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1 Department of Viticulture and Enology, University of California, Davis, CA 95616, USA
2 California Institute of Food and Agricultural Research, University of California, Davis, CA 95616, USA
Correspondence
David A. Mills
damills{at}ucdavis.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Two supplementary tables and three supplementary figures are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and other platform chemicals. Since hydrolysis of lignocellulose results in a mixture of carbohydrates, including glucose, arabinose, xylose and galactose, LAB selected to carry out this fermentation need to possess broad substrate utilization ability, tolerate a range of stresses (from end-products or inhibitors), and minimally produce by-products, among other desired traits (Galbe & Zacchi, 2002; Ingram et al., 1999). One common problem in the fermentation of lignocellulosic biomass is the inefficient use of all the sugars present (Hofvendahl & Hahn-Haegerdal, 2000). In many micro-organisms, fermentation of mixed carbohydrates is achieved sequentially, whereby the utilization of glucose, commonly the preferred carbon and energy source, represses consumption of alternative sugars. This hierarchical control, termed carbon catabolite repression (CCR), is accomplished by a complex regulatory mechanism and is a common phenomenon in LAB (Deutscher et al., 2006; Deutscher, 2008; Görke & Stülke, 2008; Saier, 1998; Stülke & Hillen, 1999; Titgemeyer & Hillen, 2002). A heat-stable phosphocarrier protein (Hpr), an Hpr kinase and a carbon catabolite protein A (CcpA) all play a role in CCR in Gram-positive bacteria. A histidine-phosphorylated Hpr (P-His-Hpr), which transfers a phosphate group onto incoming sugars, inhibits phosphoenolpyruvate : sugar phosphotransfer systems (PTS) of other carbohydrates and prevents translocation of alternative sugars into the cell (termed ‘inducer exclusion’). In addition, glycolytic intermediates such as fructose 1,6-bisphosphate and/or 6-phosphogluconate activate the Hpr kinase to phosphorylate a serine residue of Hpr. Coupled with CcpA, serine-phosphorylated Hpr represses the expression of genes involved in the catabolism of alternative sugars by binding to upstream catabolic repression element (cre) sites.
While LAB are well known to consume citrate with glucose, lactose or maltose (Kennes et al., 1991; Ramos et al., 1994), simultaneous carbohydrate utilization has been demonstrated in few species. Maltose and fructose co-fermentation has been reported in Lactobacillus brevis subsp. lindneri CB1 (Gobbetti & Corsetti, 1996).
L. brevis is commonly found on plant materials, although it has been also isolated from various other niches including beverages and animal intestinal tracts (Salminen & Wright, 1993). This species is obligately heterofermentative and employs the phosphoketolase pathway but also possesses inducible glycolytic enzymes (Saier et al., 1996). L. brevis has been shown to transport glucose, lactose, xylose and galactose via proton symport systems (Chaillou et al., 1998; Djordjevic et al., 2001; Ye et al., 1994a, b). The regulation of these proton symport systems is mediated through HPr, in which the transport mechanism is reversibly switched between proton symport and facilitated diffusion (Ye & Saier, 1995a, b; Ye et al., 1996). Previous characterization of the L. brevis xylose operon (xyl) indicated a potential upstream cre site, suggesting regulation by CCR (Chaillou et al., 1998). Indeed, when cloned and examined in Lactobacillus plantarum, the L. brevis xyl operon exhibited a decreased expression pattern in cells grown on glucose (Chaillou et al., 1998).
In this work we demonstrate an unusual phenotype of L. brevis, which unlike most LAB possesses a relaxed control of carbohydrate consumption. Numerous independent isolates of L. brevis exhibit simultaneous utilization of xylose and glucose. In fact, any carbohydrate that could be consumed by L. brevis as a sole carbon source could also be simultaneously consumed with glucose. Further mRNA and proteomic analyses indicate that the xyl operon is induced when both glucose and xylose are present in the medium.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Nucleic acid isolation.
Chromosomal DNA of L. brevis was isolated by using the Qiagen DNeasy kit according to the manufacturers' instructions. For the RNA extraction, L. brevis was cultivated in MRS medium containing 20 g glucose l–1, 20 g xylose l–1, or a 20 g l–1 glucose/xylose mixture (10 g l–1 each) and cells were collected at mid-exponential phase. Collected cells were immediately suspended in RNAlater (Ambion). Total RNA was extracted with the Qiagen RNeasy kit according to the manufacturer's instructions, with additional treatments with RNase-free DNase (Ambion).
RT-PCR analysis.
Reverse transcriptase (RT) reactions were performed by a commercially available reverse transcription system (Promega) with the recommended protocol. RT reactions for xylT and ccpA were performed in a final volume of 20 µl, which contained 5 mM MgCl2, RT buffer (10 mM Tris/HCl pH 9.0, 50 mM KCl, 0.1 % Triton X-100), 1 mM (each) deoxynucleoside triphosphates, 1 U recombinant RNasin RNase inhibitor, 15 U avian myeloblastosis virus (AMV) RT, 2 µg substrate RNA and 0.5 µM of the reverse primers, xylT-bwd primer (5'-TTTGGTCGTGCTTAGTTCAGCAGCATC-3') and ccpA-b-2 (5'- AAAACGACCACGGTGGGCGTAATCATCCCT-3'), respectively. After denaturation of RNA at 70 °C for 15 min, reaction mixtures were added and incubated at 42 °C for 15 min. Reactions were terminated by heating the mixtures at 95 °C for 5 min, followed by incubation on ice for 5 min. The cDNA products were PCR amplified in 50 µl mixtures containing 5 µl of the RT reaction mixture as the template. For the amplification of cDNA the primers xylT-fwd (5'-ATGCGAAAAGTTTCGACCGGATTTGTT-3') and ccpA-f-2 (5' CACCGCACCAATATCGTACAGTGGTTGTGT-3') were paired with the xylT-bwd and ccpA-b-2 primer, respectively.
Sample preparation and protein digestion for shotgun proteomics.
A 10 ml sample of L. brevis culture was taken at the exponential phase of growth. The amounts of initial cell mass were normalized to an OD600 of 1.0 by dilution or concentration to a final volume of 25 ml. After centrifugation, cell pellets were washed three times with PBS then resuspended in 1 ml lysis buffer containing 100 mM Tris and 8.0 M urea. The initial pH of lysis buffer was 9.0. With addition of 300 mg silica beads (Sigma-Aldrich), cells were disrupted using a bead beater (FastPrep; QBiogen) for six 30 s pulses each with a 30 s interval on ice. Beads and cell debris were removed by centrifugation and the soluble fraction was kept at –80 °C for further analysis. Protein concentration was measured by the Bio-Rad protein assay kit.
For protein reduction, 4 µl 450 mM DTT (Sigma-Aldrich) was added to 25 µl supernatant containing 100 µg protein and incubated for 45 min at 55 °C. Without alkylation, reduced protein was digested by 2.5 µg mass-spectrometry-grade trypsin (Promega) overnight at 37 °C. The tryptic peptides were purified by C18 Ziptip (Millipore) according to the manufacturer's recommendation. The Ziptip was prepared by washing with 50 % acetonitrile/H2O followed by 0.1 % (v/v) trifluoroacetic acid (TFA) in H2O. The tryptic peptide solution was then loaded onto the Ziptip and washed with 0.1 % (v/v) TFA in H2O. The peptides were eluted with 50 % acetonitrile in H2O. The purified sample was dried prior to mass spectrometry analysis.
Protein identification by mass spectrometry.
The digested samples were submitted to the Genome Center Proteomics Core at the University of California, Davis. Protein identification was performed using an Eksigent Nano LC 2-D system coupled to an LTQ ion-trap mass spectrometer (Thermo-Fisher) through a Picoview nano-spray source. Peptides were loaded on to an Agilent nanotrap (Zorbax 300SB-C18, Agilent Technologies) at a loading flow rate of 5.0 µl min–1. Peptides were then eluted from the trap and separated by a nano-scale 75 µmx15 cm New Objectives picofrit column packed in house with Michrom Magic C18 AQ packing material. Peptides were eluted using a 90 min gradient of 2–80 % buffer B (buffer A=0.1 % formic acid, buffer B=95 % acetonitrile/0.1 % formic acid). The top 10 ions in each survey scan were subjected to automatic low-energy CID.
Database searching.
Tandem mass spectra were extracted and the charge states deconvoluted by BioWorks version 3.3. Deisotoping was not performed. All MS/MS samples were analysed using X! Tandem (http://www.thegpm.org; version 2006.04.01.2). X! Tandem was set up to search against the L. brevis whole proteome. X! Tandem was searched with a fragment ion mass tolerance of 0.60 Da. Oxidation of methionine was specified as a variable modification in X! Tandem.
HPLC analysis.
Substrate and end-product concentrations were analysed by a Shimadzu HPLC system using a Bio-Rad HPX-87H column. A 1 ml sample of fermentation broth was centrifuged at 10 000 g for 10 min and the supernatant was transferred to a new microcentrifuge tube prior to analysis. For HPLC analysis of the supernatant, the Bio-Rad HPX-87H column was heated at 65 °C and a reflective index (RI) detector was used for identification of substrate(s) and product(s). As a mobile phase, 0.005 M H2SO4 was used and the flow rate was 0.6 ml min–1. The standard deviations of the HPLC concentration determinations were within ±5 mM.
To determine cell density the cell pellet was resuspended in the same volume of deionized water and the optical density (OD600) was measured using a Beckman DU 7400 spectrophotometer.
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RESULTS |
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, simultaneous utilization of glucose and xylose was observed. During 22 h of fermentation, 11.1 g l–1 of glucose and 10.5 g l–1 of xylose were consumed at the same time, and 12.5 g l–1, 4.5 g l–1 and 0.7 g l–1 of lactate, acetate and ethanol, respectively, were produced. No other significant by-products were observed by HPLC. The product yields of lactate (YL/(G+X)), acetate (YA/(G+X)) and ethanol (YE/(G+X)) from glucose and xylose were 1.01, 0.57, and 0.12, respectively, indicating that both glucose and xylose were metabolized through the heterofermentative pathway simultaneously. While CCR is a common phenomenon in LAB, derepressed strains have been developed via mutagenesis (Chaillou et al., 2001). To determine if the simultaneous utilization pattern witnessed with L. brevis ATCC 14869 was a characteristic of the species and not a trait specific to this strain, three more independent L. brevis strains were examined (NRRL 1836, NRRL 1834 and IFO3960). As shown in Fig. 1(b–d), all three strains possessed the ability to simultaneously catabolize glucose and xylose through the heterofermentative pathway, although each strain exhibited different carbohydrate consumption rates. The specific cell growth rate for each strain was similar (0.31–0.34 h–1). The product yields from glucose and xylose are summarized in Fig. 1(e). In contrast, L. pentosus ATCC 8047 examined under the same conditions (Fig. 1f) exhibited a typical sequential carbohydrate utilization pattern indicative of overt CCR by glucose.
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We then examined simultaneous carbohydrate utilization of glucose and a second carbohydrate. Monitoring substrate concentration every 6 h, it was confirmed that all sugars that could be fermented by L. brevis ATCC 14869 as a sole carbon source (Table S1) were consumed simultaneously with glucose (Fig. 2). The utilization pattern of the second carbohydrate was the same as when it was the sole carbon utilized; however, the end-product profiles were different. As expected, fermentation of pentoses or fructose as a sole carbon source resulted in the production of acetate without ethanol (Supplementary Table S2), probably due to the lesser requirement for cofactor regeneration during fermentation of those carbohydrates (Veiga da Cunha & Foster, 1992). However, a significant amount of ethanol was produced in the co-fermentation of those carbohydrates with glucose (Fig. 2a–d). Consistent with their lack of utilization as a sole carbon source, lactose, sucrose and cellobiose were not consumed in a mixture with glucose (data not shown). The co-utilization profiles of mixed sugars by L. brevis ATCC 14869 and the product yields (mM/mM) from the mixed sugars are shown in Fig. 2. Other L. brevis strains exhibited similar co-consumption patterns (Supplementary Figs S1–S3). Interestingly, some strains consumed the pentose sugars present faster than glucose; however, consumption was still simultaneous. This appears to be a strain-specific phenomenon (Figs S1–S3).
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; Zybailov et al., 2005) was then used to determine the relative expression of proteins from cells grown on xylose or on the glucose/xylose mixture compared to that observed in cells grown in glucose. As shown in Table 1(a, b), the enzymes involved in the heterofermentative pathway and encoded by the xyl operon were identified. Among eight proteins annotated as phosphoglycerate mutase (pgm) in the L. brevis genome, only one (gi:116334603) of them was expressed. Only one (gi:116333957) of four putative ldh genes was highly expressed in all conditions. Of three putative acetate kinase genes (ack) in the L. brevis genome, one (gi:116333800) was expressed in cells grown only on glucose or xylose but not on the glucose/xylose mixture. Among ten genes putatively assigned as alcohol dehydrogenase, the expression of two genes (gi:116332794 and gi:116333656) was identified in cells cultured in glucose and glucose/xylose mixture. The expression of these latter two enzymes was not detected in cells grown on xylose as a single carbon source. There are two 6-phosphogluconate dehydrogenase genes (6pgdh) in the L. brevis genome. Of these, one (gi:116332769) was expressed constitutively while the other (gi:116332818) was detected only when glucose was present in the medium.
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Expression of the xyl operon and ccpA in the presence of xylose and/or glucose
The apparent relaxed control of sugar utilization in L. brevis suggests that xylose utilization genes are expressed when xylose is available regardless of the presence or absence of glucose. As shown in Table 1(b)
, XylA (xylose isomerase) and XylB (xylulokinase) proteins were observed when L. brevis was cultivated on xylose but not when glucose was the sole carbon source in the medium. Expression of these two proteins was observed in cells grown on the glucose/xylose mixture; however, the expression level of both XylA and XylB decreased by 2.8-fold compared to that seen when xylose was used as a sole carbon source. This suggests that the presence of glucose suppresses, but does not completely inhibit, the expression of xylA and xylB.
CcpA is known to bind upstream of, and regulate expression of, the xylose operon in L. pentosus (Chaillou et al., 2001; Mahr et al., 2000; Posthuma et al., 2002). In L. brevis, CcpA was expressed consistently regardless of the carbon source; however, the expression level increased fourfold when xylose was used as a single carbon source. Expression of ccpA was also verified by RT-PCR, with a 700 bp ccpA amplicon observed from L. brevis cultured in glucose, xylose or the glucose/xylose mixture (Fig. 3a, b).
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). This suggests that xylT expression is induced by xylose, as previously proposed (Chaillou et al., 1998), and the presence of glucose does not fully inhibit its expression. Thus, the expression of xylose utilization genes in the presence of mixed sugars correlates with the observed co-utilization phenotype. |
DISCUSSION |
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; Stülke & Hillen, 1999). Relatively few bacteria have been reported to consume different sugars simultaneously. Clostridium thermohydrosulfuricum has been reported to co-metabolize mixtures of glucose and xylose as well as combinations of cellobiose, xylose and xylobiose (Cook et al., 1993; Slaff & Humphrey, 1986). Simultaneous utilization of glucose with xylose or sucrose has been observed in yeast (Kastner & Roberts, 1990; Kastner et al., 1998) and thermophilic fungi (Maheshwari & Balasubramanyam, 1988); however, it remains to be determined if this phenotype is a common trait of the species or a particular trait of the strain. Fibrolytic ruminal bacteria are able to degrade the hemicellulose of plant materials and utilize pentose sugars with glucose. Butyrivibrio fibrisolvens was reported to simultaneously consume glucose and xylose, although this effect was strain specific (Marounek & Kopecny, 1994; Strobel & Dawson, 1993). Ruminococcus albus consumed xylose and glucose simultaneously but the dominant repressor was cellobiose (Thurston et al., 1994).
The results presented here suggest that L. brevis does not possess a rigorous hierarchical control of carbohydrate utilization and can metabolize a range of fermentable carbon sources simultaneously with glucose. Simultaneous fermentation of mixed carbohydrates by L. brevis exhibited utilization rates and fermentation behaviours similar to those seen when a sole carbon source was fermented although, as expected, different end-products were observed with some sugars. The mass balance between substrates (mixed sugars) and products indicates that the xylose was metabolized via the heterofermentative pathway. Relaxed CCR was observed in several different L. brevis isolates, including the type strain, suggesting that this is a common trait for the species. In contrast, other facultatively heterofermentative LAB exhibited the classical biphasic carbohydrate consumption expected where CCR is dominant (Fig. 1f).
CCR has been previously observed in various lactobacilli including L. pentosus, L. casei, L. delbrueckii, L. plantarum and L. sakei (Mahr et al., 2000; Marasco et al., 1998; Morel et al., 1999; Schick et al., 1999; Veyrat et al., 1994; Viana et al., 2000; Zuniga et al., 1998). Such control is achieved by two independent mechanisms: inducer exclusion and genetic repression by the catabolite control protein (CcpA) and repressor protein. As shown in Fig. 3, xylT expression requires the presence of xylose. In the standard model of CCR in LAB the primary sugar, often glucose, triggers the inhibition of the inducer uptake (inducer exclusion), mediated through the action of a serine-phosphorylated Hpr on secondary sugar transporters (Saier, 1998; Ye & Saier, 1995a; Ye et al., 1996). Our results (Fig. 1a) indicate that in L. brevis, xylose can readily enter the cell even in the presence of glucose, suggesting that inducer exclusion is relaxed in this species. The carbohydrate transport systems identified in L. brevis are proton symport systems (Chaillou et al., 1998; Djordjevic et al., 2001; Ye et al., 1994a, b). Through detailed biochemical studies, L. brevis has been shown to exhibit PTS-mediated control of non-PTS permease activities for glucose, lactose and galactose (Chaillou et al., 1998; Djordjevic et al., 2001; Ye & Saier, 1995a, b). For example, when bound by a serine-phosphorylated Hpr, the L. brevis galactose : H+ symporter changes from an active symporter to a facilitated diffusion uniporter (bidirectional), prohibiting uptake of galactose against a concentration gradient (inducer exclusion) and enabling passive expulsion of galactose (inducer expulsion) (Djordjevic et al., 2001). In spite of this biochemical evidence, our results clearly suggest that secondary sugars, such as galactose, simultaneously enter, and are metabolized by, L. brevis cells in the presence of glucose. An explanation may be that galactose is not excluded but is instead metabolized, resulting in little internal buildup of galactose to drive expulsion, as would be seen with a non-metabolizable galactose analogue. While it is not known if XylT, a xylose : H+ symporter (Chaillou et al., 1998), operates in a similar fashion, it is clear from expression of the xylose operon seen in Fig. 3(c) that enough xylose enters the cell to derepress the xylT operon.
CCR is also mediated by a global transcriptional regulator, the catabolite control protein, CcpA. In other LAB, CcpA-based regulation occurs by binding of cre sites upstream of, or within, target genes (Mahr et al., 2000; Marasco et al., 1998; Saier, 1998; Schick et al., 1999; Stülke & Hillen, 1999). Two cre sites have been identified in the xyl operon, suggesting possible regulation by CcpA (Chaillou et al., 1998); however, the simultaneous utilization of xylose and glucose, along with induction of the xyl operon, belies any rigorous repression by CcpA.
Proteomic analysis clearly showed that CcpA was expressed regardless of the carbon source in the medium. However, expression of CcpA in xylose-grown cells was 4.7-fold higher than in cells grown on glucose or glucose/xylose mixture. Interestingly, there is a putative cre site upstream of ccpA, suggesting possible autoregulation of the ccpA operon by CcpA (Fig. 4).
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). A similar result was reported previously in which the activities of xylose isomerase and xylulokinase were reduced fourfold in L. brevis grown on a glucose/xylose mixture (Chaillou et al., 1998). The same authors did not detect these activities in cells grown on glucose alone, a result that parallels the lack of detectable xyl operon mRNA or proteins shown here (Fig. 3c, Table 1b). Our results clearly demonstrate that L. brevis can co-metabolize xylose, or other fermentable carbon sources, simultaneously with the glucose. Given its obligately heterofermentative mode of fermentation one might postulate that L. brevis has evolved a relaxed hierarchical control of glucose or xylose utilization because both carbohydrates are present in the environmental niches where this bacterium predominates, and both feed into the same phosphoketolase pathway. While the molecular underpinnings of this relaxed control of mixed carbohydrate utilization have yet to be elucidated, the phenotype itself is attractive for fermentation of biomass-derived substrates that possess mixtures of five and six carbon sugars. Simultaneous utilization of mixed carbohydrates is desired in order to completely ferment all available sugars, thereby maximizing potential yields and purification efficiency. Additional studies have shown that L. brevis is quite tolerant of inhibitors commonly derived from lignocellulosic biomass (data not shown). The ability to readily consume mixed sugars, combined with the available genomic sequence, suggests that L. brevis is an attractive candidate for future metabolic engineering of various bio-products.
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ACKNOWLEDGEMENTS |
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Edited by: D. A. Mills
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 4 October 2008;
revised 4 December 2008;
accepted 22 December 2008.
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, OD600; 
, glucose; 
, xylose; 
, lactic acid; 
, acetic acid; 
, ethanol. The product yields (mM/mM) based on the mixed sugar are summarized in (e): black, light grey and dark grey bars indicate the yields of lactate, acetate and ethanol, respectively. Fermentations were done in duplicate and a representative dataset is shown. The standard deviation for each chemical measured by HPLC was <0.5 mM.
, arabinose; 
, ribose; 
, fructose; 
, galactose; 
