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-PHOSPHOGLUCOMUTASE OF LACTOCOCCUS LACTIS IS UNRELATED TO THE α-D-PHOSPHOHEXOMUTASE SUPERFAMILY AND ENCODED BY THE ESSENTIAL GENE 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 phosphomannomutases within the haloacid dehalogenase superfamily. In contrast to known bacterial α-PGMs, this 28 kDa enzyme 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), genes 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 mutase. 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 enzymes 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 chromosome 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.