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Applied and Environmental Microbiology, May 2009, p. 2991-2995, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.00181-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Glycerol Utilization Gene Cluster in Streptomyces clavuligerus{triangledown} ,{dagger}

Sonia Baños,1 Rosario Pérez-Redondo,1,2 Bert Koekman,3 and Paloma Liras1,2*

Área de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain,1 Instituto de Biotecnología, INBIOTEC, Parque Científico de León, 24006 León, Spain,2 DSM Anti-Infectives B.V., Delft, The Netherlands3

Received 26 January 2009/ Accepted 4 March 2009


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ABSTRACT
 
The Streptomyces clavuligerus ATCC 27064 glycerol cluster gylR-glpF1K1D1 is induced by glycerol but is not affected by glucose. S. clavuligerus growth and clavulanic acid production are stimulated by glycerol, but this does not occur in a glpK1-deleted mutant. Amplification of glpK1D1 results in transformants yielding larger amounts of clavulanic acid in the wild-type strain and in overproducer S. clavuligerus Gap15-7-30 or S. clavuligerus {Delta}relA strains.


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INTRODUCTION
 
Streptomyces clavuligerus ATCC 27064 produces the β-lactamase inhibitor clavulanic acid (CA). This compound is formed from arginine (17) and the three-carbon molecule glyceraldehyde-3-phosphate (6) which are condensed by the carboxyethylarginine synthase, the first enzyme of the pathway, encoded by ceaS2. Mutants disrupted in this gene do not produce CA in tryptic soy broth or starch-asparagine (SA) medium but form moderate amounts of CA in glycerol-supplemented media, probably due to glycerol utilization through the duplicated CeaS1 carboxyethylarginine synthase (10).

The role of D-glyceraldehyde-3-phosphate as a CA precursor was further supported by the construction of a glyceraldehyde-3-phosphate dehydrogenase (gap1) mutant of S. clavuligerus, which produces 180 to 210% CA in comparison to the wild-type strain due to higher availability of the glyceraldehyde-3-phosphate precursor (9).

Genes for glycerol utilization in Streptomyces coelicolor form an operon, gylCABX (15, 16), containing a gene for a putative glycerol transporter, a glycerol kinase, a glycerol-3-phosphate dehydrogenase, and a gene of unknown function. They are preceded by gylR (5), which encodes a glycerol-inducible repressor controlling both gylR and the gylCABX operon. Glycerol induction and glucose catabolite repression of the glp genes are thought to be directly related to the GylR protein in S. coelicolor (5).

Due to the importance of the glycerol flow for CA production, we decided to analyze the glycerol-utilizing gene cluster of S. clavuligerus.


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Cloning and expression of the S. clavuligerus gene cluster for glycerol utilization.
 
Hybridization of S. clavuligerus total DNA with S. coelicolor gylR, glpK1, and glpD1 internal probes allowed us to obtain two closely linked BglII fragments of 5.5 and 3.0 kb (see Table S1 in the supplemental material). The sequences of these fragments revealed a gylR-glpF1-glpK1-glpD1 gene organization similar to that of S. coelicolor (gylRCABX = glpF1K1D1X) and other Streptomyces species, encoding a regulator of the IclR family, a six-transmembrane protein of the major instrinsic family for glycerol transport, a glycerol kinase, and a flavin adenine dinucleotide-dependent glyceraldehyde-3-phosphate dehydrogenase, respectively (Fig. 1A). The lengths of S. clavuligerus glpD1 and S. coelicolor glpD1 are similar, but they are 96 nucleotides (nt) shorter than the sequence deposited by the Broad Institute as part of the S. clavuligerus genome project. It encodes a 536-amino-acid protein that is 89% identical to that of S. coelicolor. There is a good synteny in the cluster and metH upstream region in different Streptomyces, but no synteny is found downstream of glpD1, wherein S. clavuligerus orf5 encodes a regulator of the GntR family.


Figure 1
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  		FIG. 1. Cluster of genes for glycerol utilization in S. clavuligerus. (A) Organization of the glp cluster. Only relevant restriction sites are indicated. S. clavuligerus 5.5- and 3.0-kb BglII fragments cloned by hybridization with S. coelicolor homologous probes are indicated with arrows. Coding and intergenic regions analyzed by RT-PCR using the indicated oligonucleotide pairs (see Table S2 in the supplemental material) are shown under the genes. (B) pBluescript KS(+) and pHZ1351-derived plasmids containing DNA fragments (black lines), obtained either by PCR or by subcloning. (C) Comparison of S. coelicolor and S. clavuligerus –35 and –10 regions in P1 and P2 glpF promoters. Common nucleotides are indicated in the middle row. The arrows show the transcription starting nucleotide in S. coelicolor.

The genome of S. clavuligerus contains a second cluster, glpF2-glpK2, for proteins that are 50 and 70% identical to GlpF1 and GlpK1 and a glpD2 gene encoding a protein that is 32% identical to GlpD1. Additional copies of glp genes have also been found in other Streptomyces species, but their functions are unknown.

Transcription of the cluster in defined SA medium (2) was analyzed at 24 h by reverse transcription-PCR (RT-PCR) (12) using the oligonucleotides indicated in Fig. 1A (also see Table S2 in the supplemental material). The gylR-glpF1 intergenic region is not amplified, indicating that gylR is expressed as a single transcript (Fig. 2A), which supports the presence of a stem-and-loop hairpin ({Delta}G = –58.9 kcal/mol) downstream of gylR. In the intergenic gylR-glpF region, sequences similar to the –35 and –10 regions of the P1 and P2 promoters of S. coelicolor (15) are detectable (Fig. 1C). The intergenic regions glpF1-glpK1 and glpK1-glpD1 are amplified, suggesting a glpF1-glpK1-glpD1 polycistronic transcript that agrees with small intergenic regions (71 and 15 nt) and a putative strong terminator ({Delta}G = –59 kcal/mol) downstream of glpD1. Transcription of glpD1 occurs in a glpK1-deleted mutant (see below) (Fig. 2C) probably from the glpF1 promoter or from a putative promoter upstream of glpK1.


Figure 2
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  		FIG. 2. Transcription of S. clavuligerus glp cluster. RNA was isolated from cells grown for 24 h in SA defined medium (lane 1), SA medium supplemented with glycerol 1.5% (lane 2) and SA medium supplemented with glycerol and glucose, 1.5% each (lane 3). (A) RT-PCR of the gylR-glpF1, glpF1-glpK1, and glpK1-glpD1 intergenic regions. (B) RT-PCR of gylR, glpK1, and glpD1. M, molecular marker. (C) RT-PCR of the glpK1 and glpD1 coding regions using RNA from 24-h cultures in SA medium supplemented with 1.5% glycerol from S. clavuligerus ATCC 27064 (left lane) and S. clavuligerus {Delta}glpK1 (right lane). A total of 200 ng of RNA and 30 amplification cycles were used in the RT-PCR experiments. WT, wild type.

Expression of glp genes is induced by glycerol and repressed by glucose in S. coelicolor (15, 16), apparently due to GylR-dependent regulation of the P1 promoter upstream of glpF1 (5). Although S. clavuligerus ATCC 27064 is unable to grow on glucose (but contains glcP and glkA genes for glucose utilization) (Pérez-Redondo et al., unpublished results), to test whether the presence of glucose exerts regulatory mechanisms, the strain was grown in SA defined medium and in SA supplemented with either glycerol (1.5%) or glycerol plus glucose (1.5% each), and internal fragments (Fig. 1A) of gylR, glpK1, and glpD1 were amplified by RT-PCR. The gylR transcript is almost undetectable in SA medium but increases in the presence of glycerol (Fig. 2B, lanes 2 and 3). Similarly, gylK1 and gylD1 transcription is stronger when the RNA was obtained from glycerol-containing cultures (Fig. 2B, lanes 2 and 3). This suggests a glycerol induction of the gene cluster as occurs in the glpFKD P1 and P2 promoters of S. coelicolor (5, 16). No differences in transcription levels of gylR, glpK1, or glpD1 were observed using RNA obtained from cultures supplemented with glycerol in relation to those supplemented with glycerol and glucose (Fig. 2B, lane 3), indicating that glucose does not affect the expression of the glp genes, as opposed to what occurs in S. coelicolor.


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Bioinformatic search of regulatory sequences.
 
Binding of GylR to the S. coelicolor glpF upstream region has been reported (3). The tetrameric protein GylR has been purified for Escherichia coli, and several binding sites have been identified by footprinting analysis (8, 18, 19). Operator sequences act cooperatively to modulate the affinity of GylR to sequences present upstream or in the coding regions of the regulated genes. Using the alignment of 15 experimentally tested E. coli operators of the glp regulon and their complementary sequences, an information theory model was created (13, 14). The logo of this model reflects the conserved positions at nt 4 and 17 and an AT-rich internal stretch (see Fig. S1A in the supplemental material). The scan program identified putative target sequences and calculated the individual information content of each sequence, which should be positive in true sites (13).

The scan program recognizes two putative operator regions (OF2 and OR) in S. clavuligerus upstream of glpF1 and gylR. Moreover, two additional flanking sequences (OF1 and OF3) have been located upstream of glpF1. Similar operator sequences are present upstream of glpF and/or gylR in S. coelicolor A3(2), Streptomyces avermitilis NRRL8165, "Streptomyces scabies" 87.22, or Streptomyces griseus IFO 13350 (see Fig. S1B in the supplemental material). The functionality of all of these proposed operator sequences requires experimental confirmation using purified GylR protein and gel shift assays.


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Construction of a mutant with the gene glpK1 deleted.
 
To see if the glp cluster is essential for growth on glycerol, plasmid p{Delta}glpK1 was introduced into S. clavuligerus. In this plasmid, the final 1.4 kb of glpK1 and 84 bp of the 5' end of glpD1 are replaced by the gene acc(3)IV for apramycin resistance (Fig. 1B). The double mutant was confirmed by hybridization with a glpK1 internal probe.

An analysis was made for the growth, measured as DNA content (12), and CA production (11) of S. clavuligerus ATCC 27064 and its derivative strain {Delta}glpK1 in glycerol-protein extract-soy (PES) medium (9) and PES medium without glycerol (Fig. 3), in which the cells use the carbon source provided by the soybean extract. Cultures were always used in triplicate, and the samples from all of them were analyzed in duplicate. Glycerol improves the growth and CA production about 2.5-fold and 3-fold, respectively, in the wild-type strain; growth in S. clavuligerus {Delta}glpK1 is slower and corresponds to the growth of the wild type in the absence of glycerol. However, CA-specific production is not different from that of the wild-type strain grown without glycerol. This suggests that the second cluster, glpF2-glpK2, and the additional glpD2 gene present in S. clavuligerus might be functional but that they do not fully complement the absence of glpK1 and glpD1 and probably have different roles in the cell, at least in the culture conditions used.


Figure 3
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  		FIG. 3. Characteristics of S. clavuligerus {Delta}glpK1. Shown are growth (A) and specific CA production (B) by S. clavuligerus ATCC 27064 (top) and S. clavuligerus {Delta}glpK1 (bottom). The cultures were grown in glycerol-deprived PES medium (open symbols) or in PES medium containing 2% glycerol (closed symbols).

The higher CA production by S. clavuligerus ATCC 27064 in the presence of glycerol prompted us to test the effect on the growth and CA production of increasing glycerol concentrations (2 to 10%) in PES medium, using PES without glycerol as a control. As indicated above, the strain grows without glycerol. However, glycerol stimulates growth 1.6- to 1.9-fold; the best glycerol concentration for growth is 2%, whereas at higher percentages growth is slightly reduced. Volumetric production of CA is increased 3-fold to 3.5-fold in the presence of increasing glycerol concentrations, and the highest specific production is found in 10% glycerol-supplemented cultures (data not shown).


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Production of CA in S. clavuligerus ATCC 27064 transformants carrying additional copies of the glp genes.
 
S. clavuligerus(pHZ1351) and the transformants S. clavuligerus(p1351-F) and S. clavuligerus(p1351-FKD) (Fig. 1B), carrying additional copies of the glp genes in plasmid pHZ1351 (7), were pregrown in defined basal medium (1) supplemented with 0.2% L-asparagine, 1% glycerol, 0.1% NH4Cl, and 0.1% yeast extract. After 24 h, the mycelium was washed and transferred to basal medium with 0.1% NH4Cl and 2% glycerol as the sole carbon source. Duplicated samples at 0 and 96 h were diluted 100-fold for glycerol content determination with free glycerol reagent (Sigma). After 96 h, S. clavuligerus(pHZ1351) had consumed only about 7% of the glycerol, but transformants carrying the glpF1 gene or the glpF1K1D1 cluster consumed 39 and 27% of the initial glycerol, respectively (data not shown). Since CA production in this medium is low, the experiment was performed with the same strains, as well as with S. clavuligerus(p1351-KD), in PES medium supplemented with glycerol up to a 10% concentration. The patterns of growth in this rich medium are similar for all the strains (Fig. 4A). In S. clavuligerus(p1351-FKD) and S. clavuligerus(p1351-KD), carrying additional copies of glpK1 and glpD1, the formation of CA started earlier in relation to that of the control strain S. clavuligerus (pHZ1351) to reach values of 3-fold to 3.5-fold by 48 to 72 h (Fig. 4B). Therefore, amplification of glpK1 and glpD1 copy number improves CA formation, while glpF1 has almost no effect. The cephamycin C production by these strains was analyzed, but no significant differences were found.


Figure 4
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  		FIG. 4. Effect of additional copies of the glp genes. Shown are growth (A) and specific CA production (B) by S. clavuligerus(pHZ1351) (black squares), S. clavuligerus(p1351-F) (white circles), S. clavuligerus(p1351-KD) (black circles), and S. clavuligerus(p1351-FKD) (white squares) in complex PES medium containing 10% glycerol.


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Effect of the amplification of glp genes in different genetic backgrounds.
 
The positive effect of the glp cluster amplification on CA production by S. clavuligerus transformants prompted us to study whether this effect acted synergistically on other CA overproducer strains. Two strains known to produce more CA than the wild type were chosen as follows: (i) S. clavuligerus {Delta}relA, which is impaired in the stringent response (4), and (ii) S. clavuligerus Gap15-7-30, in which the gap1 gene, encoding the glyceraldehyde-3-phosphate dehydrogenase, is disrupted (9).

Both strains were transformed with plasmids p1351-FKD and pHZ1351 as a control. Cultures of S. clavuligerus {Delta}relA(pHZ1351) and S. clavuligerus {Delta}relA(p1351-FKD) were grown in triplicate in 10% glycerol-supplemented PES medium. Under these conditions, the growth and CA production of S. clavuligerus {Delta}relA(p1351-FKD) were greater (Fig. 5, top), with a maximal specific production of CA in the order of 1.7- to 2.2-fold compared to that of the control transformant.


Figure 5
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  		FIG. 5. Amplification of the glp genes in different genetic backgrounds. Shown are growth (A) and specific CA production (B) by S. clavuligerus {Delta}relA(p1351-FKD) (black squares), S. clavuligerus {Delta}relA(pHZ1351) (black circles), S. clavuligerus Gap15-7-30(p1351-FKD) (white squares), and S. clavuligerus Gap15-7-30(pHZ1351) (white circles).

S. clavuligerus Gap15-7-30(pHZ1351) and S. clavuligerus Gap15-7-30(p1351-FKD) were grown in PES unsupplemented medium (which contains 2% glycerol) to compare with previous results (9). Under these conditions, S. clavuligerus Gap15-7-30(p1351-FKD) CA production was in the order of 4.2- to 4.8-fold that of S. clavuligerus Gap15-7-30(pHZ1351), reaching levels of 1,030 µg/mg DNA at 84 h of fermentation in batch cultures (Fig. 5, bottom). Higher glycerol concentrations (10%) in cultures of this strain did not significantly increase CA production (not shown).

In summary, the additive effect of high glycerol concentration, improved glycerol utilization by glpF1K1D1 carrying transformants, and different genetic backgrounds results in a specific CA production of about 7.5-fold, which confirms the validity of these approaches for obtaining overproducer strains.


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Nucleotide sequence accession numbers.
 
Sequences for metH-gylR-glpF1K1D1 (FM210760), the glycerol downstream region (FM210761), and the region around glpF2-glpK2 (FM210762) have been deposited in the EMBL database.


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ACKNOWLEDGMENTS
 
This work was supported by grants from the Spanish Ministry of Science and Technology (BIO2003-03274, BIO2006-14853), Junta de Castilla y León (LE001A07), and the European Community (Actinogen; LSHMCT-2004-005224).

We acknowledge the receipt of DNA sequences from Wilbert Heijne (DSM, The Netherlands) and the receipt of S. clavuligerus Gap15-7-30 from C. A. Townsend. We thank Antonio Rodríguez-García for help in the bioinformatic studies.


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FOOTNOTES
 
* Corresponding author. Mailing address: Área de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain. Phone: (34) 987 291504. Fax: (34) 987 291506. E-mail: paloma.liras{at}unileon.es Back

{triangledown} Published ahead of print on 13 March 2009. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


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Applied and Environmental Microbiology, May 2009, p. 2991-2995, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.00181-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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