University of Groningen

Engineering of sugar metabolism in Lactococcus lactis Pool, Weia Arianne

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record

Publication date: 2008

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA): Pool, W. A. (2008). Engineering of sugar metabolism in Lactococcus lactis. s.n.

Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment.

Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.

Download date: 02-10-2021

CHAPTER 5

THE α- OF LACTOCOCCUS LACTIS IS UNRELATED TO THE α-D-PHOSPHOHEXOMUTASE SUPERFAMILY AND ENCODED BY THE ESSENTIAL pgmH

Ana R. Neves*, Wietske A. Pool*, Rute Castro, Ana Mingote, Filipe Santos, Jan Kok, Oscar P. Kuipers and Helena Santos

Based on J. Biol. Chem. 2006, 281:36864-36873.

* Both authors contributed equally to the results described in this chapter.

Chapter 5

104 A novel α-phosphoglucomutase

SUMMARY

α-Phosphoglucomutase (α-PGM) plays an important role in carbohydrate metabolism, by catalyzing the reversible conversion of α-glucose-1-phosphate to glucose-6-phosphate. Isolation of α-PGM activity from cell extracts of Lactococcus lactis strain MG1363 led to the conclusion that this activity is encoded by yfgH, herein renamed to pgmH. Its gene product has no sequence homology to proteins in the α-D-phosphohexomutase superfamily and instead is related to the eukaryotic within the haloacid dehalogenase superfamily. In contrast to known bacterial α-PGMs, this 28 kDa is highly specific for α-glucose-1- phosphate and glucose-6-phosphate and showed no activity with mannose- phosphate. To elucidate the function of pgmH, the metabolism of glucose and galactose was characterized in mutants overproducing or with deficiency of α-PGM activity. Overproduction of α-PGM led to increased glycolytic flux and growth rate on galactose. Additionally, the intracellular concentration of UDP-glucose was decreased. Despite several attempts, we failed to obtain a deletion mutant of pgmH. The essentiality of this gene was proven by using a conditional knock-out strain, in which a native copy of the gene was provided in trans under the control of the nisin promoter. Growth of this strain was severely impaired when α-PGM activity was below the control level. Moreover, the growth-rate and biomass production were directly related to α-PGM activity. We show that the novel L. lactis α-PGM is the only enzyme mediating the interconversion of α-glucose-1-phosphate to glucose-6- phosphate in L. lactis and is essential for growth.

105 Chapter 5

INTRODUCTION

Phosphoglucomutase (E.C. 5.4.2.2) is widespread in living organisms from bacteria to humans (217). It plays various roles in carbohydrate metabolism, by catalyzing the reversible conversion of α-glucose-1-phosphate (α-G1P) to glucose-6-phosphate (G6P). In higher organisms, its major function is mediating the mobilization of sugar moieties from energy reserves (e.g. glycogen, trehalose, starch). Also, α-PGM activity is essential for the synthesis of UDP-glucose, a sugar donor for the production of glucose-containing polysaccharides. Therefore, PGM is a crucial link between catabolic and anabolic processes. The lactic acid bacterium Lactococcus lactis is used worldwide in the industrial manufacture of fermented milk products. The organism converts sugars primarily into lactic acid, thus providing an efficient means of food conservation. In L. lactis, α- phosphoglucomutase is assumed to be essential for the utilization of galactose via the Leloir pathway (105) and also for the synthesis of precursors of cell wall polysaccharides and exopolysaccharides (40, 74). In a number of Gram positive bacteria, namely Bacillus subtilis and Streptococcus pneumoniae, pgm mutants showed altered cell wall morphology and altered polysaccharide production as well as growth defects on glucose (58, 91, 100). Despite the key metabolic role of α- PGM and the wealth of knowledge on sugar metabolism of L. lactis (122), coding for this activity have not been identified in this organism. More than a decade ago the presence of two distinct phosphoglucomutase activities in L. lactis subsp. lactis with specificity for α- and β-anomers of phosphoglucose has been reported (150). A 28 kDa protein, designated β-PGM, was shown to catalyze the reversible conversion of β-G1P to G6P. The L. lactis β-PGM belongs to the haloacid dehalogenase (HAD) enzyme superfamily; its X-ray structure has been determined and the catalytic mechanism investigated (87, 88, 224). β-PGM is a catabolic enzyme in the pathway for maltose and trehalose degradation (99), which is encoded by a gene (pgmB) in the trehalose operon (4, 15). A larger protein (around 65 kDa) showing affinity for the α-anomer of glucose-1-phosphate was partially purified (150). The α-specificity and the protein size, matching the analogous parameters of bacterial α-PGMs, led the authors to conclude that it was the lactococcal α-PGM. It is pertinent noting that all α-PGMs described thus far belong to the α-D-phosphohexomutase superfamily of proteins (171, 217). Intriguingly, a BLASTp search of the available L. lactis genome sequences (15, 215) using members of the α-D-phosphohexomutase superfamily as query, or a domain

106 A novel α-phosphoglucomutase search using the highly conserved regions of proteins in this family, retrieved only FemD, a protein tentatively annotated as a phosphoglucosamine . In this work we report the identification, purification, expression and characterization of L. lactis α-PGM and its encoding gene pgmH (previously yfgH), and show that this activity is essential for growth. To our knowledge the lactococcal α-PGM is the first member of the HAD superfamily of proteins with strict specificity for α-G1P.

107 Chapter 5

EXPERIMENTAL PROCEDURES

Microbial strains and growth conditions

Strains and plasmids used throughout this study are listed in Table 1. For molecular biology procedures, L. lactis strains were cultivated as batch cultures (flasks) without aeration in M17 medium (Difco, Sparks, MD) containing 0.5% glucose (w/v) at 30°C or 38ºC. For physiological studies, L. lactis NZ9000 (84) derivatives, NZ9000[pNZ8048] (control strain), NZ9000[pNZ8048-pgmH] (hereafter designated NZ9000[pgmH+]) and NZ9000ΔpgmH[pgmH+], were grown in Chemically Defined Medium (CDM) (144) at 30ºC in 2 or 5-liter vessels (B. Braun Biostat®, MD), under anaerobic conditions and pH 6.5 as previously described (123). Glucose or galactose was added to a final concentration of 1% (w/v). Plasmid selection was achieved by addition of chloramphenicol (5 mg/l) or erythromycin (5 mg/l) to the growth medium. For overproduction of α-PGM, nisin (1 μg/l) was added to the medium when an optical density at 600 nm (OD600) of 0.5 was reached. For studies in which the nisin-inducible conditional mutant NZ9000ΔpgmH[pgmH+] was used, cells were grown overnight in M17 containing different levels of nisin and washed once with fresh M17 lacking nisin. The washed cultures were subsequently subcultured for 15 h (initial OD600 around 0.025 or 0.05 for growth on glucose or galactose, respectively) in fresh medium, either with or without nisin (0.01 to 1 µg/l). Growth was monitored by measuring the optical density at 600 nm.

General DNA techniques

General molecular techniques were performed as described by Sambrook et al. (162). Chromosomal and plasmid DNA were isolated from L. lactis according to Johansen and Kibenich (68) and Birnboim (12), respectively. L. lactis was transformed with plasmid DNA by electroporation as described by Holo and Nes (60) using a Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, CA). All DNA modification were purchased from Roche Molecular Biochemicals (Mannheim, Germany), and used according to the manufacturer’s directions. Polymerase chain reactions (PCRs) were performed using Expand DNA polymerase (Roche Molecular Biochemicals) and purified with the Roche PCR purification kit (Roche Molecular Biochemicals). Primers (listed in Table 1) were purchased from Biolegio BV (Malden, the Netherlands).

108 A novel α-phosphoglucomutase

TABLE 1: Strains, plasmids and primers used in this study Strains Description Reference

MG1363 Plasmid free and prophage cured derivative of NCDO712 (52) NZ9000 Derivative of MG1363 carrying pepN::nisRK (84) LL302 RepA+ MG1363, carrying single copy of pWV01 repA in pepX (95) NZ9000::pORI280-ΔpgmH Derivative of NZ9000 containing pORI280ΔpgmH integrated in the in pgmH This work NZ9000ΔpgmH[pgmH+] Derivative of NZ9000 containing a 711-bp deletion in pgmH and carrying pNZ8048-pgmH This work

Plasmids Description Reference

r pNZ8048 Cm ; inducible expression vector carrying PnisA (84) pNZ8048-pgmH Cmr, derivative of pNZ8048 carrying a copy of pgmH This work pORI280 Emr, LacZ+, ori+ of pWV01, replicates only in strains providing repA in trans (96) pORI280-ΔpgmH Emr; derivative of pORI280 specific for integration in the L. lactis pgmH gene This work pORI13 Emr; integration vector; ori+ repA- derivative of pWV01; promoterless lacZ (163) pORI13-pgmH’ Emr; derivative of pORI13 specific for integration in the L. lactis pgmH gene This work pVE6007 Cmr, temperature-sensitive derivative of pWV01 (107) pNZ8048-femD Cmr, derivative of pNZ8048 carrying a copy of femD This work pNZ8048-galE Cmr, derivative of pNZ8048 carrying a copy of galE This work

Primers Sequence (5’ to 3’) Restriction-site yfgH-fw CATGTCATGAAAAAAATATTAAGTTTCGACATTG RcaI yfgH-rev GCTCTAGAGAAAATTAAGCTTCTTCCATCGC XbaI yfgH-KO1 CGGAATTCCCAACGCTTCTACATCTTC EcoRI yfgH-KO2 CGGGATCCCTTATCAACGCTTACATTATAAC BamHI yfgH-KO3 CGGGATCCGAAACTGCTGCTATCCTCAAAGC BamHI yfgH-KO4 GCTCTAGACTTCACGCGTTTGGGC XbaI yfgH-fw1 GGAATTCGAAAAAAATATTAAGTTTCG EcoRI yfgH-rev1 GCTCTAGAGCTTGTTTAGCTTCAACC XbaI femD-fw CATGCCATGGGTAAATATTTTGGAACAG NcoI femD-rev GCTCTAGATTATTTCACACCAATTTCCTC XbaI galE-fw CATGTCATGACAGTTTTAGTACTTGGTGG RcaI galE-rev GGACTAGTTCAGTAGCCTTTTGGATGAC SpeI

109 Chapter 5

Construction of strains and plasmids

The genes pgmH and galE were cloned and overexpressed in L. lactis strain NZ9000 as follows. The coding regions of pgmH and galE were amplified by PCR using primers yfgH-fw/yfgH-rev and galE-fw/galE-rev (Table 1), respectively. The 0.76-kb RcaI/XbaI and the 0.98-kb RcaI/SpeI PCR-products were digested with the indicated enzymes and fragments were cloned into NcoI/XbaI or NcoI/SpeI digested pNZ8048 (84), yielding constructs pNZ8048-pgmH and pNZ8048-galE, respectively. The femD gene was amplified by PCR using primer pair femD-fw/femD-rev (Table 1). The overexpression plasmid pNZ8048-femD was constructed by cloning the 1.36-kb NcoI-XbaI resticted PCR product into similarly digested pNZ8048. The resulting construct was transformed into L. lactis strain NZ9000. L. lactis MG1363 DNA was used as template for all PCR reactions. Several strategies were employed to construct an L. lactis ΔpgmH strain. In the first strategy, a complete deletion of the pgmH gene was tried by using a two-step homologous recombination method as follows: the upstream and downstream flanking regions of pgmH were obtained by PCR using primer pairs yfgH-KO1/yfgH- KO2 and yfgH KO3/yfgH-KO4, and cloned as EcoRI/BamHI and BamHI/XbaI restriction fragments in pORI280 (96), resulting in plasmid pORI280ΔpgmH. The plasmid was obtained and maintained in L. lactis LL302 (95). pORI280ΔpgmH and pVE6007 (107) were co-transformed into L. lactis NZ9000 and this strain was taken through the temperature shift protocol for single and double-crossovers (96). No double-crossover transformants were obtained in M17 supplemented with 0.5% of the following sugars: glucose, maltose, trehalose or a mixture of trehalose (0.5%) and galactose (0.05%). In a second approach, which was used in an attempt to knock out the pgmH by single-crossover plasmid integration, a 0.46-bp fragment, containing the 5’ end of pgmH except two base pairs, was amplified by PCR using primers yfgH-fw1 and yfgH-rev1, double-digested with EcoRI/XbaI, and cloned into the similarly digested pORI13 (163). The resulting plasmid was transformed into NZ9000, but despite several attempts, no erythromycin resistant colonies were obtained. Subsequently, pNZ8048-pgmH was introduced in NZ9000::pORI280-ΔpgmH, obtained as explained above. The resulting chloramphenicol and erythromycin resistant strain was subjected to an excision strategy (96) in M17 medium with 0.5% w/v glucose and containing nisin (0.1 µg/l), yielding NZ9000ΔpgmH[pgmH+]. The addition of nisin was required to induce expression of pgmH from pNZ8048-pgmH. Integration of plasmids in the chromosome and deletions were confirmed by PCR and by Southern blotting. Probe labeling, hybridization, and detection were

110 A novel α-phosphoglucomutase performed using the ECL direct nucleic acid labeling system, according to the specifications of the manufacturer (Amersham Pharmacia Biotech, Little Chalfont, UK).

Enzyme assays

Enzymes were assayed at 30ºC immediately after mechanical disruption of a cell suspension by passage through a French press (twice at 120 MPa), and centrifugation for 15 min at 30,000 × g to remove cell debris. Protein concentration was determined by the method of Bradford (19). Routinely and during enzyme purification, α- and β-phosphoglucomutase activities were assayed as described by Qian et al. (150). The 1 ml assay mixture contained 50 mM TEA-HCl (pH 7.2), 5 mM + MgCl2, 0.5 mM NADP , 50 µM glucose-1,6-bisphosphate and 1.75 U glucose-6- phosphate dehydrogenase. Reactions were started by the addition of 1.5 mM α-G1P or β-G1P, respectively.

Purification of native α-PGM from L. lactis

The native α-PGM was purified by fast protein liquid chromatography (Amersham Biosciences). All steps were performed in the presence of 0.5 mM EDTA and 5 mM 2-mercaptoethanol, at 4ºC; 15% glycerol (w/v) was added during chromatography. Cell extracts were prepared from 135 g (wet weight) of galactose-grown MG1363 cells that were suspended in 50 mM TEA buffer

(50 mM, pH 7.2), containing 5 mM MgCl2. Precipitation steps with protamine sulfate (0.25% w/v) and with solid ammonium sulfate were essentially performed as described by Qian et al. (150). The precipitate collected in the range of 45-85%

(NH4)2SO4 saturation was dissolved in TEA buffer (50 mM, pH 7.2) containing 30 mM KCl, and dialyzed against the same buffer. The protein preparation was applied onto a gel filtration Superdex 200 (20 mM Bis-TrisPropane, pH 6.9, containing 45 mM KCl), and α-PGM activity was detected in the flow-through. Active samples were loaded onto a Resource Q column (same buffer as Superdex 200), and elution was carried out with a linear gradient of KCl (45-500 mM). Active fractions, eluted at around 240 mM KCl, were dialyzed against Tris-HCl (10 mM, pH 7.3) containing 4 mM MgCl2 and 1.6 M (NH4)2SO4. The sample was applied to a phenyl-Sepharose column, and elution was carried out with a linear gradient of (NH4)2SO4 (1.6 – 0 M).

α-PGM activity was detected at 0.5 M of (NH4)2SO4. The positive fractions were dialyzed against TEA buffer (50 mM, pH 7.5, 30 mM KCl), loaded onto a gel filtration

111 Chapter 5

Superose 6 column (same buffer as dialysis). α-PGM activity was measured in the flow-through and active fractions were evaluated by SDS-PAGE. Two putative target proteins of 28 and 37 kDa were excised from Coomassie-stained SDS-PAGE gels and the amino acid sequences of their N-termini were determined (45). ORFs encoding the two proteins were identified by BLASTp searches using the genome sequence of L. lactis subsp. lactis IL1403.

Purification and characterization of recombinant α-PGM

The α-PGM protein was purified to electrophoretic homogeneity from approximately 65 grams (wet weight) of glucose-grown nisin-induced NZ9000[pNZ8048-pgmH] cells. Cell extract preparation and chromatographic steps were essentially as described above for purification of the native enzyme, except that the gel filtration Superdex 200 (first chromatographic step) was replaced by an anion-exchange Q- Sepharose (20 mM Bis-TrisPropane, pH 6.9; 45-500 mM KCl gradient), and the last chromatographic step (gel filtration) was not required. Also, the hydrophobic step (phenyl-Sepharose) preceded the anion exchange step (Resource Q). The purified protein was stored at

-20ºC in 50 mM TEA buffer containing 5 mM MgCl2, 0.5 mM EDTA, 5 mM mercaptoethanol and 15% glycerol until further use. The pH profile of α-PGM was determined in TEA-HCl buffer in the pH range 4-9. The effect of 1 or 5 mM alternative cations (Ni2+, Zn2+, Ca2+, Mn2+, and Li+) was examined in the presence of 50 µM Mg2+. ATP and FBP were examined as potential inhibitors of α-PGM activity. Kinetic constants of the purified α-PGM were determined in the reaction direction α-G1P Æ G6P. The Vmax for the reaction direction G6P Æ α-G1P and substrate specificity were determined by 31P-NMR spectroscopy. The 3 ml reaction mixture contained 50 mM TEA-HCl buffer (pH 7.2), 2 5 mM MgCl2, 50 µM glucose-1,6-bisphosphate, 3% H2O (v/v) and 20 µg of pure enzyme. For substrate specificity, spectra were acquired before and after three hours of incubation at 30ºC in the presence of putative substrates (7.5 mM). The following compounds were examined: α-G1P, G6P, β-G1P, glucosamine-1P, glucosamine-6P, α-mannose-1P, fructose-1P, fructose-6P, FBP, FBP/α-G1P, α- galactose-1P, galactose-6P, ribose-5P, ribulose-5P, 6-phosphogluconate, UDP- galactose and UDP-glucose. For Vmax determinations, the reactions were started by the addition of G6P (50 mM) and the time course for its consumption was monitored. The rate of G6P consumption was calculated by comparison of the area of the G6P resonance to that of a known amount of methylphosphonate added as an internal

112 A novel α-phosphoglucomutase standard. One unit of enzyme activity was defined as the amount of enzyme catalyzing the conversion of 1 µmol of G6P per minute under the experimental conditions used. Molecular mass determination was performed by gel filtration on Superose 12 10/300 GL using 100 mM sodium acetate, pH 7.0.

Determination of extra- and intracellular metabolites during growth

Samples (2 ml) of L. lactis NZ9000[pNZ8048] or L. lactis NZ9000[pgmH+] cultures growing in CDM containing either glucose or galactose were collected at different points during growth, centrifuged (2000 × g, 5 min, 4ºC), and supernatant solutions were stored at –20ºC until analysis by high performance liquid chromatography. Fermentation substrates and products were quantified as described before (126). Ethanol extracts for analysis by 31P-NMR and quantification of phosphorylated metabolites in NZ9000[pgmH+] and control strains at mid-exponential growth phase were prepared as described elsewhere (151). The dried extracts were dissolved in 4 2 ml H2O containing 5 mM EDTA (final pH approximately 6.8). Assignment of resonances and quantification of phosphorylated metabolites was based on previous studies (151) or by spiking the NMR-sample extracts with the suspected, pure compounds. The reported values for intracellular phosphorylated compounds are averages of two independent growth experiments and the accuracy was around 15%.

NMR experiments and quantification of metabolites

Cells were harvested during mid-logarithmic growth phase (OD600 = 2.2), centrifuged, washed twice and re-suspended to a protein concentration of 16.5 mg/ml in 50 mM KPi buffer, pH 6.5. In vivo NMR experiments were performed as described before (123). Spectra were acquired sequentially prior to and after addition of [1-13C]-glucose or [1-13C]-galactose. After substrate exhaustion, and when no changes in the resonances due to end products and intracellular metabolites were observed, a total NMR-sample extract was prepared and used for quantification of end-products and other metabolites (125, 127). The concentration of labeled lactate determined by 1H-NMR was used as a standard to calculate the concentration of the other metabolites in the sample (124). Due to the fast pulsing conditions used for acquiring in vivo 13C-spectra, correction factors were determined to convert peak intensities into concentrations (124). The correction factors for Gal1P (0.73), α-G1P (0.73), UDP-Gal (0.67) and UDP-Glc

113 Chapter 5

(0.67) were determined as follows: an NMR-sample extract supplemented with the pure compounds was circulated through the NMR tube at a similar rate to that used for cell suspensions and 13C-NMR spectra were acquired with a 60º flip angle and a recycle delay of 1.5 s (saturating conditions) or 60.5 s (relaxed conditions). The quantitative kinetic data for intracellular metabolites were calculated as described elsewhere (125, 127). The lower limit for in vivo NMR detection of intracellular metabolites under these conditions was 3-4 mM. Intracellular metabolite concentrations were calculated using a value of 2.9 μl/mg of protein for the intracellular volume of L. lactis (146).

NMR spectroscopy

NMR-spectra of living cells were run at 30°C using a quadruple-nucleus probe head on a Bruker DRX500 spectrometer (Karlsruhe, Germany). Acquisition of 31P-NMR and 13C-NMR spectra was performed as described by Neves et al. (127). Although individual experiments are illustrated in each figure, each experiment was repeated at least twice and the results were highly reproducible. The values reported are averages of two to four experiments and the accuracy varied from ±2% (extracellular products) to ±10% in the case of intracellular metabolites with concentrations below 5 mM. The quantification of phosphorylated metabolites and the measurement of α- PGM activity were performed as described by Ramos et al. (151). Carbon and phosphorus chemical shifts are referenced to external methanol or H3PO4 (85%) designated at 49.3 ppm and 0.0 ppm, respectively.

Chemicals

[1-13C]Glucose (99% enrichment) and [1-13C]-galactose (99% enrichment) were obtained from Campro Scientific (Veenendaal, The Netherlands) and Cambridge Isotope Laboratories (Andover, MA, USA), respectively. Formic acid (sodium salt) was purchased from Merck Sharp & Dohme (Paço de Arcos, Portugal). All other chemicals were reagent grade and obtained from Sigma-Aldrich (St. Louis, MO).

114 A novel α-phosphoglucomutase

RESULTS

Purification of α-PGM activity and identification of the coding gene

A BLASTp search of L. lactis MG1363 and IL1403 genomes using sequences of proteins in the α-D-phosphohexomutase superfamily identified femD as the best hit. FemD, annotated as a putative phosphoglucosamine mutase (15), is a protein with a calculated molecular weight of 48 kDa. To ascertain whether femD encodes a protein with α-PGM activity, the gene (1356 bp) was cloned into pNZ8048, under the control of the nisin inducible promoter, and introduced in NZ9000. Cell extracts of NZ9000[pNZ8048-femD] grown on glucose-M17 and induced with nisin (1 µg/l) contained a clearly overexpressed protein with a size of 48 kDa, as evidenced by SDS-PAGE (not shown). Moreover, the activity of α-PGM in the induced strain was identical to that of control cells (0.15 U/mg protein) indicating that femD does not encode an α-PGM. A PCR strategy to clone the lactococcal α-PGM gene using degenerate primers based on highly conserved regions of phosphohexomutases (171) and the complementation of an E. coli pgm::tet mutant (102) with a genomic library of L. lactis were also attempted without success. Consequently, purification of α-PGM activity was pursued to obtain protein sequence information and identify the gene.

A B M 18202223242628 yfgH galE 97 M-+-+

67 75

5 0 45 39 37 kDa

25 Molecular mass (kDa) mass Molecular

30 28 kDa Molecular mass (kDa) 20

α-PGM α-PGM (U) 0.4 0.93.3 5.6 5.6 1.4 0.2 0.123.7 0.10 0.10 (U/mg)

Figure 1: Purification of the α-PGM activity and the identification of the coding gene. A) Protein samples loaded on SDS-PAGE; different fractions of the flow-through after elution from a Superose6 matrix are shown. Lanes from left to right: protein marker, fraction 18, 20, 22, 23, 24, 26 and 28. At the bottom the α-PGM activity of the samples in each lane is depicted. 2 potential candidates for α-PGM activity are marked with an arrow. B) Protein samples loaded on SDS-PAGE. Lanes from left to right: protein marker, L. lactis NZ9000[pgmH+] uninduced (-) and nisin-induced (+),L. lactis NZ9000[galE+] uninduced (-) and nisin-induced (+). At the bottom the α-PGM activity of the samples in each lane is depicted.

115 Chapter 5

Extracts from L. lactis MG1363 grown on M17 with galactose exhibited 2-fold higher α-PGM activity (0.3 U/mg protein), than glucose-grown cells and were used for protein isolation. α-PGM was partially purified (100 fold) (as described in the Materials and Methods section). Fractions from the last column contained several bands, as visualized by SDS-PAGE, but only two proteins (28 and 37 kDa) co-eluted with the phosphoglucomutase activity in all purification steps (Fig. 1A). These two protein bands were excised and their N-terminal amino acid sequences determined to be MKKILSFD and MTVLVLG, respectively. The corresponding open reading frames in the L. lactis IL1403 genome sequence were yfgH (NP_266726; hypothetical protein) and galE (NP_268136, UDP-glucose 4-epimerase), respectively. The product of galE was shown to be UDP-glucose 4-epimerase by others (54, 211), and cloning and overexpression of galE in NZ9000 did not lead to enhancement of α-PGM activity (Fig. 1B). The yfgH (herein renamed as pgmH) gene contains 759 bp and codes for a protein with 252 amino acids and a calculated protein mass of 28,276 kDa. The gene was amplified by PCR using MG1363 chromosomal DNA as template, cloned in pNZ8048 and expressed in strain NZ9000. Induction with nisin (1 μg/l) of a glucose-M17 growing NZ9000[pgmH+] culture resulted in a 30-fold increase of α-PGM activity (Fig. 1B), thus proving that pgmH encodes the α-PGM of L. lactis.

In the chromosome of L. lactis MG1363, pgmH is flanked by yfgL and yfgG (Fig. 2), both of unknown function (215). All three genes are preceded by Shine-Dalgarno sequences (5’-AAATAGGAGA-3’, for pgmH). A putative promoter region containing an extended -10 (5’-TGTTATAAT-3’) sequence precedes the pgmH coding sequence. Inverted repeat sequences, (5’- AAAAAGCAATCTATTTTGATTAGATTGTTTTT-3’) and (5’- AAAAAGTTGTCATTAATGACAGCTTTTT-3’) followed by AT-rich regions downstream of the yfgL and pgmH stop codons, respectively, could function as transcriptional terminators. Therefore, it is unlikely that the three genes are organized in an operon-like structure and possibly they have unrelated functions. A similar genomic organization is observed for strain L. lactis subps. lactis IL1403 (15).

116 A novel α-phosphoglucomutase

yfgF yfgG pgmH yfgL dfpA

Figure 2: Chromosomal organization of pgmH in L. lactis MG1363. Gene pgmH is located between yfgG and yfgL. Hooked arrow: putative promoter region. Lollipop: putative terminator region.

Interestingly, the product of pgmH (Fig. 3; DQ778336) had no sequence homology with proteins from the α-D-phosphohexomutase family. It showed around 37% identity with hypothetical proteins from human-colonizing Gram-positive bacteria (Bifidofacterium longum ZP_00121741 and Propionibacterium acnes YP_056695), and around 25% identity with eukaryotic phosphomannomutases (e.g. Mus musculus PMM2 NP_058577, Homo sapiens PMM2 AAH08310 and PMM1 AAC51117, and Saccharomyces cerevesiae Sec53 NP_116609) (Fig. 3). These proteins have been classified as members of the HAD superfamily. Despite the low overall identity, the α-PGM sequence contains the four conserved motifs that characterize the HAD superfamily: motif I (DXDX(T/V)), motif II (S/TXX) and motif III and IV (K (X)18-30(G/S)(D/S)) (79, 159).

117 Chapter 5

M. musculus -----MAT------LCLFDMDGTLTAPRQKITEEMDGFLQKLRQKTKIGVVGGSDFEKLQEQLG--NDV 56 H. sapiens PMM2 -----MAAPGPA------LCLFDVDGTLTAPRQKITKEMDDFLQKLRQKIKIGVVGGSDFEKVQEQLG--NDV 60 H. sapiens PMM1 -----MAVTAQAARRRERVLCLFDVDGTLTPARQKIDPEVAAFLQKLRSRVQIGVVGGSDYCKIAEQLGDGDEV 69 S. cerevisiae -----MSIAEFAYKEKPETLVLFDVDGTLTPARLTVSEEVRKTLAKLRNKCCIGFVGGSDLSKQLEQLG--PNV 67 P. acnes ------MARIIVRLLDRTSVCVISGGQFGQFRTQVVEALVD 35 B. longum MVVRSWSELDFDNVCSNAKVFGFDLDNTLASSKQPMKPAMIERFCALLDHTVVALISGGGMAVATSQVLDVLTP 74 L. lactis α-PGM ------MKKILSFDIDNTLNEPKMPIFPEMAELLATLSQKYIIAPISGQKYDQFLIQIINNLPE 58 : **:*.** . : : : : * .: : :.*· *: L. lactis β-PGM ------MFKAVLFDLDGVITDT----AEYHFRAWKALAEEIGINGVDR-QFNEQLKGVSREDSL 53 **:*. : * .. : :. :

M. musculus VEKYD--YVFPENGLVAYKDGKLLCKQNIQGHLGEDVIQDLINYCLSYIANIKLPKKR--GT---FIEFRNGML 123 H. sapiens PMM2 VEKYD--YVFPENGLVAYKDGKLLCRQNIQSHLGEALIQDLINYCLSYIAKIKLPKKR--GT---FIEFRNGML 127 H. sapiens PMM1 IEKFD--YVFAENGTVQYKHGRLLSKQTIQNHLGEELLQDLINFCLSYMALLRLPKKR--GT---FIEFRNGML 136 S. cerevisiae LDEFD--YSFSENGLTAYRLGKELASQSFINWLGEEKYNKLAVFILRYLSEIDLPKRR--GT---FLEFRNGMI 134 P. acnes APRLDRLHLLPACGTQYYRCVDGEWQRIYVEALTDDEKSRAMEAVETCARDLGLWEEHTWGP---VLEDRESQI 106 B. longum NARRGNLHVMPTSGSRYYRWDGTQWALVYAHDLSEATVAAVSESLERHARELGLWEQQVWGP---RIENRGSQI 145 L. lactis α-PGM SANLDNFHLFVAQGTQYYAHKAGEWKQVFNYALTDEQANAIMGALEKAAKELGHWDESVLLPGDEINENRESMI 132 . . : : * * * : : .. .. * * . : L. lactis β-PGM QKILD--LADKKVSAEEFKELAKRKNDNYVKMIQDVSPADVYPGILQLLKDLRSNKIKIALA----SASKNGPF 121 . . : : : : . . : . :

M. musculus NVSPIGRSCSQEERIEFYELDKKEHIRQKFVADLRKEFAGKGLTFSIGGQISIDVFPEGWDKRYCLRHLEHAG- 196 H. sapiens PMM2 NVSPIGRSCSQEERIEFYELDKKENIRQKFVADLRKEFAGKGLTFSIGGQISFDVFPDGWDKRYCLRHVENDG- 200 H. sapiens PMM1 NISPIGRSCTLEERIEFSELDKKEKIREKFVEDLKTEFAGKGLRFSRGGMISFDVFPEGWDKRYCLDSLDQDS- 209 S. cerevisiae NVSPIGRNASTEERNEFERYDKEHQIRAKFVEALKKEFPDYGLTFSIGGQISFDVFPAGWDKTYCLQHVEKDG- 207 P. acnes TFSALGQQAPVDAKKAWDPSGDK---KLKLREAVAGKLL--DLEVRAGGSTSVDITRVGRDKSFGIAKLLEMTG 175 B. longum TFSALGQFAPVAAKQAWDRDNTK---KQALVEAVKADLP--HMRVRAGGYTSVDVSECGIDKAYAVRKLTQTLG 214 L. lactis α-PGM AYSAIGQKAGVEAKQAWDPDMTK---RNEIAKLASQYAP--EFEFEVAGTTTINGFVPGQNKEFGMNHLMEELN 201 *.:*: . : : : : : : . .* :.: * :* : : : . L. lactis β-PGM LLEKMNLTGYFDAIADPAEVAASKPAPDIFIAAAH------AVGVAPSESIGLEDSQAG------IQAIKDSG- 182 . :. . : . . . .: * : : .

M. musculus --YKTIYFFGDKTMPGGNDHEIFTDPRTVGYTVTAPEDTRRIC-EGLFP------242 H. sapiens PMM2 --YKTIYFFGDKTMPGGNDHEIFTDPRTMGYSVTAPEDTRRIC-ELLFS------246 H. sapiens PMM1 --FDTIHFFGNETSPGGNDFEIFADPRTVGHSVVSPQDTVQRCREIFFPETAHEA 262 S. cerevisiae --FKEIHFFGDKTMVGGNDYEIFVDERTIGHSVQSPDDTVKILTELFNL------254 P. acnes LSKADVLFYGDRLDEHGNDYPVKAMG-IPCVAVDDWHDTLVKLEDLLSQA----- 224 B. longum IRADEMVFVGDRMTPTGNDYPAVEAG-AIGVRVENPQDTVQLLDALLARFDTPAR 268 L. lactis α-PGM VTKEEILYFGDMTQPGGNDYPVVQMG-IETITVRDWKETAAILKAIIAMEEA--- 252 : : *: ***. * .:* : L. lactis β-PGM ----ALPIGVGRPEDLGDDIVIVPDT------SYYTLEFLKEVWLQKQK--- 221 : . *:* *

Figure 3: Multiple sequence alignment of amino acid sequences of the α-PGM from L. lactis subsp. cremoris MG1363 and its putative homologs from Mus musculus, Homo sapiens (PMM1 and PMM2), Saccharomyces cerevisiae, Propionibacterium acnes and Bifidobacterium longum (for accession numbers, see text). The alignment was generated with ClustalW. The eukaryotic α-PMMs belong to the subfamily II of the HAD superfamily. The lactococcal β-PGM, a subfamily I member, is shown in grey. A signature pattern for motif I of the HAD superfamily phosphomutases -DXDXT- (metal binding and nucleophile) is highlighted by a dark grey box (31). The invariant aspartate residue is printed in yellow. The other signature patterns of the HAD superfamily subfamily II (motifs II, III and IV) are highlighted by blue dark boxes (79, 173). Light blue and light green boxes highlight the core motifs 1, 2, 3, 4 and cap loop 5, respectively, of β-PGM (3). Shaded yellow boxes indicate hinge regions connecting core and cap domains. Residues proposed to be important for substrate binding or catalysis are printed in red. The conserved serine residue in motif II is printed in light-blue (residue inversion in α-PMM and lactococcal α-PGM and its homologs).

118 A novel α-phosphoglucomutase

Biochemical characterization of recombinant α-PGM

The protein was purified about 13-fold to a specific activity of 65 U/mg protein. Biochemical and kinetic properties are listed in Table 2.

TABLE 2: Biochemical and kinetic properties of α-PGM from L. lactis Parameter Value

Apparent mol mass of enzyme (kDa) Native 84.3 Subunit 28 Calculated 28.3 Oligomeric structure α3 pH optimum 6.5 ±0.5

Km (µM) α-G1P 71.4 ±2.8 a) Kact (µM) Glucose-1,6-bisphosphate 16.8 ±1.3 Mg2+ 52.6 ±5.1

Vmax (U/mg protein) α-G1P 65.3 ±1.8 G6P 15.8 ±1.3 Substrate specificity α-G1P 7.5 mM 100%b) G6P 50 mM 23% β-G1P 7.5 mM 0%c) Effect of cations Mg2+ 0 mM < 2%d) Mg2+ 0.05 mM 49% Zn2+ 5 mM 2% Zn2+ 1 mM 28% Ca2+ 5 mM 0.3% Ca2+ 1 mM 1.6% Mn2+ 5 mM 17% Mn2+ 1 mM 21% Ni2+ 5 mM 43% Ni2+ 1 mM 41% Li+ 5 mM 42% Li+ 1 mM 39%

Potential inhibitors (I50) FBP 48±1.1 mM ATP No effect

a) Kact is the concentration of activator at which the rate of the reaction is half of Vmax. b) Activities are relative to the value determined for the conversion of α-G1P to G6P, which is set to 100%, as measured using 31P-NMR. c) The following phosphosugars were also examined but no activity was detected: glucosamine-1P, glucosamine-6P, α-mannose-1P, fructose-1P, fructose-6P, FBP, α- galactose-1P, galactose-6P, ribose-5P, ribulose-5P, 6-phosphogluconate, UDP-galactose and UDP-glucose; when a mixture of FBP and α-G1P was used, α-G1P was fully converted to G6P, but FBP was not used. d) Activities are relative to the value determined for the conversion of α-G1P to G6P, which is set to 100%, as measured using the standard coupling assay and 5 mM Mg2+.

119 Chapter 5

The purified protein was electrophoretically homogenous and SDS-PAGE revealed a single subunit with an apparent molecular mass of 28 kDa. A value of 84.3 kDa was determined by gel filtration (Superose 12 10/300 GL) suggesting a trimeric structure for α-PGM. The pH for maximal activity was 6.5; 50% of the activity was found at pHs 5.6 and 7.5. The rate dependence on α-G1P concentration followed Michaelis- Menten kinetics. Several phosphosugars were examined as putative substrates using the 31P-NMR direct assay. The enzyme catalyzes only the interconversion of

α-G1P into G6P. Furthermore, the apparent Vmax value for the reverse direction (G6P Æ α-G1P) was 3-fold lower than that of the forward direction, and the apparent

Km value for G6P was in the mM range. Neither α-mannose-1P nor β-G1P were substrates for the enzyme, despite the sequence similarity of L. lactis α-PGM to the eukaryotic phosphomannomutases (around 25%) and β-PGM/G1P phosphodismutases (around 10%). For maximal activity, α-PGM required Mg2+ and glucose-1,6-bisphosphate. In the absence of Mg2+, the activity was reduced below 2% and no activity was detected when glucose-1,6-bisphosphate was omitted from the assay. Zn2+ and Ca2+ strongly inhibited the activity, whereas Ni2+ and Li+ had only a slight inhibitory effect. Partial inhibition was also observed with Mn2+. ATP showed no regulatory effect on α-PGM activity, while FBP moderately inhibited the activity (50% was lost at 48 mM FBP).

Pools of glycolytic intermediates in non-growing cells

L. lactis NZ9000 harbouring pNZ8048-pgmH (hereafter named NZ9000[pgmH+]) or pNZ8048 (control) were grown in CDM containing glucose or galactose and induced with nisin for α-PGM production. The effect of α-PGM overproduction on the metabolism of glucose and galactose was studied by in vivo 13C-NMR in non- growing cell suspensions under an argon atmosphere and at pH 6.5 (Fig. 4). (i) [1-13C]-Glucose. The conversion of glucose was homofermentative in NZ9000[pgmH+] and NZ9000[pNZ8048] (lactate production above 91%), the glucose consumption rates being 0.37±0.02 and 0.39±0.02 μmol·min·mg protein-1, respectively. Minor end-products, acetate, ethanol and 2,3-butanediol, were detected in identical amounts in both strains(data not shown). NZ9000[pNZ8048] metabolized glucose like its parent MG1363 (123), showing that glucose metabolism in non-growing NZ9000 cells was not affected by the presence of pNZ8048. The dynamics of intracellular metabolite pools was not appreciably affected by the substantial increase in α-PGM activity from 0.07 U·mg protein-1 in the control to 2.6 U·mg protein-1 in NZ9000[pgmH+]. The maximal concentration of FBP decreased by

120 A novel α-phosphoglucomutase about 10% and 3-PGA increased slightly. Moreover, the UDP-glucose pool decreased from 6.7±0.5 mM in the control to 4.2±0.3 mM in NZ9000[pgmH+] (Fig. 4A & 4B).

A 50 B 50 + G 0.37 NZ9000[pNZ048] GCR 0.39 NZ9000[pgmH ] CR 40 40

30 30

20 20 Concentration (mM) Concentration (mM) 10 10

0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Time (min) Time (min)

CD40 40 + NZ9000[pNZ048] GalCR 0.16 NZ9000[pgmH ] GalCR 0.21

30 30

20 20

10 10 Concentration (mM) Concentration (mM)

0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Time (min) Time (min)

Figure 4: Metabolism of glucose or galactose in non-growing cell suspensions of L. lactis NZ9000[pNZ8048] and NZ9000[pgmH+]. Time course for substrate consumption and pools of intracellular metabolites in non-growing cultures of strains NZ9000[pNZ8048] (A, C) and NZ9000[pgmH+] (B, D) during the metabolism of [1-13C]-glucose (A, B) or [1-13C]-galactose (C, D) as monitored by 13C-NMR. Cells were grown in a bioreactor vessel on glucose (A, B) or galactose (C, D) in de-aerated

CDM at pH 6.5, and induced with nisin (1 µg/l) when the OD600 was 0.5. Lactate, ethanol, acetate and 2,3-butanediol were also detected, but their time courses were omitted from the graphs for the sake of simplicity. The glucose or galactose consumption rate (GCR or GalCR respectively) in μmol·min-1·mg protein-1 is depicted in the top-right corner of each graph. Symbols: glucose ( ); galactose ( ); FBP ( ); α-G1P ( ); Gal1P ( ); 3-PGA ( ); PEP ( ); UDP-Glc ( ); UDP-Gal ( ). Fitted lines are simple interpolations.

121 Chapter 5

(ii) [1-13C]-Galactose. As expected for galactose, the metabolism of pyruvate was shifted to products other than lactate, namely ethanol (2.2±0.2 mM) and acetate (2.4±0.4 mM), but their concentrations were about 35% lower in NZ9000[pgmH+] compared to the control strain NZ9000[pNZ8048] (data not shown). Induction with nisin resulted in a 7-fold higher (0.3 to 2.1 U·mg protein-1) α-PGM activity in the strain harbouring the pgmH construct, a modest change when compared to the 37- fold increase observed in glucose grown cells. Curiously, the galactose consumption rate was 25% greater (from 0.16±0.1 to 0.21±0.1 μmol·min·mg protein-1) in the strain overproducing α-PGM, compared to the control strain. Addition of galactose to a “starved” cell suspension of the control strain resulted in the build-up of the Leloir pathway phosphorylated intermediates, Gal1P and α-G1P, reaching maximal concentrations of 18.0±0.5 and 18.7±0.7 mM, respectively (Fig. 4C). Accumulation of FBP, the predominant metabolite during glucose metabolism, was slightly delayed and reached a maximal concentration of 24.0±1.5 mM. At the onset of galactose depletion, 3-PGA and PEP pools rose to 28.6±2.1 and 11.0±2.2 mM, respectively. These high levels of 3-PGA and PEP denote the utilization of an uptake system for galactose other than a PEP:PTS. Overproduction of α-PGM had a considerable impact on the concentrations of intracellular metabolite pools. As in the control strain, the accumulation of FBP was slightly delayed, but in NZ9000[pgmH+] its maximal concentration was clearly higher. A remarkable reduction in the size of Gal1P and α-G1P pools to 2.4 and 2.2 mM, respectively, revealed α-PGM as the main bottleneck during galactose metabolism in L. lactis. UDP-glc (3.9±0.4 mM) and UDP-gal (3.2±0.3 mM) were detected and these pools were 1.4-fold lower in the strain with increased α-PGM activity (Fig. 4C & 4D). In summary, the overproduction of α-PGM caused notable changes in the dynamics and levels of glycolytic intermediates derived from galactose, whereas no major differences were observed when glucose was used, reflecting the central role of α- PGM in the degradation of galactose. The observations in resting cells raised the question as to how a growing culture would respond to an increase in α-PGM activity. Therefore, we investigated the effect of overexpression of pgmH in growing cells.

Impact of α-PGM overproduction in growing cells

(i) Growth characteristics. The effect of overproduction of α-PGM on the growth properties of L. lactis was evaluated using either glucose or galactose as carbon source (Table 3 and Fig. 5).

122 A novel α-phosphoglucomutase

The specific activity of α-PGM measured in cells before addition of nisin was consistently higher in the strain carrying pNZ8048-pgmH, most likely due to low basal PnisA expression. Galactose per se induced α-PGM activity 2- and 4-fold in NZ9000[pgmH+] and the control strain, respectively (Table 3). Up-regulation of pgmH expression on galactose was also apparent when the strains were grown on galactose without nisin induction.

TABLE 3: Effect of pgmH overexpression on some growth properties during glucose or galactose fermentation by L. lactis. NZ9000[pgmH+] and NZ9000[pNZ8048] were grown in CDM supplemented with chloramphenicol (5 mg/l) and 1% (w/v) glucose or galactose. Nisin (1 µg/l) was added when the culture OD600 reached a value of 0.5. Growth rate constants for the entire growth phase were calculated using linear regressions. α-PGM activity was measured before (OD600, 0.5) and after induction (OD600, 0.5).

Glucose Galactose pNZ8048 pgmH+ pNZ8048 pgmH+

Carbon balance (%) 95 ± 0.5 95 ± 0.5 94 ± 0.3 94 ± 0.1 Biomass yield (g/mol) 29.9 ± 0.3 29.1 ± 0.3 26.5 ± 0.2 26.5 ± 0.4 a) ATP yield (mol/mol subs.) 1.9 ± 0.01 1.9 ± 0.01 2.1 ± 0.01 2.1 ± 0.01 b) YATP 15.6 ± 0.2 15.2 ± 0.2 12.5 ± 0.2 12.9 ± 0.2 c) Growth rate (µ1) 0.80 ± 0.01 0.82 ± 0.01 0.36 ± 0.01 0.45 ± 0.01 d) Growth rate (µ2) 0.58 ± 0.01 0.56 ± 0.02 0.37 ± 0.01 0.50 ± 0.01 c) α-PGM activity (U/mg)1 0.07 ± 0.01 0.16 ± 0.01 0.29 ± 0.01 0.36 ± 0.02 d) α-PGM activity (U/mg)1 0.07 ± 0.01 2.63 ± 0.20 0.34 ± 0.02 2.05 ± 0.16 Lactate/substrate (%) 92 ± 0.4 91 ± 0.4 71 ± 1.0 79 ± 0.8 Other products/substrate (%) 2 ± 0.5 3 ± 0.7 23 ± 0.3 19 ± 0.3

a) The global ATP yield was calculated from the fermentation products assuming that all ATP was synthesized by substrate-level phosphorylation b) YATP, biomass yield relative to ATP production c) before addition of nisin d) after addition of nisin (1 µg/l)

The growth rate was affected by nisin addition, but the magnitude and the sign of the effect was sugar-dependent (Table 3). In the absence of nisin, the growth rate on glucose was about twice as high as that on galactose. Furthermore, the growth rate on glucose decreased considerably upon addition of nisin, but this negative effect was unrelated to α-PGM overproduction (16-fold increase). Only a 7 fold higher α- PGM activity was achieved during growth on galactose; however, the growth rate of NZ9000[pgmH+] was substantially greater than in the control strain (0.50 versus 0.36

123 Chapter 5 h-1), reaching a value close to that on glucose (0.56 h-1). The results show that α- PGM activity in the control strain is limiting during growth on galactose, and that this bottleneck was overcome by pgmH overexpression. In glucose-grown cells, increased α-PGM activity had no impact on product formation, with lactate accounting for over 90% of the end-products. When galactose was used as carbon source, the lactate yield increased slightly in the strain overproducing α-PGM (Table 3, Fig. 5), in line with the increased growth rate of strain NZ9000[pgmH+]. Biomass yield, ATP yield, and biomass yield on ATP were dependent on the sugar used and not affected by overproduction of α-PGM (Table 3).

5 B 120 A Glucose Galactose

100 4

) 80 600 3 60 2 40 Growth (OD

1 Concentration (mM) 20

0 0 0 5 10 15 pgmH+ pNZ8048 pgmH+ pNZ8048 Time (h)

Figure 5: Growth of L. lactis NZ9000[pNZ8048] and NZ9000[pgmH+] on glucose or galactose in CDM at 30°C and pH 6.5. A) Biomass formation was spectrophotometrically followed during growth on glucose (open symbols) or galactose (closed symbols). Symbols: squares (open and closed) NZ9000[pNZ8048]; triangles (open and closed), NZ9000[pgmH+]. B) End products from the fermentation of glucose (59.3±0.2 mM) and galactose (57.7±0.2 mM). To induce expression of pgmH, nisin (1 µg/l) was added at an OD600 of 0.5. Symbols: lactate ( ); acetate ( ); ethanol ( ). Data are from a representative experiment where the error in each point ≤ 10%.

(ii) Pools of phosphorylated metabolites. Table 4 shows pool sizes for glycolytic intermediates and sugar-nucleotides determined by 31P-NMR in cell extracts derived from mid-exponential phase cultures. FBP was the major metabolite on glucose, whereas on galactose 3-PGA and PEP were also present in high amounts. The size of the FBP pool varied to a small extent and in a sugar-dependent way in response to increased α-PGM activity. As expected, the Leloir pathway intermediate Gal1P was below the detection limit on glucose; on galactose its concentration was slightly

124 A novel α-phosphoglucomutase lower (20%) in the α-PGM overproducing strain. The concentration of α-G1P decreased notably in NZ9000[pgmH+] regardless the sugar used.

It is thought that in L. lactis UDP-sugars and UDP-aminosugars are derived from α- G1P and F6P, respectively, hence their concentrations could respond to changes in α-PGM activity. Overproduction of α-PGM resulted in reduction or constancy of UDP-sugars on galactose or glucose grown cells, respectively. Concentrations of UDP-aminosugars and 5-phosphorylribose 1-pyrophosphate in mid-exponential phase cells of L. lactis were not significantly affected by pgmH overexpression, except for UDP-N-acetylglucosamine, the first cytoplasmatic precursor of peptidoglycan, which level responded inversely to an increase in the activity of α- PGM (Table 4).

TABLE 4: Effect of pgmH overexpression on the pools of phosphorylated metabolites and UDP-sugars during growth on glucose or galactose. NZ9000[pgmH+] and NZ9000[pNZ8048] were grown in CDM supplemented with choramphenicol (5 mg/l) and 1% (w/v) glucose or galactose. Nisin (1 µg/l) was added when the culture OD600 reached a value of 0.5. Phosphorylated metabolites (mM) were measured in cell extracts obtained during mid-exponential growth phase (OD600 2.2). The average accuracy ±15%.

Glucose Galactose pNZ8048 pgmH+ pNZ8048 pgmH+

α-G1P (α-glucose-1-phosphate) 0.4 0.2 2.4 1.0 Gal1P (galactose-1-phosphate) 0.0 0.0 3.7 3.0 FBP (fructose-1,6-bisphosphate) 13.9 11.8 9.7 11.0 G6P (glucose-6-phosphate) 3.7 3.8 3.3 1.8 3-PGA (3-phosphoglycerate) 2.0 2.8 9.2 7.5 PEP (phosphoenolpyruvate) 1.0 1.6 3.3 3.6 2-PGA (2-phosphoglycerate) 0.1 0.2 0.9 0.6

UDP-Gal (UDP-galactose) 0.5 0.4 1.4 1.0 UDP-Glc (UDP-glucose) 2.4 2.4 4.0 2.8 UDP-GalNa) 1.1 1.2 0.6 0.8 UDP-GlcNAc 3.2 2.6 2.0 2.4 UDP-N-AcMur-pPep 1.2 1.3 1.8 1.9 UDP-GlcN 0.4 0.6 0.7 0.6 PRPP 0.9 0.7 1.6 1.4

α-PGM activity (U/mg) 0.07 2.63 0.34 2.05

a) Resonance at -11.98 tentatively assigned to UDP-galactosamine (136, 151). Abbreviations: UDP-GlcNAc, UDP-N-acetylglucosamine; UDP-N-AcMur-pPep, UDP-N-acetylmuromoyl- pentapeptide; UDP-GlcN, UDP-glucosamine; PRPP, 5-phosphorylribose 1-pyrophosphate.

125 Chapter 5

Inactivation of the chromosomal pgmH gene and its effect on L. lactis growth

To investigate whether the α-PGM activity is essential for growth of L. lactis on glucose and galactose we decided to inactivate the pgmH gene (see Experimental Procedures). Several attempts to disrupt pgmH by single-crossover plasmid integration with pORI13-pgmH’ or by a two-step homologous recombination method (96) failed. These results suggest that pgmH plays an essential role in L. lactis. Integration of pORI280ΔpgmH in NZ9000 resulted in erythromycin resistant colonies harboring a disrupted as well as an integral copy of pgmH. Only when pgmH was expressed in trans (under nisin control in pNZ8048) in NZ9000::pORI280ΔpgmH, was it possible to delete the chromosomal copy of pgmH, as confirmed by PCR and Southern analysis (not shown). The resulting strain, NZ9000ΔpgmH[pgmH+] was constructed and maintained in the presence of 0.1 µg/l nisin. To ascertain whether α-PGM was limiting during growth on glucose-M17, low nisin (0.01 µg/l) overnight grown-cultures (α-PGM activity 0.11 U·mg protein-1) were subcultured in fresh medium with increasing concentrations of nisin (0 to 1 µg/l). In the absence of nisin, the mutant strain showed poor growth with an average growth constant of 0.20 h-1 (Fig. 6A); at time-point 12 h, α-PGM activity had decreased to 0.025 U·mg protein-1. Under the same conditions, the control strain NZ9000[pNZ8048] showing a growth -1 rate of 0.55 h , reached a biomass concentration five times higher (OD600 of 3.2 as compared to 0.6) and a steady α-PGM activity of 0.15 U·mg protein-1. Modulation of expression of pgmH by varying the nisin concentration in the range 0.01 to 1 µg/l resulted in a series of cultures with α-PGM activity between 63 and 4000% of the control level (Fig. 6B). Nisin concentrations as low as 0.05 µg/l already resulted in overexpression of pgmH (Fig. 6B). A reduction of the growth rate to 70% of that of the control was observed at the lowest α-PGM activity corresponding to 63% of the control level (nisin 0.01 µg/l). The data show that α-PGM activities below the control level do not sustain maximal growth of L. lactis on glucose-M17.

126 A novel α-phosphoglucomutase

A B 4 C

1 ) 3

600 0.5 g/l) μ 2 0.1

0.05 Nisin (

Growth (OD 1 0.01

0 0 0246810121416 01234567 Time (h) α-PGM (U·mg protein-1)

Figure 6: Effect of α-PGM activity on the growth kinetics of L. lactis in glucose-M17. A) Overnight cultures of NZ9000ΔpgmH[pgmH+] grown in the presence of 0.01 µg/l nisin were subcultured in fresh glucose-M17 containing 0 µg/l ( ), 0.01 µg/l ( ), 0.05 µg/l ( ) and 1 µg/l ( ) of nisin; NZ9000[pNZ8048] was used as the control strain ( ). Growth was monitored for

15 h by measuring the optical density at 600 nm (OD600). B) For each of the growth curves, α- PGM specific activity at time 12 h of culturing is shown. α-PGM specific activity was measured in cell-free extracts as described in Experimental Procedures. Activity in the control strain NZ9000[pNZ8048] is shown as a dashed bar. Data is shown from a representative experiment where the error in each point ≤ 10%.

The effect of controlled limitation of pgmH expression was evaluated during growth on glucose and galactose in M17 medium. Strain NZ9000ΔpgmH[pgmH+] was grown overnight in medium containing different concentrations of nisin and subsequently subcultured in nisin-free medium (Fig. 7A & 7B). α-PGM activity in the overnight cultures at the time of subculturing (17 h after inoculation) showed a similar pattern as in Fig. 6B. In glucose-grown cells, nisin concentrations of 0.1, 0.01 and 0 µg/l in the overnight cultures resulted in 16, 65 and 74% reduction in the growth rates, respectively. In contrast, nisin concentrations of 0.5 and 1 µg/l in the inocula supported growth constants similar to those of the control strain. The effect on growth rates correlated well with the decline of α-PGM activity (Fig. 7A). Moreover, the stepwise reduction in the final optical density was consistent with the nisin amounts used in the overnight cultures. These results show that pgmH is essential for growth of L. lactis on glucose.

127 Chapter 5

A 4 B C

) 3 1 600 0.5 g/l)

2 μ 0.1 Growth (OD 1 Nisin ( 0.01

0 0 0246810121416 0.00 0.05 0.10 0.15 0.20 Time (h) α-PGM (U·mg protein-1)

C 4 D C

) 3 1 600 0.5 g/l)

2 μ 0.1 Nisin ( Growth (OD 1 0.01

0 0 02468101214 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Time (h) α-PGM (U·mg protein-1)

Figure 7: Growth dependence of NZ9000ΔpgmH[pgmH+] on α-PGM activity. Overnight cultures of NZ9000ΔpgmH[pgmH+] grown on glucose-M17 (A) or galactose-M17 (C) containing nisin 0 µg/l ( ), 0.01 µg/l ( ), 0.1 µg/l ( ), 0.5 µg/l ( ) and 1 µg/l ( ) were subcultured in fresh M17 medium without nisin on glucose (A) or galactose (C). NZ9000[pNZ8048] was used as the control strain ( ). Growth was monitored for 15 h by measuring the optical density at 600 nm (OD600). For each of the growth curves in graphs A and C, α-PGM specific activity at time 12 h of culturing are shown in diagrams (B) and (D), respectively. α-PGM specific activity was measured in cell-free extracts as described in the Experimental Procedures. Activity in the control strain NZ9000[pNZ8048] is shown as a dashed bar. The dashed line indicates the wild-type level α-PGM activity. Data is shown from a representative experiment where the error in each point ≤ 10%.

When galactose was used as sole carbon source, the effect of limiting the expression of pgmH on the growth rates and final optical density was more pronounced than on glucose (Fig. 7C). Regardless of the nisin concentration used (0 to 1 µg/l), final biomass and growth rate constants were below 60% of the respective -1 parameters in the control strain (OD600, 3.5 and µ, 0.41 h ) (Fig. 7C). An α-PGM

128 A novel α-phosphoglucomutase activity of 1.38 U·mg protein-1 was measured at time-point 12 h in cells grown overnight and subcultured in fresh medium with nisin at 0.5 µg/l, but the growth parameters did not improve. Although no final explanation for this intriguing behavior can be put forward, it is possible that complex regulatory mechanisms resulting from cross-reactions involving galactose metabolism and nisin-induction are implicated (29, 80). The severe growth defects shown here for L. lactis NZ9000ΔpgmH[pgmH+] validate the conclusion that pgmH is essential for growth of L. lactis on galactose.

129 Chapter 5

DISCUSSION

In this report we describe the identification and functional analysis of L. lactis α- phosphoglucomutase, which represents the first characterized member of a novel α- PGM family.

Sequence comparison and properties

All α-PGMs characterized so far belong to the α-D-phosphohexomutase superfamily of proteins, comprising α-PGMs (mostly eukaryotic), bacterial and archaeal phosphomannomutases/phosphoglucomutases (PMM/PGMs), phosphoglucosamine (mostly bacterial) and the strictly eukaryotic phosphoacetylglucosamine mutases (171). The α-PGM encoded by L. lactis pgmH did not show any similarity to the members of the α-D phosphohexomutase superfamily nor did it contain the family’s consensus motifs. Instead, it showed sequence homology to eukaryotic phosphomannomutases (Fig. 3 and Fig. 8), an unrelated group of proteins that despite their phosphohexomutase activity belong to the HAD superfamily (PF03332, http://www.sanger.ac.uk/cgi-bin/Pfam). Saccharomyces cerevisiae SEC53 was the first member studied (71), but thereafter several other enzymes have been characterized to some extent (130, 137, 179). These proteins have about 260 amino acids and feature the conserved sequence motifs characteristic of the HAD superfamily (3, 31, 79, 159), which are all present in the pgmH product (Fig. 3). The HAD superfamily comprises two branches that have acquired phosphohexomutase function, the eukaryotic α-phosphomannomutases (PF03332) and β- phosphoglucomutases (PF00702) (87). As all known α-PGMs fall into the α-D- phosphohexomutase superfamily, we propose that the L. lactis α-PGM represents a novel line of α-D-phosphohexomutase evolution (Fig. 8). Unlike the eukaryotic phosphomannomutases, which in general use both mannose- 1-phosphate and glucose-1-phosphate, L. lactis α-PGM shows strict specificity for α- G1P. Narrow substrate specificity has been described for the eukaryotic α-PGMs that fall into the α-D-phosphohexomutase superfamily (154), in contrast to the bacterial α-PGMs (PMM/PGM), which have a rather broad substrate preference (222). Within the α-D-phosphohexomutase superfamily, substrate specificity has been related to subtle residue variance in the catalytic domains (171). In the HAD superfamily, like in the α-D-phosphohexomutase superfamily, the catalytic cycle proceeds via a bisphosphorylated sugar intermediate to the reversible conversion of 1-phospho to 6-phosphosugars and requires Mg2+ as cofactor (Table 2, (137, 138, 150, 171)). However, kinetic studies showed that a different reaction mechanism

130 A novel α-phosphoglucomutase operates, in which the active residue is phosphoaspartyl (110, 138). The recent publication of the X-ray structures of HAD superfamily phosphohexomutases, lactococcal β-PGM and human PMM1 sheds some light on the mechanism used

(88, 89, 173, 224). In addition to the hexose C1 configuration (α- or β-anomer) specificity, the position and fold of the cap domain allow distinguishing eukaryotic α- PMMs from bacterial β-PGMs (Fig. 3, (3)). The position and fold of the cap domain (Fig. 3) places the L. lactis α-PGM in the HAD superfamily subclass II, whereas β- PGM is a subclass I protein. Sequence identities between L. lactis α-PGM (query sequence) and human α-PMM1 or L. lactis β-PGM are 25% and 10%, respectively. Taken together the structural and sequence similarities and the anomer specificity suggest that the L. lactis α-PGM is mechanistically closer to the α-PMMs than to the β-PGMs. This hypothesis is strengthened by the presence in the α-PGM sequence of the residues that are involved in the catalytic process of α-PMM1, in particular the nucleophile Asp8, the acid/base Asp10 and the Gln51 (Asp19, Asp21 and Gln62 in human α PMM1). However, only some of the conserved positively charged residues at the interface of the cap and core domains in α-PMMs are present in L. lactis α- PGM (Fig. 3), suggesting a mechanism of action different from that of the electrostatic wedge proposed for the α-PMM (173). To analyze the phylogenetic relationship of proteins with phosphohexomutase activity, of which the L. lactis α-PGM was characterized here, the neighbour-joining tree construction method was used (200). A phylogram including both characterized and putative phosphohexomutases from eukaryotic and bacterial sources is given in Fig. 8. The depicted topology clearly separates members of the α-D- phosphohexomutase and HAD superfamilies despite their similar function. Topological organization within the α-D-phosphohexomutase superfamily is identical to that observed by others and has been thoroughly discussed elsewhere (171, 217), but the topology within the HAD branch is intriguing. The reconstruction presented here reflects the family division in the HAD superfamily, with bacterial β- PGMs and eukaryotic α-PMMs divided into two distinct groups that are supported by good bootstrap values. The L. lactis α-PGM is included in a cluster comprising proteins with unknown function of the human-associated organisms Bifidobacterium longum and Propionibacterium acnes and a plant pathogen, Gibberella zeae, that branches from the line leading to the eukaryotic α-PMMs. This suggests a common origin of these proteins and the eukaryotic α-PMMs, which is also supported by the common α-anomeric specificity. A possible explanation relies on independent lateral gene transfer from eukarya to their commensal or pathogenic organisms. Surprisingly, of all the bacteria with available genome sequences, L. lactis appears

131 Chapter 5 to be unique insofar as it lacks an α-PGM of the α-D-phosphohexomutase superfamily.

Sthe_PMM/PGM Spne_PMM/PGM Spyo_PMM/PGM Bsubt_PMM/PGM Blon_PGM Gxyl_PGM 782 Spne_PNGM Llac_femD Spyo_PNGM Cglu_PGM 1000 Bsub_PNGM Vfis_PGM 1000 Ecol_PGM Ecol_PNGM 1000

1000 Ecol_PMM 508 1000 Scer_PGM 924 1000 1000 Paer_PMM/PGM Hsap_PGM 1000 Ocun_PGM 1000 1000 978 1000 Hsap_PAGM 924 1000 568 837 Mmus_PAGM 1000 1000 1000 Dmel_PAGM 1000 1000 1000 Llac_yhfA 1000 1000 Spyo_PGMB 847 724

780 1000 Lpla_PGMB 678 Llac_PGM 724 590 508 Pacn_HP Bsub_PGMB 617 Efae_PGMB 996 Blon_HP Nmen_PGMB Gzea_PMM Llac_PGMB 1000

1000 Orys_PMM Ecun_PMM Mmus_PMM Cpar_PMM Hsap_PMM2 0.1 Scer_PMM Hsap_PMM1 Calb_PMM

Figure 8: Unrooted phylogenetic tree based on available amino acid sequences of phosphomutases. The ClustalX program (200) was used for sequence alignments and to generate the phylogenetic tree. The significance of the branching order was evaluated by bootstrap analysis of 1,000 computer-generated trees. The bootstrap values are indicated. Bar, 0.1 changes/site. Abbreviations and GenBank accession numbers: Ocun, Oryctolagus cuniculus PGM P00949; Scer, Saccharomyces cerevisiae: PGM NP_013823, PMM NP_116609; Hsap, Homo sapiens: PGM AAH67763, PAGM AAD55097, PMM1 AAC51117, PMM2 AAH08310; Llac, Lactococcus lactis: FemD NP_266580, α-PGM (pgmH) DQ778336 , PGMB NP_266585; YhfA NP_266899; Spne, Streptococcus pneumoniae: PGNM AAL00221, PMM|PGM AAD56627; Spyo, Streptococcus pyogenes: PGNM YP_280210, PMM|PGM YP_282301, PGMB YP_281891; Bsub, Bacillus subtilis: PGNM NP_388058, PMM/PGM CAH04980, PGMB NP_391335; Ecol, Escherichia coli: PNGM AAC76208, PMM O85343, PGM AAC73782; Mmusc, Mus musculus: PAGM NP_082628, PMM NP_058577; Dmel, Drosophila

132 A novel α-phosphoglucomutase melanogaster: PAGM NP_648588; Pacn, Propionibacterium acnes; HP YP_056695; Blon, Bifidobacterium longum: HP ZP_00121741, PGM NP_696782; Gzea, Gibberella zeae: PMM EAA71459; Orys, Oryza sativa: PMM XP_474395; Cpar, Cryptosporidium parvum: PMM EAK87737; Ecun, Encephalitozoon cuniculi: PMM CAD26542; Calb; Candida albicans: PMM EAL02637; Paer, Pseudomonas aeruginosa: PMM/PMG NP_254009; Sthe, Steptococcus thermophilus: PMM/PGM AAV62380; Gxyl, Gluconacetobacter xylinus: PGM P38569; Cglu, Corynebacterium glutamicum: PMM/PGM CAF21203; Vfis, Vibrio fischeri: PGM AAM77720; Nmen, Neisseria meningitides: PGMB CAB85309; Efae, Enterococcus faecalis: PGM NP_814693; Lplan, Lactobacillus plantarum: PGM NP_783891. PGM, phosphoglucomutase; PGNM; phosphoglucosamine mutase; PAGM; phosphoacetylglucosamine mutase; PMM, ; HP, hypothetical protein; PGMB; beta-phosphoglucomutase. α-D phosphohexomutase and haloacid dehalogenase superfamilies are highlighted by light and dark grey boxes, respectively.

Physiological function of the L. lactis α-PGM

In bacteria, the interconversion of α-G1P to G6P catalyzed by α-D- phosphohexomutases is a key step in the production of UDP-Glc, the glucosyl donor for the synthesis of glucose-containing polysaccharides (cell wall polysaccharides, capsules and exopolysaccharides) and in the catabolism of galactose. We demonstrated that the product of femD, the only gene in the L. lactis chromosome with homology to known α-D-phosphohexomutases, does not display α-PGM activity. Thus far, pgmB was the only gene identified in L. lactis encoding phosphoglucomutase activity (15, 149), but the enzyme is specific for the β-anomer of phosphoglucose, an intermediate in the catabolism of the disaccharides maltose and trehalose (224). In this work we show that the highly specific α-PGM encoded by the pgmH gene mediates the reversible conversion of α-G1P into G6P in L. lactis. Remarkably, all strategies attempted to disrupt pgmH failed. In bacteria, inactivation of α-PGM generally results in altered or defective lipopolysaccharides (49, 111, 118, 216) and/or reduced or abolished polysaccharide production (32, 213) mainly due to a deficient UDP-Glc supply. However, in some Gram-positives growth defects and altered cell morphology have also been associated with disruption of PGM genes (22, 57, 91, 100). Our experiments with varying nisin concentration or progressive decrease of α-PGM activity by using the pgmH conditional knock-out showed dramatic effects on growth rate and final biomass when the activity was below the control level. In the absence of nisin, a residual α-PGM activity, probably due to leakage of PnisA in a high copy system, could still sustain modest growth. Under these conditions, cell division and morphology were affected as denoted by the appearance of long chains comprising cells that had lost their typical lactococcal shape (data not shown). L. lactis strains deficient in UDP-galactose-4-epimerase

133 Chapter 5

(galE) (14), the major autolysin (acmA) (180) or the lipoteichoic acids d-alanylation (181) showed a similar behaviour, suggesting altered or deficient biosynthesis of cell wall polysaccharides. Earlier attempts to inactivate L. lactis MG1363 galU, encoding the enzyme that catalyzes the conversion of α-G1P to UDP-Glc, were also unsuccessful (14), and as for α-PGM, no other genes coding for a galactose uridyl transferase were found in the available genome sequences (15, 215). Therefore, it is reasonable to conclude that UDP-Glc synthesis in L. lactis relies entirely on pgmH and galU gene products. Moreover, we can speculate that α-PGM exerts considerable flux control in the synthesis of glucose-containing polysaccharides, since we showed that the size of the UDP-Glc pool responds to variations in the level of this enzyme. Under an applied point of view, this knowledge could be exploited to develop strains with improved exopolysaccharide production or altered lytic capacity, both industrially relevant traits. The involvement of pgmH in galactose metabolism became clear from the observed changes in the dynamics of intracellular metabolites in cells overproducing α-PGM. The lower concentration of α-G1P and Gal1P in the strain overexpressing pgmH identifies the step catalyzed by α-PGM as a bottleneck in galactose metabolism. This view is further substantiated by the improved growth and galactose consumption rates in the strain overexpressing pgmH as well as the growth impairment in mutants with reduced α-PGM activity. Overproduction of α-PGM affected the levels of glycolytic metabolites and the glycolytic flux from galactose, but not from glucose, reflecting the different role of pgmH in the metabolism of these two sugars: α-PGM is required for galactose degradation, whereas its main function is providing precursors for biosynthetic pathways during growth on glucose.

Altogether, the results presented here support the conclusion that pgmH is the only gene in the L. lactis genome coding for α-PGM activity. Therefore, the purification of a 65 kDa protein with α-PGM activity reported earlier (150) can only be explained on the basis of the different genetic backgrounds used. This work revealed a novel α-D- phoshoglucomutase that falls into the haloacid dehalogenase family, unlike all other known α-PGM. The demonstration of the essentiality of pgmH is a final confirmation of the crucial role played by the enzyme in the physiology of L. lactis. To further understand the unique features unraveled by the present study, the structural determination of this new α-D-phoshoglucomutase is in progress.

134 A novel α-phosphoglucomutase

ACKNOWLEDGEMENTS

This work was supported by contract QLK1-CT-2000-01376 of the Commission of the European Communities and contract POCTI/BIO/48333/2002 of Fundação para a Ciência e a Tecnologia (FCT). A. R. Neves acknowledges a post-doctoral fellowship of FCT. We thank Claudia Sanchéz for technical assistance with the HPLC measurements and Thijs Kouwen for cloning the pgmH gene.

135