Unusual production of glutathione in Actinobacteria

Abstract

Most Actinobacteria produce mycothiol as the major thiol. In addition to mycothiol Rhodococcus AD45 generates a substantial level of glutathione possibly using genes acquired in a lateral transfer. Instead of mycothiol, Rubrobacter radiotolerans and Rubrobacter xylanophilus produce glutathione, whose synthesis appears to involve enzymes substantially different from those in other organisms.

Low molecular weight thiols, like glutathione, are critical for aerobic cells, since they maintain the required redox balance for normal cellular processes and protect against various toxins and oxidative stress (Ghezzi, 2005; Masip et al., 2006). While most eukaryotes and gram-negative bacteria contain glutathione as the dominant thiol, prior surveys of Actinobacteria have demonstrated that mycothiol, not glutathione, is the main thiol in an overwhelming majority of these organisms (Newton et al., 1996). Previously, the only actinomycetes reported to contain glutathione were Nocardiopsis flava ATCC 29533 (Newton et al., 1996) and Rhodococcus AD45 (van Hylckama Vlieg et al., 1998). Also, there have been reports of glutathione dependent enzymes (Grund et al., 1990; van Hylckama Vileg et al., 1999, Dhar et al., 2003) in certain strains of Actinobacteria. Herein, we examine the thiol composition of selected Actinobacteria and report some unexpected findings.

Cultures were grown on Tryptic Soy (TS) broth, since this medium contains only traces of glutathione (Newton et al., 1996), shaken at 220 rpm until the cells reached mid-log phase growth when they were harvested by centrifugation for 10 min at 4,500 × g at 4°C. The exception was Kineococcus radiotolerans, which was grown on TS agar plates, removed by scraping and pelleted for thiol analysis. All strains were incubated at 30°C, except Mycobacterium smegmatis and Rubrobacter radiotolerans, which were incubated at 37°C, and Rubrobacter xylanophilus, which was incubated at 55 °C. Before harvesting, the purity of the cultures was checked by examining colony morphology and gram stain. Thiol derivatization with monobromobimane (mBBr) and analytical high-performance liquid chromatography were performed according to protocols previously described for mycothiol determination (Rawat et al., 2003) and CoA analysis (Fahey & Newton, 1987). Purified derivatized mycothiol and glutathione derivatized with mBBr served as standards.

As a reference, we redetermined the thiol content of M. smegmatis and obtained values consistent with those reported earlier (Table 1). We also reanalyzed the thiol content of Nocardiopsis flava ATCC 29533 and were unable to detect any glutathione although the level of mycothiol was in the same order of magnitude (1.3 ± 0.3 μmol/g dry weight versus 2.7 μmol/g dry weight as previously reported) (Table 1). The discrepancy in the glutathione content between the two studies may have been due to contamination of the N. flava culture in the previous study (Newton et al., 1996). Other mycothiol dominant species are included in Table 1 and a careful examination failed to detect glutathione. These include Streptomyces griseus ATCC 13273, from which two glutathione S-transferase isoenzymes with activity against the standard glutathione S-transferase substrate, 1-chloro-2,4-dinitrobenzene (CDNB), have been purified (Dhar et al., 2003), and Streptomyces ghanensis, which requires glutathione for growth on m-hydroxybenzoate, presumably for the isomerization of maleylpyruvate (Grund et al., 1990). In both these strains, mycothiol content and enzymatic activity using mycothiol as a substrate were not measured; since glutathione is absent in this genus, the in vivo thiol substrate for these enzymatic reactions is unlikely to be glutathione.

Table 1.

Thiol content of Actinomycetes.

	Thiol content (μmol/ g residual dry weight)
Strain	Mycothiol	Glutathione
Mycobacterium smegmatis mc2155*	11.3 ± 0.2 (4)	<0.01
Streptomyces ghanensis ATCC 14672 1	1.4 ± 0.1 (28)	<0.01
Streptomyces griseus ATCC 13273 1	3.0 ± 0.4 (28)	<0.01
Nocardiopsis flava ATCC 29533	1.3 ± 0.3 (4)	<0.01
Rhodococcus erythropolis ATCC 4277	3.9 ± 0.3 (4)	<0.01
Rhodococcus RHA1 2*	3.0 - 9.0 (4)	<0.01
Rhodococcus AD45 2*	15 – 20 (4)	1 - 3 (4)
Arthrobacter histodinolovorans*	0.3 ± 0.0 (3)	<0.01
Arthrobacter aurescens*	1.0 ± 0.2 (3)	<0.01
Rubrobacter radiotolerans ATCC 51242	<0.01	5.3 ± 0.6 (3)
Rubrobacter xylanophilus DSM 9941*	<0.01	2.4 ± 0.2 (4)
Kineococcus radiotolerans ATCC BAA149	4.0 ± 0.4 (3)	<0.01
Escherichia coli DH5	<0.01	7. 7 ± 1.1 (3)

Samples were harvested during log phase growth. Values are means ± SEM (n).

¹Mean of quadruplicate samples harvested during 7 days of growth

²Range of MSH levels during log phase of growth

^*Rhodococcus Rha1 (a gift from by Dr. Julian Davies), Rhodococcus AD45 (a gift from Dr. D. Jansen), Rubrobacter xylanophilus DSM 9941(a gift from Dr. J. Batista), Mycobacterium smegmatis mc²155 (a gift from Dr. W. R. Jacob, Jr.), Arthrobacter histodinolovorans and Arthrobacter aurescens (gifts from Dr. A. Wright).

Rhodococcus sp. AD45 is a strain that can use isoprene as the sole source of carbon and energy and can oxidize many epoxides and chlorinated ethenes to chlorinated epoxyethanes (van Hylckama Vlieg et al., 1998; van Hylckama Vlieg et al., 1999). A glutathione S-transferase, IsoI, involved in the metabolism of these compounds has been purified (van Hylckama Vlieg et al., 1999). We find that Rhodococcus sp. AD45 produces a very high level of mycothiol but also a significant level of glutathione throughout its growth cycle (Table 1). The level of glutathione (2.0 μmol per g residual dry weight) is comparable to that reported by Van Hycklama Vlieg et al. (1998) based upon the determination of the adduct formed with 1,2-epoxyhexane. In contrast to strain AD45, only mycothiol and no glutathione was detected in Rhodococcus sp. Rha1 (Table 1) or Rhodococcus erythropolis (Table 1). A gene sequence encoding the two glutathione biosynthesis enzymes, two glutathione S-transferases, and other enzymes required for isoprene metabolism has been identified in Rhodococcus AD45 by van Hylckama Vlieg et al. (2000). This suggests that in conjunction with a possible horizontal gene transfer of isoprene degradation genes, there has been a transfer of the glutathione biosynthetic pathway to support the glutathione S-transferase activity required for this pathway.

Given the high concentration of mycothiol found in Rhodococcus sp. AD45 it is surprising that no mycothiol adduct was seen in the earlier study (van Hylckama Vlieg et al., 1998). Mycothiol S-conjugate amidase cleaves an amide bond in mycothiol toxin adducts to produce mercapturic acids and 1O-(2-amino-2-deoxy-±-D-glucopyranosyl)-D-myo-inositol, which is recycled back to mycothiol. We determined the mycothiol amidase activity by reacting 100 μg of cell extract protein in 25 mM HEPES (pH 7.5), 3 mM β-mercaptoethanol with 30 μM of mycothiol-monobromobimane adduct (MS-MB) in a 50 μl reaction volume. The samples were incubated for 30 min at 30°C, and then the reaction was stopped by adding 50 μl of 40 mM methanesulfonic acid and the samples were subjected to HPLC analysis for detection of the bimane derivative of N-acetylcysteine. The mycothiol S-conjugate amidase activity (2.5 ± 0.1 nmol/min/mg protein) in Rhodococcus AD45 cell extracts was twice that of M. smegmatis (Newton et al., 2000). Thus, any mycothiol adducts in Rhodococcus AD45 would have been further metabolized to produce mercapturic acids (Newton et al., 2000). Alternatively, the thiol composition may have favored glutathione in that study where cells were grown on isoprene.

The most surprising finding of the present study is that Rubrobacter radiotolerans (Yoshinaka et al., 1973; Suzuki et al., 1998) and Rubrobacter xylanophilus (Carreto et al., 1996), both strongly radiation resistant (Ferreira et al., 1999), contain only glutathione (Table 1). This result was confirmed by analyzing thiol content with two different high-performance liquid chromatography protocols. Glutathione is not uniquely associated with radiation resistance since the radiation resistant Kineococcus radiotolerans, an actinomycete discovered at the Savannah River nuclear waste site (Phillips et al., 2002), contains mycothiol but no glutathione (Table 1). Interestingly, in the genus Arthrobacter in which R. radiotolerans was originally classified (Suzuki et al., 1998), there are species that do not contain either mycothiol or glutathione such as A. globiformis (Newton et al., 1996) and species such as A. histodinolovorans and A. aurescens that contain mycothiol (Table 1).

To identify the glutathione biosynthesis genes, the R. xylanophilus genome sequence (GenBank CP000386; Comprehensive Microbial Resource, www.cmr.org ) was Blastp (Altschul et al., 1997) searched using representatives of the four major classes (Copley & Dhillon, 2002; Janowiak & Griffith, 2005) of glutathione synthetase (GshB). The GshB sequences from Homo sapiens, Escherichia coli, and Nostoc sp. produced no significant hits (E-value < 0.1). However, the C-terminal 390 amino acid sequence of the bifunctional Streptococcus agalactiae GshF protein (Janowiak & Griffith, 2005), that encodes the GshB equivalent, produced one weak hit, a 425 amino acid phosphoribosylamine:glycine ligase (Rxyl_0991; YP_643769), with E-value of 6e⁻⁴ that exhibited 29% identity in a 101 amino acid overlap. None of the R. xylanophilus annotated genes, including Rxyl_0991, contained the signature for prokaryotic GshBs, PFAM02951 (http://pfam.sanger.ac.uk/), although Rxyl_0991 and fifteen other proteins did possess the glutathione synthetase ATP binding domain, PFAM02955.

A Blastp search (Altschul et al., 1997) for possible γ-glutamylcysteine synthetase (GshA) sequences using the sequences from E. coli, Homo sapiens, Xylella fastidiosa, or the N-terminal 520 residues of the GshF protein sequence from S. agalactiae (May & Leaver, 1994; Janowiak & Griffith, 2005) produced no hits with E-values below 0.1. A search using the E. coli YbdK protein, which does not provide the principal GshA activity in E. coli but has been shown to have γ-glutamylcysteine synthetase activity (Lehmann et al., 2004), produced three hits, having E-values ranging from 3.8e⁻³¹ to 3.2e⁻⁴¹ and exhibiting 27-29% identity (44-49% similarity) over 343-360 residue overlaps with the 372 residues of the YbdK protein. A ClustalW alignment of the YbdK and the three Rubrobacter genes is shown in Fig. 1. These three proteins, Rxyl_0030 (YP_642823), Rxyl_1350 (YP_644125) and Rxyl_1127 (YP_643905), also contain the GCS2 domain (PF04107) that is present in all GshAs. However, members of the GCS2 superfamily are present in many strains, such as M. smegmatis, which lack glutathione and thus these genes may simply code for a carboxylate amine ligase of unknown function. Moreover, Blastp analysis with Rxyl_0030, Rxyl_1350 and Rxyl_1127 indicated that these genes are most similar to genes in Gloeobacter violaceus, an unicellular cyanobacterium with high GC content (62%), and the mycothiol containing GC rich actinomycetes, such as Mycobacterium species, which likely do not contain glutathione. Conversely, a gene, Rxyl_0031, annotated as a redoxin, with a thioredoxin superfamily domain (http://supfam.mrc-lmb.cam.ac.uk/), which encompasses glutathione S-transferases, glutathione peroxidase, and thiol tranferase domains, is present upstream of Rxyl_0030 and forms a bicistronic operon with it. Similarly, a gene annotated as a redoxin is present upstream but on the complementary strand of Rxyl_1127. Future experiments consisting of gene inactivation or recombinant protein expression are clearly needed to ascribe a role in glutathione biosynthesis for Rxyl_0030, Rxyl_1127, or Rxyl_1350.

Fig. 1.

ClustalW alignment of YbdK from Escherichia coli W3110 (BAA35221); Rxyl_0030 (YP_642823), Rxyl_1350 (YP_644125) and Rxyl_1127 (YP_643905) from Rubrobacter xylanophilus.

Intriguingly, the R. xylanophilus genome contains two genes, Rxyl_0300 (YP_643088) and Rxyl_0239(YP_643028), which have both the N-terminal signature (PF02798) and a C-terminal signature (PF00043) required for glutathione S-transferase activity. Blastp analysis of Rxyl_0300 indicated that this gene is similar to many genes annotated as putative glutathione S-transferases. The top four hits with 61-70% sequence identity (77-79% similarity) and E-values ranging from 1.3e⁻⁷⁵ to 5.9e⁻⁸⁰ were from Burkholderia and Ralstonia. Blastp analysis of Rxyl_0239 also produced hits in genes annotated as glutathione S-transferases in the genera, Burkholderia, Bordetella and Pseudomonas. Burkholderia, Ralstonia and Pseudomonas strains are omnipresent and metabolically diverse and thus contribute to such processes as biodegradation of pollutants and biocontrol of root diseases; some, along with Bordetella species, also cause disease in plants, animals and humans. The need for glutathione detoxification activity may have provided the selective pressure for evolution of glutathione metabolism in Rubrobacter.

Rubrobacter branched early from the main line of descent leading to Actinobacteria (Kunisawa, 2007; Gao & Gupta, 2005), possibly before the evolution of mycothiol biosynthesis; it may have independently evolved the ability to synthesize glutathione or acquired the ability via a lateral gene transfer. Identification of the glutathione biosynthesis genes should clarify this issue and shed light on the evolution of glutathione in these organisms. The acquisition of genes involved in glutathione biosynthesis along with the complete isoprene degradation pathway in Rhodococcus AD45 suggests that lateral transfer of genes involved in glutathione biosynthesis may be important in polluted environments where bacteria need glutathione requiring degradation pathways for survival.