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Keywords:

  • membrane lipid;
  • abiotic stress;
  • Brucella

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Distribution and structure of OLs
  5. Biosynthesis of OLs
  6. OL-modifying activities
  7. Prediction of OL distribution
  8. Regulation of OL biosynthesis and modification
  9. OL functions
  10. Perspectives
  11. Acknowledgements
  12. References
  13. Supporting Information

Ornithine lipids (OLs) are phosphorus-free membrane lipids that are widespread in eubacteria, but absent from archaea and eukaryotes. They contain a 3-hydroxy fatty acyl group attached in amide linkage to the α-amino group of the amino acid ornithine. A second fatty acyl group is ester-linked to the 3-hydroxy position of the first fatty acid. About 25% of the bacterial species whose genomes have been sequenced are predicted to have the capacity to form OLs. Distinct OL hydroxylations have been described in the ester-linked fatty acid, the amide-linked fatty acid, and the ornithine moiety. These modifications often seem to form part of a bacterial stress response to changing environmental conditions, allowing the bacteria to adjust membrane properties by simply modifying already existing membrane lipids without the need to synthesize new lipids.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Distribution and structure of OLs
  5. Biosynthesis of OLs
  6. OL-modifying activities
  7. Prediction of OL distribution
  8. Regulation of OL biosynthesis and modification
  9. OL functions
  10. Perspectives
  11. Acknowledgements
  12. References
  13. Supporting Information

The permeability barrier of cells is formed by amphipathic lipids, which consist of a hydrophobic and a hydrophilic portion. The hydrophobic moieties have the propensity to self-associate, and the hydrophilic moieties have the tendency to interact with each other and the aqueous environment, leading to the formation of membrane structures. In general, glycerophospholipids such as phosphatidylglycerol, phosphatidylethanolamine, cardiolipin, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol are the primary building blocks of membranes, but several other lipid classes can be also important and essential membrane components. Almost all Gram-negative bacteria have the lipid-A-containing lipopolysaccharide in the outer layer of the outer membrane (Raetz et al., 2007), but several other lipid classes such as hopanoid and steroid lipids, sphingolipids, glycosylated diacylglycerols, sulfolipids, betaine lipids, and ornithine lipids (OLs) have been described that can be formed only by certain bacterial groups or under specific stress conditions. For example, under phosphorus-limiting growth conditions, some bacteria replace the majority of their glycerophospholipids with phosphorus-free membrane lipids such as sulfolipids, betaine lipids, glycolipids, and OLs (Benning et al., 1995; Geiger et al., 1999; Weissenmayer et al., 2002; Gao et al., 2004). Challenging of some bacteria with low pH conditions can cause the modification of already existing membrane lipids, such as the formation of lysyl-phosphatidylglycerol from phosphatidylglycerol or the hydroxylation of OLs (Rojas-Jiménez et al., 2005; Sohlenkamp et al., 2007; González-Silva et al., 2011; Vences-Guzmán et al., 2011).

Distribution and structure of OLs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Distribution and structure of OLs
  5. Biosynthesis of OLs
  6. OL-modifying activities
  7. Prediction of OL distribution
  8. Regulation of OL biosynthesis and modification
  9. OL functions
  10. Perspectives
  11. Acknowledgements
  12. References
  13. Supporting Information

The capacity to form OLs is apparently widely distributed in eubacteria, but so far, OLs have not been detected in archaea and eukaryotes (López-Lara et al., 2003; Geiger et al., 2010). They contain a 3-hydroxy fatty acyl group that is attached in amide linkage to the α-amino group of ornithine. A second fatty acyl group, the so-called piggy-back fatty acid, is ester-linked to the 3-hydroxy position of the first fatty acid (Knoche & Shively, 1972; Geiger et al., 1999). In some bacteria, OLs can be modified by hydroxylation in one or more positions. In recent years, several genes coding for OL hydroxylases have been identified. OLs can be hydroxylated in the ester-linked fatty acid, the amide-linked fatty acid, and the ornithine moiety (Rojas-Jiménez et al., 2005; González-Silva et al., 2011; Vences-Guzmán et al., 2011). In Gluconobacter cerinus, OLs hydroxylated in the C-2 position of the ester-linked fatty acid can be modified with a taurine residue that is amide-linked to the α-carboxy group of ornithine. This tauro-OL is also called cerilipin after the bacterial species from which it was isolated (Tahara et al., b). Although OLs are present in both membranes of Gram-negative bacteria, they are more abundant in the outer membrane (Dees & Shively, 1982; Palacios-Chaves et al., 2011; Vences-Guzmán et al., 2011).

Structurally similar lipids in which other amino acids are present instead of ornithine have been described. A lysine lipid has been described in an Agrobacterium tumefaciens strain (Tahara et al., b), glycine lipids were detected in Cytophaga johnsonae and Cyclobacterium marinus (Kawazoe et al., 1991; Batrakov et al., 1999), glutamine lipids were described in Rhodobacter sphaeroides (Zhang et al., 2009, 2011), and serineglycine lipids (SGLs) were isolated from the opportunistic pathogen Flavobacterium meningosepticum (Kawai et al., 1988; Shiozaki et al., b).

Biosynthesis of OLs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Distribution and structure of OLs
  5. Biosynthesis of OLs
  6. OL-modifying activities
  7. Prediction of OL distribution
  8. Regulation of OL biosynthesis and modification
  9. OL functions
  10. Perspectives
  11. Acknowledgements
  12. References
  13. Supporting Information

The biosynthesis of the unmodified OL (sometimes also called S1 (Rojas-Jiménez et al., 2005)) occurs in two steps. The genes coding for the acyltransferase activities OlsB and OlsA required for OL biosynthesis were first discovered in the α-proteobacterium Sinorhizobium meliloti (Weissenmayer et al., 2002; Gao et al., 2004). In the first step, the N-acyltransferase OlsB is responsible for the transfer of a 3-hydroxy fatty acyl group from 3-hydroxy fatty acyl-acyl carrier protein (ACP) to the α-amino group of ornithine, thereby forming lyso-ornithine lipid (LOL) (Gao et al., 2004). In the second step, the O-acyltransferase OlsA catalyzes the transfer of an acyl group from acyl-ACP to the 3-hydroxyl group of LOL group forming OL (Weissenmayer et al., 2002) (Fig. 1).

image

Figure 1. OlsBA-dependent biosynthesis of OLs. AcpP, constitutive acyl carrier protein.

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The N -acyltransferase OlsB

OlsB-deficient mutants have been isolated in S. meliloti, Rhodobacter capsulatus, Brucella abortus, and Burkholderia cenocepacia, and they are in all cases unable to form OLs (Gao et al., 2004; Aygun-Sunar et al., 2006; González-Silva et al., 2011; Palacios-Chaves et al., 2011). The analysis of molecular species of OLs present in different organisms suggests that the distinct OlsB proteins apparently present strong substrate specificity for specific fatty acid chain lengths. Apparently, OlsB enzymes from Rhizobium tropici and S. meliloti almost exclusively attach a 3-hydroxylated C18 fatty acid to ornithine (Geiger et al., 1999; Vences-Guzmán et al., 2011), whereas OlsB from B. cenocepacia almost exclusively transfers a 3-hydroxylated C16 fatty acid (González-Silva et al., 2011). In contrast, OLs from Pseudomonas aeruginosa present a variety of chain lengths in the amide-linked fatty acid (Lewenza et al., 2011), indicating that OlsB from P. aeruginosa shows laxer substrate specificity and can transfer a variety of 3-hydroxy fatty acids to ornithine.

The O -acyltransferase OlsA

OlsA-deficient mutants of S. meliloti, R. capsulatus, B. abortus, and P. aeruginosa are unable to form OLs (Weissenmayer et al., 2002; Aygun-Sunar et al., 2006; Lewenza et al., 2011; Palacios-Chaves et al., 2011). In some cases, an accumulation of LOL has been observed in OlsA-deficient mutants that can be exacerbated by OlsB overexpression (Gao et al., 2004). In contrast to what has been observed for OlsB, OlsA seems to be less selective for specific fatty acids. More details relating to OlsA and OlsB can be found in Geiger et al. (2010).

OL-modifying activities

  1. Top of page
  2. Abstract
  3. Introduction
  4. Distribution and structure of OLs
  5. Biosynthesis of OLs
  6. OL-modifying activities
  7. Prediction of OL distribution
  8. Regulation of OL biosynthesis and modification
  9. OL functions
  10. Perspectives
  11. Acknowledgements
  12. References
  13. Supporting Information

Once the unmodified OL S1 has been synthesized by the acyltransferases OlsB and OlsA, it can be modified in some organisms by introducing hydroxyl groups in the different moieties of the OL structure or by transfer of taurine to the α-carboxy group of ornithine (Tahara et al., 1978). So far, three different OL hydroxylases have been described: OlsC, OlsD, and OlsE (Rojas-Jiménez et al., 2005; González-Silva et al., 2011; Vences-Guzmán et al., 2011) (Fig. 2). The gene/enzyme responsible for the taurine modification of OLs in G. cerinus has not been identified.

image

Figure 2. Hydroxylation of OLs. Three different OL hydroxylases have been described so far. The unmodified OL S1 can be hydroxylated by OlsC, leading to the formation of the OL P1; it can be hydroxylated by OlsD, leading to the formation of OL NL1; or it can be hydroxylated by OlsE, leading to the formation of OL S2. Also, hydroxylated OLs can be subject to a second hydroxylation: for example, OL P2 is a double-hydroxylated OL hydroxylated by OlsC and OlsE (structure of P2 is not shown in figure). The brackets indicate that the exact position of the introduced hydroxyl group is not known.

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Mutants lacking OlsB activity and thereby deficient in the first step of OL biosynthesis have been shown to lack modified OLs also, indicating that there is no alternative to the OlsBA pathway in the organisms studied so far.

The OL hydroxylase OlsC

In some species of the genus Burkholderia (González-Silva et al., 2011), Flavobacterium (Kawai et al., 1988; Asselineau, 1991), Thiobacillus (Knoche & Shively, 1972), Gluconobacter (Tahara et al., 1976a, 1976b), Streptomyces (Asselineau, 1991), Ralstonia (Galbraith et al., 1999), and Rhizobium (Vences-Guzmán et al., 2011), OLs hydroxylated in C-2 position of the ester-linked fatty acid have been described.

These 2-hydroxylated fatty acids are not formed during normal fatty acid biosynthesis, and specific enzyme activities are therefore necessary to introduce the hydroxyl group at this position. Gibbons et al. (2000) had isolated a gene from Salmonella responsible for the introduction of a 2-hydroxyl group into a lipid-A-bound myristic acid residue. The hydroxylation reaction is catalyzed by the Fe2+/O2/α-ketoglutarate-dependent LpxO dioxygenase. Rojas-Jiménez et al. (2005) had identified a gene called olsC in R. tropici encoding an LpxO homolog responsible for the synthesis of hydroxylated OLs. Later, it was shown that OlsC is responsible for the introduction of a hydroxyl group in the C-2 position of the piggy-back fatty acid of OLs (Vences-Guzmán et al., 2011).

A prediction indicates that OlsC of R. tropici CIAT899 is a water-soluble protein of 281 amino acids (Rojas-Jiménez et al., 2005). Owing to its homology to LpxO from Salmonella, it can be expected that OlsC-dependent hydroxylation of the ester-linked fatty acid will also be Fe2+/O2/α-ketoglutarate dependent.

Genes encoding OlsC homologs can be found in Agrobacterium vitis, Agrobacterium radiobacter, Ochrobactrum anthropi, Ochrobactrum intermedium, Aurantimonas manganoxydans, Fulvimarina pelagi, Roseomonas cervicalis, Chelativorans sp., Mycobacterium rhodesiae, and several Brucella species (Supporting Information, Table S1). Interestingly, in the so-called classical Brucella such as Brucella ovis, Brucella suis, Brucella melitensis, or B. abortus, which are intracellular pathogens, the olsC gene is present only as pseudogene containing a frameshift mutation. As a consequence, the olsC gene is translated into two ORFs, making the gene olsC nonfunctional (Palacios-Chaves et al., 2011). In the genomes of several atypical Brucella strains such as Brucella microti, Brucella sp. BO1, or Brucella sp. BO2 which share several characteristics with the opportunistic soil pathogen Ochrobactrum, olsC genes lacking the frameshift can be detected that are probably functional. This observation implies that organisms like Ochrobactrum, R. tropici, and nonclassical Brucella such as Brucella isolated from soil that present both (De et al., 2008;Scholz et al., 2008a, 2008b, 2009, 2010) an intracellular and a free-living lifestyle have preserved a functional copy of olsC, whereas the classical Brucella strains that are strictly intracellular pathogens present only a nonfunctional copy of olsC (Palacios-Chaves et al., 2011). A functional OlsC might confer a selective advantage in adverse abiotic stress conditions, but might not be of use or even have a negative impact when the bacteria are inside a host.

Recently, Vences-Guzmán et al. (2011) reported a more detailed study of an olsC-deficient Rtropici mutant. Strains lacking the OL hydroxylase OlsC showed a growth defect at increased temperatures (37 and 42 °C) and under acid pH conditions (4.5 and 4.0). Strain 899-olsCΔ1 lacking OlsC formed underdeveloped nodules on bean plants 21 days after inoculation with the bacteria. The nodules lacked lenticels and fixed two times less nitrogen (Rojas-Jiménez et al., 2005). The three R. tropici mutants (ΔolsC, ΔolsE, and ΔolsCΔolsE) lacking OL hydroxylases established their symbiosis only poorly (Vences-Guzmán et al., 2011). As R. tropici is challenged by low pH conditions inside its host plant (Udvardi et al., 1991; Udvardi & Day, 1997), it can be speculated that the observed symbiotic phenotype is a consequence of the mutants' increased acid sensitivity.

The OL hydroxylase OlsD

The OL hydroxylase OlsD was first isolated from B. cenocepacia J2315, a β-proteobacterium known as an opportunistic pathogen of humans. González-Silva et al. (2011) originally suggested that 2-hydroxylation of OLs in B. cenocepacia might be performed by an LpxO homolog called OlsD (BCAM2401). OlsD indeed hydroxylated OL, but the hydroxylation did not occur on the ester-linked fatty acid. Surprisingly, data obtained by mass spectrometry suggested that OlsD modifies the amide-linked fatty acid of OLs with a hydroxyl group (Fig. 2), a modification that was previously unknown. Unfortunately, their analysis did not allow for the determination of the exact position of the hydroxyl group. OlsD from B. cenocepacia is a 249-amino-acid protein, apparently lacking transmembrane helices (González-Silva et al., 2011). It is widely distributed within the genus Burkholderia, but homologs are also present in three Serratia strains. The gene coding for the 2-hydroxylase activity hydroxylating the ester-linked fatty acyl residue in the C-2 position in B. cenocepacia has not been identified yet.

The OL hydroxylase OlsE

Rojas-Jiménez et al. (2005) had described the presence of four different OLs in R. tropici CIAT899. The presence of OlsC alone could not explain this number of distinct structures. Using a functional expression screen conjugating a cosmid bank from R. tropici into S. meliloti, Vences-Guzmán et al. (2011) identified the gene olsE coding for the hydroxylase OlsE. Mass spectrometry analysis showed that OlsE introduced a hydroxyl group in the ornithine moiety. So far, the exact position of the hydroxylation could not be determined, but ninhydrin staining of the different OLs shows that the hydroxyl group affects the reactivity of the lipid to ninhydrin.

Bioinformatic predictions indicate that the OlsE protein (331 amino acids) from R. tropici CIAT899 is highly hydrophobic and might form between 4 and 6 transmembrane helices. OlsE belongs to the fatty acyl hydroxylase superfamily (cl01132), which is characterized by the presence of two copies of the HXHH motif. This superfamily includes fatty acid and carotene hydroxylases, sterol desaturases, and C-4 sterol methyl oxidase (Arthington et al., 1991; Bard et al., 1996; Mitchell & Martin, 1997; Kennedy et al., 2000). A similar motif can be found in membrane-bound fatty acid desaturases such as OLE1 from Saccharomyces cerevisiae and in bacterial alkane hydroxylase and xylene monooxygenase (Kok et al., 1989; Suzuki et al., 1991). In these proteins, the conserved histidine residues act to co-ordinate an oxo-bridged di-iron cluster (Fe-O-Fe) that functions as part of the reaction center (Fox et al., 1993; Shanklin et al., 1994). The closest OlsE homologs are present in all the sequenced Agrobacterium strains, Rhodospirillum centenum, Parvibaculum lamentivorans, Verrucomicrobium spinosum, Micavibrio aeruginosavorus, and Azospirillum amazonense. More distant homologs are present in several actinomycetes, a few Gammaproteobacteria, and a few other Alphaproteobacteria (Table S1).

No growth phenotype was observed for the OlsE--deficient mutant at increased temperatures or under pH stress conditions. Bean plants infected with OlsE-deficient mutants presented less red nodules and more white nodules than plants infected with the wild type. Nitrogen fixation of nodules from OlsE mutant-infected plants was clearly reduced (Vences-Guzmán et al., 2011).

Taurine transfer to OLs

In G. cerinus, a taurine residue can be amide-linked to the α-amino group of the ornithine moiety of OL (Tahara et al., b). It has been shown that a cell-free protein crude extract from G. cerinus contains an enzymatic activity responsible for the transfer of taurine to OL hydroxylated in the 2-position of the piggy-back fatty acid. This taurine transfer activity depends on the presence of ATP and bivalent cations (Tahara et al., b). As no G. cerinus strain has been sequenced so far, a bioinformatic search for candidate genes/proteins has not been possible.

Prediction of OL distribution

  1. Top of page
  2. Abstract
  3. Introduction
  4. Distribution and structure of OLs
  5. Biosynthesis of OLs
  6. OL-modifying activities
  7. Prediction of OL distribution
  8. Regulation of OL biosynthesis and modification
  9. OL functions
  10. Perspectives
  11. Acknowledgements
  12. References
  13. Supporting Information

The wealth of genome sequence information that has been produced in recent years allows for an accurate analysis of the distribution of OL biosynthesis genes. Genes coding for OlsB have a high predictive value, and it should be possible to predict the capacity of an organism to synthesize OL from the presence of the olsB gene. In many cases, where the olsB gene is phylogenetically less well conserved, the fact that olsB often occurs in an operon with olsA is of help. For the purpose of predicting the distribution of OLs, we analyzed all sequenced bacterial genomes for the presence of a gene encoding an OlsB homolog. BLAST searches with OlsB sequences from S. meliloti and B. cenocepacia pick up OlsB homologs in about 25% of the sequenced bacterial species which belong to the Alpha-, Beta-, Gamma-, Deltaproteobacteria, Actinomycetales, spirochetes, green nonsulfur bacteria, verrucomicrobia, firmicutes, Aquificales, and cyanobacteria (Table S1). Within the class Alphaproteobacteria, OlsB homologs can be detected in most sequenced species belonging to the orders Rhizobiales, Rhodobacterales, and Rhodospirillales, but are generally absent from species belonging to the orders Caulobacterales, Rickettsiales, and Sphingomonadales. OlsB can also be detected in the majority of sequenced Betaproteobacteria, including most Burkholderiales and many Neisseriales, but are absent from the Nitrosomonales. In the Gammaproteobacteria, OlsB homologs are absent from Enterobacterales, Vibrionales, Pasteurellales, Legionellales, and Aeromonadales. Many organisms presenting OlsB homologs belong to the orders Acidithiobacillales, Chromatiales, Pseudomonadales, Methylococcales, and Thiotrichales. In this context, it has to be mentioned that OLs have been described in Serratia marcescens, which belongs to the Enterobacteriaceae (Miyazaki et al., 1993). Unfortunately, no complete genome sequence of S. marcescens has been published so far. Within the Deltaproteobacteria, OlsB homologs are encoded in the genomes of Stigmatella aurantiaca, Bacteriovorax marinus, and Bdellovibrio bacteriovorus. Interestingly, OLs have been detected in the Deltaproteobacterium Sorangium cellulosum So ce56 (Keck et al., 2011), but no gene encoding an OlsB homolog is present in the genome. The best hit when searching the S. cellulosum genome with OlsB from B. cenocepacia is the gene rimI1, which is predicted to encode a ribosomal protein alanine acetyltransferase (sce1382). This suggests that a second unrelated family of N-acyl transferases might be responsible for LOL formation in S. cellulosum and possibly in other bacteria. Among the actinomycetes are several species encoding OlsB homologs. Most of them can be classified into the families Gordoniaceae, Micromonosporaceae, Mycobacteriaceae, Nocardiaceae, Pseudonocardiaceae, and Streptomyceteae. Among the spirochetes, several species from the genus Leptospira present a gene encoding an OlsB homolog. Only very few species belonging to other taxonomical groups present a gene encoding an OlsB homolog in their genomes. Compared to the large number of bacterial species that have been shown to form OL or that are predicted to be able to form OL, only few bacterial species have the now known OL-modifying enzymes. The identified OL hydroxylases belong either to the Fe2+/O2/α-ketoglutarate-dependent superfamily of hydroxylases (OlsC and OlsD) or to the di-iron fatty acid hydroxylase superfamily (OlsE) (Table S1). The phylogenetic distribution of these OL hydroxylases is described in the sections The OL hydroxylase OlsC'The OL hydroxylase OlsC ', The OL hydroxylase OlsD'The OL hydroxylase OlsD ' and The OL hydroxylase OlsE'The OL hydroxylase OlsE ' and in Table S1. The 2-hydroxylase from Burkholderia species has not been isolated yet, so it is not known whether it belongs to the already mentioned superfamilies or to yet another superfamily such as the cytochrome P450-dependent enzymes (Matsunaga et al., 2000; Lee et al., 2003; Girhard et al., 2007; Fujishiro et al., 2011). As possible OL modifications might occur only under specific stress conditions, it is possible that additional modifications with their respective responsible enzymatic activities and genes will be found in the future in other organisms.

Regulation of OL biosynthesis and modification

  1. Top of page
  2. Abstract
  3. Introduction
  4. Distribution and structure of OLs
  5. Biosynthesis of OLs
  6. OL-modifying activities
  7. Prediction of OL distribution
  8. Regulation of OL biosynthesis and modification
  9. OL functions
  10. Perspectives
  11. Acknowledgements
  12. References
  13. Supporting Information

It has been observed that the biosynthesis of OLs is regulated by the presence of certain nutrients in the growth medium. Some organisms such as S. meliloti synthesize only very minor amounts of OLs under phosphate-replete conditions (Geiger et al., 1999). If the same organism is cultivated in a medium with limiting phosphate concentrations, then olsB gene transcription, which is regulated by the transcriptional regulator PhoB (Geiger et al., 1999; Krol & Becker, 2004), is increased. It seems that at least in S. meliloti OlsB is the limiting factor for OL formation because constitutive expression of OlsB in S. meliloti 1021 causes the accumulation of OLs whether the bacteria are grown in high or low concentrations of phosphate (Gao et al., 2004). However, many other bacteria such as Brucella species, Burkholderia species, Agrobacterium species, Mesorhizobium loti (Devers et al., 2011), and R. tropici synthesize OLs constitutively in relatively high amounts even when grown in rich culture media containing high phosphate concentrations (González-Silva et al., 2011; Palacios-Chaves et al., 2011; Vences-Guzmán et al., 2011). The reason for this difference occurring even in closely related bacterial species is not understood.

The OL biosynthesis genes olsA and olsB are separated by more than ten genes in S. meliloti, whereas in P. aeruginosa and many other organisms, they form an operon. These differences in gene organization might indicate differences in the regulation of gene expression. This is consistent with the observation that phosphate starvation induces olsB expression, but not olsA expression in S. meliloti (Gao et al., 2004; Krol & Becker, 2004), whereas in P. aeruginosa also olsA is induced by phosphate limitation (Lewenza et al., 2011).

A different nutritional condition, low magnesium ion concentration, has been shown to repress OL biosynthesis in Pseudomonas fluorescens (Minnikin & Abdolrahimzadeh, 1974).

The frequency of OL hydroxylation seems to correlate in some cases with abiotic stress conditions. In B. cenocepacia and R. tropici, increased temperatures (42 °C) caused the accumulation of OL species hydroxylated in the C-2 position of the piggy-back fatty acid (Taylor et al., 1998; Vences-Guzmán et al., 2011). Under acidic growth conditions, both the OlsD-dependent hydroxylation and the OlsC-dependent hydroxylation seem to be induced in B. cenocepacia and R. tropici, respectively (González-Silva et al., 2011; Vences-Guzmán et al., 2011).

OL functions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Distribution and structure of OLs
  5. Biosynthesis of OLs
  6. OL-modifying activities
  7. Prediction of OL distribution
  8. Regulation of OL biosynthesis and modification
  9. OL functions
  10. Perspectives
  11. Acknowledgements
  12. References
  13. Supporting Information

Although several mutants deficient in OL biosynthesis have been constructed and characterized, the roles that OLs play are still not clear. In Gram-negative bacteria, OLs are enriched in the outer membrane (Dees & Shively, 1982; Lewenza et al., 2011; Vences-Guzmán et al., 2011), and owing to their zwitterionic nature, it had been proposed that they play an important role in the stabilization of negative charges of LPS and therefore in outer membrane stability (Freer et al., 1996). One common observation seems to be that OLs are involved in stress response. For the case of 2-hydroxylated lipid A, it has been suggested that the additional hydroxyl groups might increase the extent of hydrogen bonding between the lipid molecules, thereby decreasing the outer membrane fluidity while at the same time making it less permeable (Gibbons et al., 2000). These changes should be of advantage under abiotic stress conditions such as increased temperature or low pH. The introduction of a 2-hydroxyl group into OLs should have similar consequences as described above for lipid A hydroxylation. Interestingly, both B. cenocepacia and R. tropici show an increase in OL 2-hydroxylation under thermal stress conditions (Taylor et al., 1998; Vences-Guzmán et al., 2011), and R. tropici mutants deficient in the OL hydroxylase OlsC show a severe growth defect under this condition.

Earlier studies have reported an increase in resistance to antimicrobial peptides correlating with OL accumulation in some bacteria (Minnikin & Abdolrahimzadeh, 1974; Dorrer & Teuber, 1977). Recently, however, it has been demonstrated that OLs are not required to increase the resistance to antimicrobial peptides in B. abortus and P. aeruginosa (Lewenza et al., 2011; Palacios-Chaves et al., 2011).

During the last year, two more OL hydroxylations have been described (González-Silva et al., 2011; Vences-Guzmán et al., 2011). As OLs from some bacteria can present multiple hydroxylations within the same molecule, it probably can be assumed that different modifications affect membrane properties in different ways. Accordingly, the responsible hydroxylase activities should be regulated differentially. At high temperature or in acid pH, conditions under which the OlsC-modified OL P1 accumulated in R. tropici CIAT899 (Vences-Guzmán et al., 2011), the OlsE-hydroxylated OLs S2 and P2 could not be detected. Consistent with this idea, we have observed in A. tumefaciens that the relative amount of the OlsE-hydroxylated OL S2 increases at lower growth temperature (Vences-Guzmán et al., preparation). This indicates that the OlsE-dependent hydroxylation might increase, for example, membrane fluidity, which would be opposite to the predicted effect of the OlsC-dependent hydroxylation.

In the purple nonsulfur facultative phototroph R. capsulatus, it has been shown that OL biosynthesis and the steady-state amounts of some extracytoplasmic proteins, including various c-type cytochromes, are interrelated. In the absence of OLs, R. capsulatus does not contain a full complement of c-type cytochromes under certain physiological conditions (Aygun-Sunar et al., 2006). One possible explanation is that protein–lipid interactions between OLs and certain membrane proteins are required for the localization, folding, stability, assembly, and/or enzymatic activity of certain integral membrane proteins (Aygun-Sunar et al., 2006).

Interestingly, OLs also serve functions outside the membrane in some organisms. It has been reported that OLs are used as emulsifiers for crude oil in the marine bacterium Myroides sp. (Maneerat et al., 2006).

OLs as bioactive lipids

The lipid A moiety of LPS is detected by the TLR4/MD2 receptor of the mammalian innate immune response, causing macrophages to synthesize potent mediators of inflammation, such as TNF-α and IL-1β (Beutler & Cerami, 1988; Dinarello, 1991); especially, its phosphate and acyloxyacyl groups are needed to trigger full TLR4/MD2 activation in human cells (Rietschel et al., 1994). OLs and SGLs also contain the acyloxyacyl structure present in lipid A.

It has been shown that OLs and SGLs can be used as adjuvants (Kato & Goto, 1997; Kawai et al., 1999, 2002) and when injected into mice before lipid A can prevent the lethal effects of the latter. It was speculated that the OLs and SGLs might function as antagonistic blockers of events triggered by lipid A (Kawai et al., 1991). The components involved in the translation of the signal induced by OLs have not yet been identified. The structural similarity of the OLs with lipid A and the SGLs suggests that OLs will probably use the same components as the lipid A and SGLs.

A recent study showed that B. abortus OLs do not stimulate cytokine secretion in murine macrophages, whereas OLs from Bordetella pertussis notably stimulated TNF-α and IL-6 secretion (Palacios-Chaves et al., 2011). At first glance, the only difference between OLs from B. abortus and OLs from B. pertussis seems to be with respect to fatty acyl chain lengths (Palacios-Chaves et al., 2011). An alternative explanation might be that B. pertussis presents hydroxylated OLs under specific growth conditions. Most studies failed to detect hydroxylated OL in B. pertussis, but Thiele & Schwinn (1973) clearly detected the presence of a ninhydrin-positive lipid migrating similarly as a hydroxylated OLs from B. cenocepacia or R. tropici (Taylor et al., 1998; Rojas-Jiménez et al., 2005; González-Silva et al., 2011; Vences-Guzmán et al., 2011).

Perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Distribution and structure of OLs
  5. Biosynthesis of OLs
  6. OL-modifying activities
  7. Prediction of OL distribution
  8. Regulation of OL biosynthesis and modification
  9. OL functions
  10. Perspectives
  11. Acknowledgements
  12. References
  13. Supporting Information

The recent decade has brought many advances in our knowledge about OL biosynthesis and function. In 2002 and 2004, Geiger and coworkers identified two acyltransferases required for OL biosynthesis. The general idea is that both proteins are sufficient for OL biosynthesis. However, the expression of sinorhizobial OlsBA in Escherichia coli is not sufficient to convert this host into an OL producer (O. Geiger and I.M. López-Lara, unpublished data). Our combined analysis of the scientific literature with respect to OLs and the presence of OlsB-encoding genes in bacterial genome sequences indicates that in addition to the OlsBA-dependent pathway, other pathways for OL biosynthesis must exist at least in S. cellulosum and Flavobacterium sp. More recently, three OL hydroxylases have been discovered, two of which catalyzing modifications that were not known previously. Still, the gene encoding the 2-hydroxylase from Burkholderia, one of the first organisms where the 2-hydroxylation of the piggy-back fatty acid has been described, is still unknown.

The exact functions of the different OL modifications have not been defined yet, although several OL biosynthesis genes are now known and mutants deficient in these activities have been constructed and partially characterized. The analysis of the mutants should continue, especially with respect to changes in membrane properties caused by the presence and absence of the distinct modifications. Hand in hand should go a structural determination of the products of the OlsD- and OlsE-catalyzed reactions. The exact structure of both modifications is required to understand the function/properties of the different lipids on a biophysical level.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Distribution and structure of OLs
  5. Biosynthesis of OLs
  6. OL-modifying activities
  7. Prediction of OL distribution
  8. Regulation of OL biosynthesis and modification
  9. OL functions
  10. Perspectives
  11. Acknowledgements
  12. References
  13. Supporting Information

M.Á.V.-G. is a PhD student from the Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México, and is a recipient of a scholarship from the Consejo Nacional de Ciencia y Tecnología, México. Work in our laboratory has been financed by grants from CONACyT-Mexico (46020-N and 153200) and DGAPA/UNAM (IN217907 and IN201310) to C.S.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Distribution and structure of OLs
  5. Biosynthesis of OLs
  6. OL-modifying activities
  7. Prediction of OL distribution
  8. Regulation of OL biosynthesis and modification
  9. OL functions
  10. Perspectives
  11. Acknowledgements
  12. References
  13. Supporting Information
  • Arthington BA , Bennett LG , Skatrud PL , Guynn CJ , Barbuch RJ , Ulbright CE & Bard M (1991) Cloning, disruption and sequence of the gene encoding yeast C-5 sterol desaturase. Gene 102: 3944.
  • Asselineau J (1991) Bacterial lipids containing amino acids or peptides linked by amide bonds. Fortschr Chem Org Naturst 56: 185.
  • Aygun-Sunar S , Mandaci S , Koch HG , Murray IV , Goldfine H & Daldal F (2006) Ornithine lipid is required for optimal steady-state amounts of c-type cytochromes in Rhodobacter capsulatus . Mol Microbiol 61: 418435.
  • Bard M , Bruner DA , Pierson CA , Lees ND , Biermann B , Frye L , Koegel C & Barbuch R (1996) Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase. P Natl Acad Sci USA 93: 186190.
  • Batrakov SG , Nikitin DI , Mosezhnyi AE & Ruzhitsky AO (1999) A glycine-containing phosphorus-free lipoaminoacid from the gram-negative marine bacterium Cyclobacterium marinus WH. Chem Phys Lipids 99: 139143.
  • Benning C , Huang ZH & Gage DA (1995) Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation. Arch Biochem Biophys 317: 103111.
  • Beutler B & Cerami A (1988) Tumor necrosis, cachexia, shock, and inflammation: a common mediator. Annu Rev Biochem 57: 505518.
  • De BK , Stauffer L , Koylass MS et al. (2008) Novel Brucella strain (BO1) associated with a prosthetic breast implant infection. J Clin Microbiol 46: 4349.
  • Dees C & Shively JM (1982) Localization of quantitation of the ornithine lipid of Thiobacillus thiooxidans . J Bacteriol 149: 798799.
  • Devers EA , Wewer V , Dombrink I , Dörmann P & Hölzl G (2011) A processive glycosyltransferase involved in glycolipid synthesis during phosphate deprivation in Mesorhizobium loti . J Bacteriol 193: 13771384.
  • Dinarello CA (1991) Interleukin-1 and interleukin-1 antagonism. Blood 77: 16271652.
  • Dorrer E & Teuber M (1977) Induction of polymyxin resistance in Pseudomonas fluorescens by phosphate limitation. Arch Microbiol 114: 8789.
  • Fox BG , Shanklin J , Somerville C & Munck E (1993) Stearoyl-acyl carrier protein delta 9 desaturase from Ricinus communis is a diiron-oxo protein. P Natl Acad Sci USA 90: 24862490.
  • Freer E , Moreno E , Moriyon I , Pizarro-Cerda J , Weintraub A & Gorvel JP (1996) Brucella-Salmonella lipopolysaccharide chimeras are less permeable to hydrophobic probes and more sensitive to cationic peptides and EDTA than are their native Brucella sp. counterparts. J Bacteriol 178: 58675876.
  • Fujishiro T , Shoji O , Nagano S , Sugimoto H , Shiro Y & Watanabe Y (2011) Crystal structure of H2O2-dependent cytochrome P450SPalpha with its bound fatty acid substrate: insight into the regioselective hydroxylation of fatty acids at the alpha position. J Biol Chem 286: 2994129950.
  • Galbraith L , Jonsson MH , Rudhe LC & Wilkinson SG (1999) Lipids and fatty acids of Burkholderia and Ralstonia species. FEMS Microbiol Lett 173: 359364.
  • Gao JL , Weissenmayer B , Taylor AM , Thomas-Oates J , López-Lara IM & Geiger O (2004) Identification of a gene required for the formation of lyso-ornithine lipid, an intermediate in the biosynthesis of ornithine-containing lipids. Mol Microbiol 53: 17571770.
  • Geiger O , Röhrs V , Weissenmayer B , Finan TM & Thomas-Oates JE (1999) The regulator gene phoB mediates phosphate stress-controlled synthesis of the membrane lipid diacylglyceryl-N,N,N-trimethylhomoserine in Rhizobium (Sinorhizobium) meliloti . Mol Microbiol 32: 6373.
  • Geiger O , González-Silva N , López-Lara IM & Sohlenkamp C (2010) Amino acid-containing membrane lipids in bacteria. Prog Lipid Res 49: 4660.
  • Gibbons HS , Lin S , Cotter RJ & Raetz CR (2000) Oxygen requirement for the biosynthesis of the S-2-hydroxymyristate moiety in Salmonella typhimurium lipid A. Function of LpxO, A new Fe2+/alpha-ketoglutarate-dependent dioxygenase homologue. J Biol Chem 275: 3294032949.
  • Girhard M , Schuster S , Dietrich M , Durre P & Urlacher VB (2007) Cytochrome P450 monooxygenase from Clostridium acetobutylicum: a new alpha-fatty acid hydroxylase. Biochem Biophys Res Commun 362: 114119.
  • González-Silva N , López-Lara IM , Reyes-Lamothe R , Taylor AM , Sumpton D , Thomas-Oates J & Geiger O (2011) The dioxygenase-encoding olsD gene from Burkholderia cenocepacia causes the hydroxylation of the amide-linked fatty acyl moiety of ornithine-containing membrane lipids. Biochemistry 50: 63966408.
  • Kato H & Goto N (1997) Adjuvanticity of an ornithine-containing lipid of Flavobacterium meningosepticum as a candidate vaccine adjuvant. Microbiol Immunol 41: 101106.
  • Kawai Y , Yano I & Kaneda K (1988) Various kinds of lipoamino acids including a novel serine-containing lipid in an opportunistic pathogen Flavobacterium. Their structures and biological activities on erythrocytes. Eur J Biochem 171: 7380.
  • Kawai Y , Kaneda K , Morisawa Y & Akagawa K (1991) Protection of mice from lethal endotoxemia by use of an ornithine-containing lipid or a serine-containing lipid. Infect Immun 59: 25602566.
  • Kawai Y , Nakagawa Y , Matuyama T , Akagawa K , Itagawa K , Fukase K , Kusumoto S , Nishijima M & Yano I (1999) A typical bacterial ornithine-containing lipid Nalpha-(D)-[3-(hexadecanoyloxy)hexadecanoyl]-ornithine is a strong stimulant for macrophages and a useful adjuvant. FEMS Immunol Med Microbiol 23: 6773.
  • Kawai Y , Watanabe M , Matsuura M , Nishijima M & Kawahara K (2002) The partially degraded lipopolysaccharide of Burkholderia cepacia and ornithine-containing lipids derived from some Gram-negative bacteria are useful complex lipid adjuvants. FEMS Immunol Med Microbiol 34: 173179.
  • Kawazoe R , Okuyama H , Reichardt W & Sasaki S (1991) Phospholipids and a novel glycine-containing lipoamino acid in Cytophaga johnsonae Stanier strain C21. J Bacteriol 173: 54705475.
  • Keck M , Gisch N , Moll H et al. (2011) Unusual outer membrane lipid composition of the gram-negative, lipopolysaccharide-lacking myxobacterium Sorangium cellulosum So ce56. J Biol Chem 286: 1285012859.
  • Kennedy MA , Johnson TA , Lees ND , Barbuch R , Eckstein JA & Bard M (2000) Cloning and sequencing of the Candida albicans C-4 sterol methyl oxidase gene (ERG25) and expression of an ERG25 conditional lethal mutation in Saccharomyces cerevisiae . Lipids 35: 257262.
  • Knoche HW & Shively JM (1972) The structure of an ornithine-containing lipid from Thiobacillus thiooxidans . J Biol Chem 247: 170178.
  • Kok M , Oldenhuis R , van der Linden MP , Raatjes P , Kingma J , van Lelyveld PH & Witholt B (1989) The Pseudomonas oleovorans alkane hydroxylase gene. Sequence and expression. J Biol Chem 264: 54355441.
  • Krol E & Becker A (2004) Global transcriptional analysis of the phosphate starvation response in Sinorhizobium meliloti strains 1021 and 2011. Mol Genet Genomics 272: 117.
  • Lee DS , Yamada A , Sugimoto H , Matsunaga I , Ogura H , Ichihara K , Adachi S , Park SY & Shiro Y (2003) Substrate recognition and molecular mechanism of fatty acid hydroxylation by cytochrome P450 from Bacillus subtilis. Crystallographic, spectroscopic, and mutational studies. J Biol Chem 278: 97619767.
  • Lewenza S , Falsafi R , Bains M , Rohs P , Stupak J , Sprott GD & Hancock RE (2011) The olsA gene mediates the synthesis of an ornithine lipid in Pseudomonas aeruginosa during growth under phosphate-limiting conditions, but is not involved in antimicrobial peptide susceptibility. FEMS Microbiol Lett 320: 95102.
  • López-Lara IM , Sohlenkamp C & Geiger O (2003) Membrane lipids in plant-associated bacteria: their biosyntheses and possible functions. Mol Plant Microbe Interact 16: 567579.
  • Maneerat S , Bamba T , Harada K , Kobayashi A , Yamada H & Kawai F (2006) A novel crude oil emulsifier excreted in the culture supernatant of a marine bacterium, Myroides sp. strain SM1. Appl Microbiol Biotechnol 70: 254259.
  • Matsunaga I , Sumimoto T , Ueda A , Kusunose E & Ichihara K (2000) Fatty acid-specific, regiospecific, and stereospecific hydroxylation by cytochrome P450 (CYP152B1) from Sphingomonas paucimobilis: substrate structure required for alpha-hydroxylation. Lipids 35: 365371.
  • Minnikin DE & Abdolrahimzadeh H (1974) Replacement of phosphatidylethanolamine and acidic phospholipids by an ornithine-amide lipid and a minor phosphorus-free lipid in Pseudomonas fluorescens Ncmb 129. FEBS Lett 43: 257260.
  • Mitchell AG & Martin CE (1997) Fah1p, a Saccharomyces cerevisiae cytochrome b5 fusion protein, and its Arabidopsis thaliana homolog that lacks the cytochrome b5 domain both function in the alpha-hydroxylation of sphingolipid-associated very long chain fatty acids. J Biol Chem 272: 2828128288.
  • Miyazaki Y , Oka S , Hara-Hotta H & Yano I (1993) Stimulation and inhibition of polymorphonuclear leukocytes phagocytosis by lipoamino acids isolated from Serratia marcescens . FEMS Immunol Med Microbiol 6: 265271.
  • Palacios-Chaves L , Conde-Álvarez R , Gil-Ramírez Y et al. (2011) Brucella abortus ornithine lipids are dispensable outer membrane components devoid of a marked pathogen-associated molecular pattern. PLoS ONE 6: e16030.
  • Raetz CR , Reynolds CM , Trent MS & Bishop RE (2007) Lipid A modification systems in gram-negative bacteria. Annu Rev Biochem 76: 295329.
  • Rietschel ET , Kirikae T , Schade FU et al. (1994) Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J 8: 217225.
  • Rojas-Jiménez K , Sohlenkamp C , Geiger O , Martínez-Romero E , Werner D & Vinuesa P (2005) A ClC chloride channel homolog and ornithine-containing membrane lipids of Rhizobium tropici CIAT899 are involved in symbiotic efficiency and acid tolerance. Mol Plant Microbe Interact 18: 11751185.
  • Scholz HC , Hubalek Z , Nesvadbova J et al. (2008a) Isolation of Brucella microti from soil. Emerg Infect Dis 14: 13161317.
  • Scholz HC , Hubalek Z , Sedlacek I et al. (2008b) Brucella microti sp. nov., isolated from the common vole Microtus arvalis . Int J Syst Evol Microbiol 58: 375382.
  • Scholz HC , Hofer E , Vergnaud G , Le Fleche P , Whatmore AM , Al Dahouk S , Pfeffer M , Kruger M , Cloeckaert A , Tomaso H (2009) Isolation of Brucella microti from mandibular lymph nodes of red foxes, Vulpes vulpes, in lower Austria. Vector Borne Zoonotic Dis 9: 153156.
  • Scholz HC , Nockler K , Gollner C et al. (2010) Brucella inopinata sp. nov., isolated from a breast implant infection. Int J Syst Evol Microbiol 60: 801808.
  • Shanklin J , Whittle E & Fox BG (1994) Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase. Biochemistry 33: 1278712794.
  • Shiozaki M , Deguchi N , Ishikawa T , Haruyama H , Kawai Y & Nishijima M (1998a) Revised structure of flavolipin and synthesis of its isomers. Tetrahedron Lett 39: 44974500.
  • Shiozaki M , Deguchi N , Mochizuki T et al. (1998b) Revised structure and synthesis of flavolipin. Tetrahedron 54: 1186111876.
  • Sohlenkamp C , Galindo-Lagunas KA , Guan Z , Vinuesa P , Robinson S , Thomas-Oates J , Raetz CR & Geiger O (2007) The lipid lysyl-phosphatidylglycerol is present in membranes of Rhizobium tropici CIAT899 and confers increased resistance to polymyxin B under acidic growth conditions. Mol Plant Microbe Interact 20: 14211430.
  • Suzuki M , Hayakawa T , Shaw JP , Rekik M & Harayama S (1991) Primary structure of xylene monooxygenase: similarities to and differences from the alkane hydroxylation system. J Bacteriol 173: 16901695.
  • Tahara Y , Yamada Y & Kondo K (1976a) New lysine-containing lipid isolated from Agrobacterium tumefaciens . Agric Biol Chem 40: 14491450.
  • Tahara Y , Kameda M , Yamada Y & Kondo K (1976b) New lipid – ornithine and taurine-containing cerilipin. Agric Biol Chem 40: 243244.
  • Tahara Y , Shinmoto K , Yamada Y & Kondo K (1978) Enzymatic-synthesis of tauro-ornithine lipid in Gluconobacter cerinus . Agric Biol Chem 42: 205206.
  • Taylor CJ , Anderson AJ & Wilkinson SG (1998) Phenotypic variation of lipid composition in Burkholderia cepacia: a response to increased growth temperature is a greater content of 2-hydroxy acids in phosphatidylethanolamine and ornithine amide lipid. Microbiology 144: 17371745.
  • Thiele OW & Schwinn G (1973) The free lipids of Brucella melitensis and Bordetella pertussis . Eur J Biochem 34: 333344.
  • Udvardi MK & Day DA (1997) Metabolite transport across symbiotic membranes of legume nodules. Annu Rev Plant Physiol Plant Mol Biol 48: 493523.
  • Udvardi MK , Lister DL & Day DA (1991) ATPase activity and anion transport across the peribacteroid membrane of isolated soybean symbiosomes. Arch Microbiol 156: 362366.
  • Vences-Guzmán MA , Guan Z , Ormeño-Orrillo E , González-Silva N , López-Lara IM , Martínez-Romero E , Geiger O & Sohlenkamp C (2011) Hydroxylated ornithine lipids increase stress tolerance in Rhizobium tropici CIAT899. Mol Microbiol 79: 14961514.
  • Weissenmayer B , Gao JL , López-Lara IM & Geiger O (2002) Identification of a gene required for the biosynthesis of ornithine-derived lipids. Mol Microbiol 45: 721733.
  • Zhang X , Ferguson-Miller SM & Reid GE (2009) Characterization of ornithine and glutamine lipids extracted from cell membranes of Rhodobacter sphaeroides . J Am Soc Mass Spectrom 20: 198212.
  • Zhang X , Hiser C , Tamot B , Benning C , Reid GE & Ferguson-Miller SM (2011) Combined genetic and metabolic manipulation of lipids in Rhodobacter sphaeroides reveals non-phospholipid substitutions in fully active cytochrome c oxidase. Biochemistry 50: 38913902.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Distribution and structure of OLs
  5. Biosynthesis of OLs
  6. OL-modifying activities
  7. Prediction of OL distribution
  8. Regulation of OL biosynthesis and modification
  9. OL functions
  10. Perspectives
  11. Acknowledgements
  12. References
  13. Supporting Information
FilenameFormatSizeDescription
fml2623-sup-0001-TableS1.docxWord document78KTable S1. List of OlsB, OlsC, OlsD and OlsE homologs present in eubacteria.

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