Abstract
We compared the cellular fatty acid profiles of Myxococcus xanthus cells grown in either a Casitone-based complex medium or a chemically defined medium. The cells grown in the complex medium had a much higher content of the abundant branched-chain fatty acid iso-15:0 and several other branched-chain species. The higher branched-chain fatty acid content of the cells grown in the complex medium was dependent on the esg locus, which encodes the E1α and E1β components of a branched-chain keto acid dehydrogenase (BCKAD) multienzyme complex involved in branched-chain fatty acid biosynthesis. Cells grown in the complex medium were also found to have a higher level of esg transcription and more BCKAD enzyme activity than cells from the chemically defined medium. The level of esg transcription appears to be an important factor in the growth medium-dependent regulation of the M. xanthus branched-chain fatty acid content.
The fruiting myxobacteria are unusual among gram-negative bacteria in that branched-chain fatty acids (BCFA) constitute the majority of the cellular fatty acids (12). In Myxococcus xanthus, the most extensively studied myxobacterium, the BCFA have been reported to constitute about 65% of the total (21, 22). A single branched-chain species, iso-15:0, accounts for nearly 50% of the M. xanthus fatty acid. The BCFA are also the predominant fatty acid species in several gram-positive genera, such as Bacillus, Micrococcus, and Sarcina (6, 12, 20).
Our current understanding of the biosynthesis of BCFA is based primarily on work carried out with Bacillus species (6, 11, 12, 24). The pathway for BCFA synthesis begins with the three branched-chain amino acids (BCAA) leucine, isoleucine, and valine. These amino acids are deaminated and decarboxylated to produce short BCFA coenzyme A derivatives which serve as primers in the fatty acid elongation reactions. Elongation occurs in two carbon steps analogous to those used in straight-chain fatty acid synthesis.
An evolutionarily conserved multienzyme complex, the branched-chain keto acid dehydrogenase (BCKAD), is responsible for decarboxylation of the three branched-chain keto acids (produced by transamination reactions involving the BCAA) and the formation of the three coenzyme A derivatives of the resulting short BCFA (12). The BCKAD complex is composed of four polypeptide chains referred to as E1α, E1β, E2, and E3. In M. xanthus, the esg locus encodes the E1α and E1β BCKAD components (21). This conclusion is based on the observations that the predicted amino acid sequences of the esg open reading frames share significant similarity to those of proteins belonging to these conserved protein families, and that esg transposon insertion mutants have reduced levels of the BCFA and reduced BCKAD enzyme activity. Significantly, the esg mutants retain a reduced capacity for BCFA synthesis, indicating the existence of an esg-independent pathway or pathways for production of these fatty acid species. The esg mutants also fail to produce fruiting bodies, multicellular structures produced in response to nutrient depletion in M. xanthus (4, 19). Developmental studies of esg mutants have suggested the involvement of the BCFA in a cell-cell signaling system used to regulate developmental gene expression (5).
Growth medium-dependent fatty acid content of M. xanthus cells.
Since we were interested in using the chemically defined A1 medium (1) in labeling studies designed to identify the lipid species derived from the BCAA in M. xanthus, the total fatty acid content of wild-type (DZF1) and esg mutant (JD275) cells grown in A1 medium was compared with that of cells grown in the complex medium CTT medium (1% Casitone, 10 mM Tris-hydrochloride [pH 7.6], 1 mM KHPO4, 8 mM MgSO4) (8) as follows. The cells were grown to 70 to 100 Klett units, as measured with a Klett-Summerson colorimeter with the red filter. The cells were harvested by centrifugation at 8,000 × g for 10 min, and then the cell pellets were stored frozen until required for fatty acid analysis. Equal amounts (45 mg) of the cell pellets were used in the fatty acid analysis. Whole-cell fatty acid analysis was performed by Microcheck, Inc. (Burlington, Vt.) with sulfuric acid-methanol-treated lipid extracts and high-resolution gas chromatography. Note that CTT medium was used in an earlier study in which the fatty acid content of M. xanthus cells was determined (21). The primary carbon and energy sources in A1 medium are pyruvate and aspartate, and A1 medium also contains relatively low concentrations of the three BCAA that are essential for growth. M. xanthus grows much more slowly in A1 medium than in CTT medium. The generation times in the A1 and CTT media are 24 h and 4 to 5 h, respectively. The comparison of the fatty acid contents of cells grown in the two media showed that the levels of certain of the abundant BCFA were lower in the A1 medium-grown M. xanthus cells than in the CTT medium-grown cells (Table 1). Most significantly, the relative abundance of iso-15:0 declined about 40% in the A1 medium-grown cells (from 45.3% to 26.7%). The levels of the iso-13:0, iso-17:0, and iso-17:0 3OH species declined as well, and only one of the BCFA, iso-14:0 3OH, was found in greater quantities in A1 medium-grown cells. In combination with the overall decline in the BCFA content found with the A1 medium-grown cells, these cells were found to have a greatly increased level of the straight-chain saturated fatty acid 16:0 (palmitic acid) (from 3.9% in CTT medium to 22.3% in A1 medium). There was also a small increase in the level of the straight-chain unsaturated fatty acid 16:1 ω5c (from 16.4% to 20.6%). Clearly, in the M. xanthus wild-type cells, the fatty acid content varied widely, depending on the growth medium.
TABLE 1.
Fatty acid profiles of M. xanthus wild-type and esg mutant cells grown in CTT and A1 media
Since the esg locus encodes components of the BCKAD complex, which is used to produce BCFA, the fatty acid content of an esg mutant strain was also investigated. As observed previously (21), the esg mutant had lower levels of the BCFA than the wild type when cells were grown in CTT medium (Table 1). As an indication of this general pattern, the iso-15:0 content was only 16.2% in the CTT medium-grown esg mutant cells, compared with 45.3% in CTT medium-grown wild-type cells. However, in contrast to the pattern observed with wild-type cells, the BCFA content for A1 medium-grown esg mutant cells was similar to that found in CTT medium-grown mutant cells. For example, the levels of iso-15:0 were 20.8% in the A1 medium-grown esg cells and 16.2% for CTT medium-grown cells, while the values for iso-17:0 were 8.5 and 4.2% in the A1- and CTT medium-grown cells, respectively. In place of high levels of the BCFA, the esg mutant cells contained a large amount of the unsaturated fatty acid 16:1 ω5c (approximately 40%) in addition to a substantial level of palmitic acid (approximately 10%). The levels of these abundant straight-chain fatty acids in the esg mutant cells differed significantly from those in the wild type. Comparison of the wild-type and esg mutant fatty acid profiles in the two media clearly indicates that the esg locus is involved in the growth medium-dependent alteration in fatty acid composition observed in M. xanthus cells.
esg expression in different growth media.
One explanation for the esg-dependent regulation of the fatty acid profile that was observed is that expression of the esg locus is regulated in response to the growth medium. If this were the case, then it would be expected that there would be a low level of esg expression in A1 medium and a higher level in CTT medium. This gene expression pattern might result in a low level of the BCKAD in A1 medium-grown cells and correspondingly low production of the BCFA, while the CTT medium-grown cells would be expected to have relatively high levels of the BCKAD and a high BCFA content. To investigate esg expression, we utilized M. xanthus JD306 (4), which contains a Tn5lac (13) insertion within the esg locus, such that production of the β-galactosidase from the lacZ gene of Escherichia coli is placed under esg transcriptional control. This strain also has an unaltered copy of the esg locus and has a wild-type phenotype. JD306 was first grown for several generations in A1 medium before cells were collected by centrifugation and transferred to fresh CTT medium. The CTT medium culture was incubated at 32°C with vigorous agitation. The cell density was measured during incubation in CTT medium with a Klett-Summerson colorimeter, and 1.0-ml samples were removed for determination of the amount of β-galactosidase activity. The measurement of β-galactosidase activity has been described previously (17). Expression of the esg locus was found to be low in the A1 medium-grown cells (approximately 30 U) and, after a lag of about 5 h, was found to increase dramatically in CTT medium (Fig. 1). The greatest amount of esg-driven β-galactosidase activity was 550 U after 20 h of incubation in CTT medium. The level of β-galactosidase activity plateaued in the mid-log phase of growth (Fig. 1) and did not change when cells entered stationary phase (data not shown). A very similar pattern of esg expression was observed when the A1 medium-grown cells were transferred to Casitone-yeast extract medium (2), another commonly used M. xanthus complex medium, instead of CTT medium (data not shown).
FIG. 1.
Expression of the esg locus following transfer of M. xanthus cells from the chemically defined A1 medium to CTT medium. Expression of the esg locus was monitored with M. xanthus JD306 (4). This strain contains an esg-lacZ transcriptional fusion that was used to monitor the level of esg transcription. Following the transfer of the JD306 cells to CTT medium, the cell density was measured (Klett units [open squares]) at the indicated times and samples were collected for the determination of the amount of esg-driven β-galactosidase specific activity (Miller units [solid squares]).
Growth medium-dependent BCKAD activity.
To determine if increased esg expression in CTT medium was accompanied by increased BCKAD enzyme activity, assays were performed with crude extracts from A1- or CTT medium-grown cells. This was an important experiment, because the esg locus encodes only the E1α and E1β components of the BCKAD, and the genes for the unlinked E2 and E3 components have not yet been identified. Crude extracts from wild-type cells were assayed as described previously (21) with the three branched-chain keto acid substrates α-ketoisovaleric acid (KIV), α-keto-β-methyl-n-valeric acid, and α-ketoisocaproic acid. Assays with all three substrates indicated that higher levels of BCKAD activity are found in cells after growth in the CTT medium (Fig. 2). Most significantly, the CTT-grown cells had about 10-fold greater BCKAD activity than the A1 medium-grown cells with KIV as the substrate. In M. xanthus crude extracts, the highest levels of BCKAD activity have been consistently observed with KIV. Previous studies with an esg mutant strain have suggested that there may be a second BCKAD enzyme in M. xanthus, but the esg-encoded BCKAD has been shown to be responsible for at least 80% of the activity with the KIV substrate in CTT medium-grown cells (21). Thus, the results of the enzyme activity study show that the increased expression of esg that was observed in CTT medium-grown cells is correlated with an increased level of BCKAD activity.
FIG. 2.
BCKAD activity in M. xanthus cells grown in A1 (open bars) or CTT (solid bars) medium. The BCKAD specific activity was determined in extracts of the wild-type M. xanthus strain DZF1 grown vegetatively in the complex medium CTT or in the chemically defined medium A1. The three branched-chain keto acids KIV, α-keto-β-methyl-n-valeric acid (KMV), and α-ketoisocaproic acid (KIC) were used as substrates. The specific activity is presented in nanomoles of 2,6-dichlorophenolindolphenol reduced per minute per milligram of crude extract protein. The values reported are the averages of two separate determinations. The range between the values obtained in the separate determinations was less than 10% of the average.
Time course of the growth medium-dependent change in fatty acid content.
The time course of the change in the cellular fatty acid composition of M. xanthus cells was also investigated. Wild-type cells were transferred from A1 medium to CTT medium, and samples of the growing cells were removed at 10-h intervals. These samples were then assayed for esg-driven β-galactosidase activity, and the total cellular fatty acid composition was determined. The changes in the relative amounts of the three most abundant fatty acids in M. xanthus, iso-15:0, 16:0, and 16:1 ω5c, are shown in Fig. 3B. The most rapid change in the fatty acid composition was observed during the first 10 h in CTT medium, an interval during which esg expression also increased rapidly (Fig. 3A). The amount of iso-15:0 increased from 32% to 50%, and there was a corresponding decrease in the level of 16:0 from 18% to 6%. The amount of the unsaturated fatty acid 16:0 ω5c gradually declined. The cells exhibited a lag in CTT medium before beginning logarithmic growth, with a generation time of 4 to 5 h. During the 10- to 30-h interval, there were only a small increase in the iso-15:0 level and a small decrease in the 16:0 level. The amount of esg-driven β-galactosidase activity plateaued during this interval, although the cells continued to grow logarithmically.
FIG. 3.
Time course for the change in the relative amounts of the iso-15:0, 16:1 ω5c, and 16:0 fatty acid species during growth of M. xanthus cells in CTT medium. The esg-lacZ fusion strain JD306 was grown for more than six generations in A1 medium and than transferred to CTT medium. The cell density of the culture was monitored (A [open squares]), and samples were harvested from the culture at the indicated times and assayed for esg-driven β-galactosidase specific activity (A [solid squares]). The relative levels of three of the most abundant fatty acids, iso-15:0, 16:1 ω5c, and 16:0, were also determined by fatty acid analysis of cells harvested at the indicated times (B). Fatty acid analysis was performed as described in the text and as shown in Table 1.
esg expression in growth media with various concentrations of BCAA.
Attempts to determine the specific medium component(s) responsible for increased esg expression in complex media have been unsuccessful. Since the BCAA are used to produce the branched-chain keto acids which serve as the substrates for the BCKAD, these amino acids would be expected to be involved in esg regulation. This is the case in Pseudomonas putida, where transcription of the BCKAD proteins has been shown to respond to the availability of the BCAA (14, 16). However, the three BCAA are components of the chemically defined A1 medium, and altering the levels of the BCAA in A1 medium, either individually or as a group, did not result in increased esg expression (data not shown). Similarly, the use of other defined media that have been utilized for growth of M. xanthus and that contain different combinations of amino acids as the carbon and energy sources (7, 25) did not stimulate esg expression. Presently, there is no evidence that esg expression responds directly to the concentration of the BCAA in the growth medium, and it is unclear why complex media containing partially hydrolyzed protein induce high levels of esg expression.
Conclusions.
In this study, we have shown that the fatty acid composition of M. xanthus cells can vary widely, depending on the growth medium. The ability of bacteria to regulate the cellular fatty acid composition in response to environmental conditions has been documented previously. For example, E. coli has been shown to alter fatty acid composition in response to changes in temperature (10, 15) or exposure to alcohols (10). E. coli is also known to alter cellular fatty acid content upon entry into stationary phase (9). Regulation involving the BCFA, fatty acids not found in E. coli, has been observed in species of Bacillus (23) and Thermus (18) grown at different temperatures. Relatively little is known about the biological function of regulation of fatty acid content, but it is generally believed that increased concentrations of unsaturated fatty acids or BCFA relative to saturated straight-chain fatty acids helps to maintain bacterial membrane fluidity at low growth temperatures. The biological significance of the growth medium-dependent alteration of the BCFA composition found in M. xanthus is unknown.
Our results strongly suggest that the regulation of M. xanthus fatty acid content involves the regulation of esg expression. esg expression was found to be high in CTT medium-grown cells with high BCFA levels and low in A1 medium-grown cells with low BCFA content. The importance of the esg locus in the growth medium-dependent regulation of fatty acid content was demonstrated by the finding that esg mutant cells exhibited low BCFA levels in a growth medium-independent fashion. Since the esg locus encodes the E1α and E1β components of a BCKAD used in BCFA synthesis, it was not surprising to find that the level of BCKAD activity found in CTT medium-grown cells was greater than that found in A1 medium-grown cells. These results are compatible with the idea that regulation of the amount of the esg BCKAD is at least one of the mechanisms involved in regulation of the M. xanthus fatty acid composition and suggest that the genes encoding the E2 and E3 components of this enzyme complex may be regulated similarly. Previous studies have suggested that there may be another BCKAD in M. xanthus (21), but our results argue that growth medium-dependent regulation of fatty acid content is primarily associated with the esg pathway for BCFA synthesis and not with the other pathway(s) that appears to exist.
With the exception of E. coli, little is known about the molecular mechanisms employed by bacteria to control the cellular fatty acid content. In E. coli, the unsaturated fatty acid cis-vaccenic acid is found in greater amounts in cells grown at low temperature. In this case, the activity of the enzyme involved in the specific production of cis-vaccenic acid, β-ketoacyl acyl carrier protein synthase II, is greatest at low temperature, leading to a high cellular content of cis-vaccenic acid (3). Thus, in contrast to what we have observed in M. xanthus, transcriptional regulation of fatty acid biosynthesis enzymes does not appear to be involved in the E. coli system. The identification of the esg locus as an important component in the M. xanthus system for regulated BCFA synthesis opens the way to further analysis of the biological importance of the fatty acid content in M. xanthus.
Acknowledgments
Financial support from the Oklahoma Center for the Advancement of Science and Technology (OCAST) is gratefully acknowledged.
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