,25). LuxR then induces expression of the lux operon, which causes bioluminescence (47,48). This model has recently become slightly more complicated with the identification of a second autoinducer synthase, AinS, and the regulatory protein LuxO, both of which serve to modulate quorum-sensing-induced luminescence in P. fischeri (25,26,34). On the other hand, P. aeruginosa has two quorum-sensing systems (lasR/lasI and rhlR/rhlI) organized into a complex hierarchy, which together regulate numerous genes required for virulence (2, 36).The most common and well-characterized gram-negative bacterial autoinducers are N-acyl homoserine lactones (AHLs) (see above reviews). AHLs consist of a variable acyl chain attached to a conserved homoserine lactone head group. The acyl chains can vary in length from 4 to 14 carbons. They can also vary in the nature of the substituent on the third carbon, from hydrogen to a hydroxyl or oxo group. One last characteristic that can confer variability is the presence of a do...

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Section: Discussionmentioning

confidence: 99%

Characterization of theSinorhizobium meliloti sinR/sinILocus and the Production of NovelN-Acyl Homoserine Lactones

et al. 2002

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“…For example, in the case of the rhizobial carrier proteins, four ACPs each have specific nodulation functions (17). Thus, each of these carrier proteins is targeted to their respective site of action.…”

Section: Discussionmentioning

confidence: 99%

d -Alanylation of Lipoteichoic Acid: Role of the d -Alanyl Carrier Protein in Acylation

Kiriukhin

Neuhaus

2001

J Bacteriol

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(11,12). Heaton and Neuhaus (12) showed that D-alanyl-Dcp donates its D-alanyl residue to the poly(Gro-P) moiety of membrane-associated LTA. Neither the mechanism nor the topology of this D-alanylation reaction is known. To address this key reaction, several groups have identified the genes in a number of organisms containing the dlt operon (11,21,23). In addition to the genes encoding Dcl (dltA) and Dcp (dltC), dltB and dltD encode a putative transport protein (20) and a protein which facilitates the binding of Dcl and Dcp for ligation with Dalanine and has thioesterase activity for mischarged D-alanyl acyl carrier proteins (ACPs) (5), respectively. A gene encoding an enzyme which catalyzes the transfer of the D-alanine residue from D-alanyl-Dcp to membrane-associated LTA has not been identified.Dcp provides the essential link between the ligase (Dcl) and the incorporation of D-alanine into LTA. This carrier protein is a homologue of those ACPs which function in fatty acid biosynthesis and metabolism (4, 26). However, it was unexpected to find that Dcl will ligate D-alanine to ACPs from Escherichia coli, Vibrio harveyi, Saccharopolyspora erythraea, and Bacillus subtilis (12). Nevertheless, only Dcp participates in the D-alanylation of LTA. These observations suggested that there are at least two determinants for interaction of Dcp with its cognate partners, one of which is recognized by Dcl and one of which is recognized by the membrane acceptor LTA. It is this second determinant which is the focus of structural studies on Dcp (B. F. Volkman, Q. Zhang, D. Debabov, E. Rivera, G. Kresheck, and F. C. Neuhaus, unpublished results), and the biochemical studies to be reported here.For Staphylococcus aureus, it was found that growth in the presence of NaCl resulted in a lower D-alanine ester content in LTA (8). The mechanism by which the ester content was reduced during growth in NaCl is unknown and was not correlated with the reaction catalyzed by DltD (5). Instead, an NaCl-activated, thioesterase-like activity specific for D-alanylDcp which is distinct from that catalyzed by DltD was discovered (5, 20).Our goal here was to establish the mechanism of D-alanine transfer from D-alanyl-Dcp to LTA. To accomplish this goal, the thioesterase-like activity of LTA specific for D-alanyl-Dcp was examined in incubations containing LTA in different microenvironments: (i) purified, (ii) membrane associated, and (iii) membrane associated (salt treated). The results suggested that complex formation between D-alanyl-Dcp and LTA is one of the features resulting either in the transfer of D-alanine from D-alanyl-Dcp to LTA when this amphiphile is membrane associated or in the hydrolysis of D-alanyl-Dcp when the LTA is not membrane associated.

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“…For example, there are four different ACPs in Rhizobia, AcpP, NodF, RkpF, and AcpXL (29,30). M. tuberculosis has a biosynthetic AcpM and two other putative ACPs (11).…”

Section: Downloaded Frommentioning

confidence: 99%

The application of computational methods to explore the diversity and structure of bacterial fatty acid synthase

Zhang

Marrakchi

White

et al. 2003

Journal of Lipid Research

100

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BACTERIAL FATTY ACID SYNTHESISFatty acid metabolism is a fundamental component of the cellular metabolic network. Fatty acids are the essential building blocks for membrane phospholipid formation. Most bacteria synthesize fatty acids using a series of discrete monofunctional proteins, each catalyzing one reaction in the pathway [reviewed in ref. (1-3)]. The bacterial system, also known as the dissociated, type II fatty acid synthase (FAS II), contrasts with the yeast and animal type I fatty acid synthases (FAS I). The type II system is a collection of individual enzymes encoded by separate genes, whereas the type I system is a polypeptide about 260 kDa in size with multiple active sites that perform all the catalytic reactions in the pathway. Although the structural organizations of FAS I and FAS II are different, the chemical reactions and the catalytic mechanisms for fatty acid synthesis are essentially the same.Escherichia coli FAS II has been extensively studied and the biochemical properties of the individual enzymes are the paradigm for type II fatty acid synthase (1). Figure 1 outlines the major steps in the bacterial FAS II. Acetyl-CoA carboxylase catalyzes the first committed reaction of fatty acid biosynthesis. The product of the reaction is malonylCoA and the malonate group is then transferred to ACP by malonyl-CoA:ACP transacylase (FabD) to form malonyl-ACP. Fatty acid synthesis is initiated by the Claisen condensation of malonyl-ACP with acetyl-CoA catalyzed by ␤ -ketoacyl-ACP synthase III (FabH) to form ␤ -ketobutyryl-ACP. Four enzymes catalyze each cycle of elongation. The ␤ -keto group is reduced by the NADPH-dependent ␤ -ketoacyl-ACP reductase (FabG), and the resulting ␤ -hydroxy intermediate is dehydrated by the ␤ -hydroxyacyl-ACP dehydratase (FabA or FabZ) to an enoyl-ACP. Next, the reduction of the enoyl chain by the NAD(P)H-dependent enoyl-ACP reductase (FabI, FabK, or FabL) produces an

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Cited by 23 publications

References 41 publications

Characterization of theSinorhizobium meliloti sinR/sinILocus and the Production of NovelN-Acyl Homoserine Lactones

Characterization of theSinorhizobium meliloti sinR/sinILocus and the Production of NovelN-Acyl Homoserine Lactones

d -Alanylation of Lipoteichoic Acid: Role of the d -Alanyl Carrier Protein in Acylation

The application of computational methods to explore the diversity and structure of bacterial fatty acid synthase

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