A LacI-Family Regulator Activates Maltodextrin Metabolism of Enterococcus faecium

et al. (2013) A LacI-Family Regulator Activates Maltodextrin Metabolism of Enterococcus faecium. PLoS ONE 8(8): e72285. doi:10.1371/journal.pone.0072285 Editor: Riccardo Manganelli A LacI-Family Regulator Activates Maltodextrin Metabolism of Enterococcus faecium Xinglin Zhang 0 Malbert Rogers 0 Damien Bierschenk 0 Marc J. M. Bonten 0 Rob J. L. Willems 0 Willem van Schaik 0 0 Department of Medical Microbiology, University Medical Center Utrecht , Utrecht , The Netherlands Enterococcus faecium is a gut commensal of humans and animals. In the intestinal tract, E. faecium will have access to a wide variety of carbohydrates, including maltodextrins and maltose, which are the sugars that result from the enzymatic digestion of starch by host-derived and microbial amylases. In this study, we identified the genetic determinants for maltodextrin utilization of E. faecium E1162. We generated a deletion mutant of the mdxABCD-pulA gene cluster that is homologous to maltodextrin uptake genes in other Gram-positive bacteria, and a deletion mutant of the mdxR gene, which is predicted to encode a LacI family regulator of mdxABCD-pulA. Both mutations impaired growth on maltodextrins but had no effect on the growth on maltose and glucose. Comparative transcriptome analysis showed that eight genes (including mdxABCD-pulA) were expressed at significantly lower levels in the isogenic mdxR mutant strain compared to the parental strain when grown on maltose. Quantitative real-time RTPCR confirmed the results of transcriptome analysis and showed that the transcription of a putative maltose utilization gene cluster is induced in a semi-defined medium supplemented with maltose but is not regulated by MdxR. Understanding the maltodextrin metabolism of E. faecium could yield novel insights into the underlying mechanisms that contribute to the gut commensal lifestyle of E. faecium. - Enterococci are facultative anaerobic Gram-positive bacteria commonly found in the gastrointestinal tracts of humans and animals [1]. In the last twenty years, E. faecium has emerged as a clinical pathogen of major importance. This development has been linked to its ability to efficiently acquire antibiotic resistance genes and genetic elements that may contribute to virulence [2,3]. The ability of both commensal and clinical E. faecium strains to effectively colonize the intestinal tract determines the ecological success of this species. Therefore, understanding the mechanisms of successful host colonization is important for the development of novel strategies to prevent or treat infections with these opportunistic pathogens. The metabolism of carbohydrates in the complicated food webs of the mammalian intestinal tract is crucially important for gut colonization of commensals and opportunistic pathogens [48]. Carbohydrate utilization of E. faecium remains poorly understood despite its potential importance in colonization and adaptation to healthy individuals [9] and hospitalized patients [10]. One of the main energy and carbon sources for bacteria in the intestine originates from complex polysaccharides, such as starch [4]. Starch is a plant storage glycan that consists of glucose monomers joined via -1,4 glycosidic linkages with additional branches introduced by -1,6 linked glucose moieties. In the human intestinal tract, starch is digested by host-derived and microbial amylases. Its breakdown products (mainly maltose and maltodextrins) can be absorbed by the host small intestine [11], but can also reach the colon [12,13] where they can be metabolized by bacteria from several genera [14,15]. The metabolism of maltodextrin has been investigated in Escherichia coli [16,17] and in several Grampositive bacteria, including Bacillus subtilis [18,19], Listeria monocytogenes [20] and Streptococcus pyogenes [21,22]. The maltose/maltodextrin regulon in E. coli consists of ten genes encoding four glycoside hydrolases, a maltodextrin phosphorylase, a maltodextrin glucosidase, a periplasmic amylase, together with an ATP-binding cassette (ABC) transporter [16,17]. In B. subtilis, maltose and maltodextrin are separately transported by a maltose-specific phosphotransferase system and a maltodextrin-specific ABC transporter, respectively [18], while in L. monocytogenes both maltose and maltodextrin are taken up by the same ABC transporter [20]. In this study, we identified the determinants of maltodextrin uptake and metabolism in E. faecium. Materials and Methods Bacterial strains, plasmids and growth conditions E. faecium strains, E. coli strains and plasmids used or generated in this study are listed in Table 1. The E. faecium strain E1162 (with sequence type 17) was used throughout this study. This strain was isolated from a bloodstream infection in France in 1996 and its genome has previously been sequenced [23]. Unless otherwise mentioned, E. faecium was grown in brain heart infusion broth (BHI; Oxoid) at 37 C. The E. coli strains DH5 (Invitrogen) and EC1000 [24] were grown in Luria-Bertani medium. Where necessary, antibiotics were used at the following concentrations: gentamicin at 300 g ml1 for E. faecium and 25 g ml1 for E. coli, spectinomycin at 300 g ml1 for E. faecium and 100 g ml 1 for E. coli. All antibiotics were obtained from Sigma-Aldrich (Saint Louis, MO). Growth of cultures was determined by measuring the optical density at 660 nm (OD660). Construction of deletion mutants and in trans complementation Markerless gene deletion mutants in the mdxR gene (locustag: EfmE1162_2133) and the mdxABCD-pulA gene cluster (locustag: EfmE1162_0366 - EfmE1162_0370) were created via the Cre-lox recombination system as previously described [25,26]. Briefly, the 5 and 3 flanking regions (approximately 500 bp each) of the target genes were PCR amplified with the primers in Table 2. The two flanking regions were then fused together by fusion PCR (generating an EcoRI site between both fragments) and cloned into pWS3 [9], resulting in pDEL1a and pDEL2a. Then a gentamicinresistance cassette which was flanked by lox66- and lox71sites [26] was cloned into the EcoRI site that was generated between the 5 and 3 flanking regions in pDEL1a and pDEL2a, respectively. The resulting plasmids pDEL1b and pDEL2b were then electrotransformed into E. faecium E1162. Marked mutants were obtained by growing the gentamicin-resistant transformants at appropriate temperatures supplemented with appropriate antibiotics [26]. The plasmid pWS3-Cre [26], carrying a gene encoding Cre recombinase, was introduced into the marked mutant by electroporation and further culturing for the removal of the gentamicin resistance cassette and subsequent loss of pWS-Cre was performed as previously described [26]. Excision of the gentamicin resistance cassette and loss of pWS3-Cre was verified by PCR using primers listed in Table 2. An in trans complementated strain (mdxR+mdxR) of the mdxR deletion mutant (mdxR) was generated as previously described [26,27]. The gene (...truncated)