细菌糖转运系统

Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus Rodolphe Barrangou*, Eric Altermann*, Robert Hutkins†, Raul Cano‡, and Todd R. Klaenhammer*§ *Genomic Sciences Program and Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, NC 27695; †Department of Food Science and Technology, University of Nebraska, Lincoln, NE 68583-0919; and ‡California Polytechnic State University, Environmental Biotechnology Institute, San Luis Obispo, CA 93407 Contributed by Todd R. Klaenhammer, May 8, 2003 Lactobacillus acidophilus is a probiotic organism that displays the ability to use prebiotic compounds such as fructooligosaccharides (FOS), which stimulate the growth of beneficial commensals in the gastrointestinal tract. However, little is known about the mecha- nisms and genes involved in FOS utilization by Lactobacillus spe- cies. Analysis of the L. acidophilus NCFM genome revealed an msm locus composed of a transcriptional regulator of the LacI family, a four-component ATP-binding cassette (ABC) transport system, a fructosidase, and a sucrose phosphorylase. Transcriptional analysis of this operon demonstrated that gene expression was induced by sucrose and FOS but not by glucose or fructose, suggesting some specificity for nonreadily fermentable sugars. Additionally, expres- sion was repressed by glucose but not by fructose, suggesting catabolite repression via two cre-like sequences identified in the promoter–operator region. Insertional inactivation of the genes encoding the ABC transporter substrate-binding protein and the fructosidase reduced the ability of the mutants to grow on FOS. Comparative analysis of gene architecture within this cluster re- vealed a high degree of synteny with operons in Streptococcus mutans and Streptococcus pneumoniae. However, the association between a fructosidase and an ABC transporter is unusual and may be specific to L. acidophilus. This is a description of a previously undescribed gene locus involved in transport and catabolism of FOS compounds, which can promote competition of beneficial microorganisms in the human gastrointestinal tract. The ability of select intestinal microbes to use substratesnondigested by the host may play an important role in their ability to successfully colonize the mammalian gastrointestinal (GI) tract. A diverse carbohydrate catabolic potential is associ- ated with cariogenic activity of Streptococcus mutans in the oral cavity (1), adaptation of Lactobacillus plantarum to a variety of environmental niches (2), and residence of Bifidobacterium longum in the colon (3), illustrating the competitive benefits of complex sugar utilization. Prebiotics are nondigestible food ingredients that selectively stimulate the growth and�or activity of beneficial microbial strains residing in the host intestine (4). Among sugars that qualify as prebiotics, fructooligosaccharides (FOS) are a diverse family of fructose polymers used commer- cially in food products and nutritional supplements that vary in length and can be either derivatives of simple fructose polymers or fructose moieties attached to a sucrose molecule. The linkage and degree of polymerization can vary widely (usually between 2 and 60 moieties), and several names such as inulin, levan, oligofructose, and neosugars are used accordingly. The average daily intake of such compounds, originating mainly from wheat, onion, artichoke, banana, and asparagus (4, 5), is fairly signifi- cant, with �2.6 g of inulin and 2.5 g of oligofructose consumed in the average American diet (5). FOS are not digested in the upper GI tract and can be degraded by a variety of lactic acid bacteria (6–9), residing in the human lower GI tract (4, 10). FOS and other oligosaccharides have been shown in vivo to benefi- cially modulate the composition of the intestinal microbiota and specifically to increase bifidobacteria and lactobacilli (4, 10, 11). A variety of Lactobacillus acidophilus strains in particular have been shown to use several polysaccharides and oligo- saccharides such as arabinogalactan, arabinoxylan, and FOS (6, 9). Despite the recent interest in FOS utilization, little information is available about the metabolic pathways and enzymes responsible for transport and catabolism of such com- plex sugars in lactobacilli. In silico analysis of a particular locus within the L. acidophilus North Carolina Food Microbiology (NCFM) genome revealed the presence of a gene cluster encoding proteins potentially involved in prebiotic transport and hydrolysis. This specific cluster was analyzed computationally and functionally to reveal the genetic basis for FOS transport and catabolism by L. acidophilus NCFM. Materials and Methods Bacterial Strain and Media Used in This Study.The strain used in this study is L. acidophilus NCFM (12). Cultures were propagated at 37°C, aerobically in deMan, Rogosa, Sharpe broth (Difco). A semisynthetic medium consisted of: 1% bactopeptone (wt�vol) (Difco), 0.5% yeast extract (wt�vol) (Difco), 0.2% dipotassium phosphate (wt�vol) (Fisher), 0.5% sodium acetate (wt�vol) (Fisher), 0.2% ammonium citrate (wt�vol) (Sigma), 0.02% mag- nesium sulfate (wt�vol) (Fisher), 0.005% manganese sulfate (wt�vol) (Fisher), 0.1% Tween 80 (vol�vol) (Sigma), 0.003% bromocresol purple (vol�vol) (Fisher), and 1% sugar (wt�vol). The carbohydrates added were either glucose (dextrose) (Sig- ma), fructose (Sigma), sucrose (Sigma), or FOS. Two types of complex sugars were used as FOS: a GFn mix (manufactured by R. Hutkins, University of Nebraska), consisting of glucose monomers linked �-1,2 to two, three, or four fructosyl moieties linked �-2,1, to form kestose (GF2), nystose (GF3), and fructo- furanosyl-nystose (GF4), respectively; and an Fn mix, Raftilose, derived from inulin hydrolysis (Orafti). Without carbohydrate supplementation, the semisynthetic medium was unable to sus- tain bacterial growth above OD600 nm � 0.2. Computational Analysis of the Putative Multiple Sugar Metabolism (msm) Operon. A 10-kbp DNA locus containing a putative msm operon was identified from the L. acidophilus NCFM genome sequence. ORF predictions were carried out by four computa- tional programs: GLIMMER (13, 14), CLONE MANAGER (Scientific and Educational Software, Durham, NC), the National Center for Biotechnology Information ORF finder (www.ncbi.nlm.nih. gov�gorf�gorf.html), and GENOMAX (InforMax, Frederick, Abbreviations: ABC, ATP-binding cassette; cre, catabolite response element; FOS, fructoo- ligosaccharides; MSM, multiple sugar metabolism; PTS, phosphotransferase system; LGT, lateral gene transfer; GI, gastrointestinal; NCFM, North Carolina Food Microbiology. Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AY172019, AY172020, and AY177419). §To whom correspondence should be addressed. E-mail: klaenhammer@ncsu.edu. www.pnas.org�cgi�doi�10.1073�pnas.1332765100 PNAS � July 22, 2003 � vol. 100 � no. 15 � 8957–8962 M IC RO BI O LO G Y MD). GLIMMER was previously trained with a set of L. acidophi- lus genes available in public databases. The predictedORFs were translated into putative proteins that were submitted to BLASTP analysis (15). RNA Isolation and Analysis. Total RNA was isolated by using TRIzol (GIBCO�BRL), following the supplier’s instructions. Cells in the exponential phase were harvested by centrifugation (2 min, 15,800 � g) and cooled on ice. Pellets were resuspended in TRIzol by vortexing and underwent five cycles of 1-min bead beating and 1 min on ice. Nucleic acids were subsequently purified by using three chloroform extractions and precipitated by using isopropanol and centrifugation for 10 min at 11,600 � g. The RNA pellet was washed with 70% ethanol and resus- pended into diethyl pyrocarbonate-treated water. RNA samples were treated with DNAse I according to the supplier’s instruc- tions (Boehringer Mannheim). First-strand cDNA was synthe- sized by using the Invitrogen RT-PCR kit according to the supplier’s instructions. cDNA products were subsequently am- plified by using PCR with primers internal to genes of interest. For RNA slot blots, RNA samples were transferred to nitrocel- lulose membranes (Bio-Rad) using a slot-blot apparatus (Bio- Dot SF, Bio-Rad), and the RNAs were UV crosslinked to the membranes. Blots were probed with DNA fragments generated by PCR that had been purified from agarose gels (GeneClean III kit, Midwest Scientific, St. Louis). Probes were labeled with �-32P with the Amersham Pharmacia Multiprime Kit and con- sisted of 700- and 750-bp fragments internal to the msmE and bfrA genes, respectively. Hybridization and washes were carried out according to the supplier’s instructions (Bio-Dot Microfil- tration Apparatus, Bio-Rad), and radioactive signals were de- tected by using a Kodak Biomax film. Primers are listed in Table 2, which is published as supporting information on the PNAS web site, www.pnas.org. Comparative Genomic Analysis. A gene cluster bearing a fructosi- dase gene was selected after computational data-mining of the L. acidophilus NCFM genome. Additionally, microbial clusters containing fructosidase EC 3.2.1.26 orthologs or bearing an ATP-binding cassette (ABC) transport system associated with an �-galactosidase EC 3.2.1.22 were selected from public data- bases (National Center for Biotechnology Information, The Institute for Genomic Research). The sucrose operon is a widely distributed cluster consisting of either three or four elements, namely: a regulator, a sucrose phosphotransferase (PTS) trans- porter, a sucrose hydrolase, and occasionally a fructokinase. Two gene cluster alignments were generated: (i) a PTS alignment representing similarities over the sucrose operon, bearing a PTS transport system associated with a sucrose hydrolase; and (ii) an ABC alignment representing similarities over the multiple sugar metabolism cluster, bearing an ABC transport system usually associated with a galactosidase. Sequence information is avail- able in Table 3, which is published as supporting information on the PNAS web site. Phylogenetic Trees. Nucleotide and protein sequences were aligned computationally by using the CLUSTALW algorithm (16). The multiple alignment outputs were used for generating un- rooted neighbor-joining phylogenetic trees by using MEGA2 (17). In addition to a phylogenetic tree derived from 16S rRNA genes, trees were generated for ABC transporters, PTS transporters, transcription regulators, fructosidases, and fructokinases. Gene Inactivation. Gene inactivation was conducted by site- specific plasmid integration into the L. acidophilus chromosome via homologous recombination (18). Internal fragments of the msmE and bfrA genes were cloned into pORI28 by using Escherichia coli as a host (19), and the constructs were subse- quently purified and transformed into L. acidophilus NCFM. The ability of the mutant strains to grow on a variety of carbohydrate substrates was investigated by using growth curves. Strains were grown on semisynthetic medium supplemented with 0.5% wt�vol carbohydrate. Results Computational Analysis of the msm Operon. Analysis of the msm locus using four ORF-calling programs revealed the presence of seven putative ORFs. Because most of the encoded proteins were homologous to those of the msm operon present in S. mutans (20), a similar gene nomenclature was used. The analysis of the predicted ORFs suggested the presence of a transcrip- tional regulator of the LacI repressor family, MsmR; a four- component transport system of the ABC family, MsmEFGK; and two enzymes involved in carbohydrate metabolism, namely a fructosidase EC 3.2.1.26, BfrA; and a sucrose phosphorylase EC 2.4.1.7, GtfA. A putative Shine–Dalgarno sequence 5�AG- GAGG3� was found within 10 bp upstream of the msmE start codon. A dyad symmetry analysis revealed the presence of two stem–loop structures that could act as putative Rho-independent transcriptional terminators: one between msmK and gtfA (be- tween base pairs 6,986 and 7,014), free energy�13.6 kcal�mol�1, and one 20 bp downstream of the last gene of the putative operon (between base pairs 8,500 and 8,538), free energy �16.5 kcal�mol�1. The operon structure is shown in Fig. 1. The regulator contained two distinct domains: a DNA-binding domain at the N terminus with a predicted helix-turn-helix motif (pfam00354), and a sugar-binding domain at the C terminus (pfam00532). The transporter elements consisted of a periplas- mic solute-binding protein (pfam01547), two membrane- spanning permeases (pfam00528), and a cytoplasmic nucleotide- binding protein (pfam 00005), characteristic of the different subunits of a typical ABC transport system (21). A putative anchoring motif LSLTG was present at the N terminus of the substrate-binding protein. Each permease contained five trans- membrane regions predicted computationally (22). Analyses of ABC transporters in recently sequencedmicrobial genomes have defined four characteristic sequence motifs (23, 24). The pre- dicted MsmK protein included all four ABC conserved motifs, namely: Walker A: GPSGCGKST (consensus GxxGxGKST or [AG]xxxxGK[ST]); Walker B: IFLMDEPLSNLD (consensus hhhhDEPT or DExxxxxD); ABC signature sequence: LSGG; and Linton and Higgins motif: IAKLHQ (consensus hhhhH�, with h, hydrophobic and �, charged residues). The putative fructosidase showed high similarity to glycosyl hydrolases (pfam 00251). The putative sucrose phosphorylase shared 63% residue identity with that of S. mutans. Sugar Induction and Coexpression of Contiguous Genes. Transcrip- tional analysis of the msm operon by using RT-PCR and RNA slot blots showed that sucrose and both types of oligofructose (GFn and Fn) were able to induce expression of msmE and bfrA (Fig. 2A). In contrast, glucose and fructose did not induce Fig. 1. Operon layout. The start and stop codons are shaded, the putative ribosome binding site is boxed, and the cre-like elements are underlined. Terminators are indicated by hairpin structures. 8958 � www.pnas.org�cgi�doi�10.1073�pnas.1332765100 Barrangou et al. transcription of those genes, suggesting specificity for nonreadily fermentable sugars and the presence of a regulation system based on carbohydrate availability. In the presence of both FOS and readily fermentable sugars, glucose repressed expression of msmE, even if present at a lower concentration, whereas fructose did not (Fig. 2B). Analysis of the transcripts induced by oligo- fructose indicated that all genes within the operon are coex- pressed (Fig. 6, which is published as supporting information on the PNAS web site) in a manner consistent with the S. mutans msm operon (25). Mutant Phenotype Analysis. The ability of the bfrA (fructosidase) andmsmE (ABC transporter) mutant strains to grow on a variety of carbohydrates was monitored by both optical density at 600 nm and colony-forming units. The mutants retained the ability to grow on glucose, fructose, sucrose, galactose, lactose, and FOS-GFn, in a manner similar to that of the control strain (Fig. 7, which is published as supporting information on the PNAS web site), a lacZmutant of the L. acidophilus parental strain also generated by plasmid integration (18). This strain was chosen because it also bears a copy of the plasmid used for gene inactivation integrated in the genome. In contrast, both the bfrA and msmE mutants halted growth on FOS-Fn prematurely (Fig. 3), likely on exhaustion of simple carbohydrate from the semi- synthetic medium. After one passage, the msmE mutant dis- played slower growth on FOS-Fn, whereas the bfrAmutant could not grow (Fig. 3). Additionally, terminal cell counts from overnight cultures grown on FOS-Fn were significantly lower for the mutants, especially after one passage (Fig. 7). Comparative Genomic Analyses and Locus Alignments. Comparative genomic analysis of gene architecture between L. acidophilus, S. mutans, Streptococcus pneumoniae, Bacillus subtilis, and Bacillus halodurans revealed a high degree of synteny within the msm cluster, except for the core sugar hydrolase (Fig. 4A). In contrast, gene content was consistent, whereas gene order was not well conserved for the sucrose operon (Fig. 4B). The lactic acid bacteria exhibit a divergent sucrose operon, where the regulator and hydrolase are transcribed opposite the transporter and the fructokinase. In contrast, gene architecture was variable among the proteobacteria. Phylogenetic Trees. Phylogenetic trees were generated to investi- gate whether there was a correlation between protein similarity, gene architecture, and the phylogenic relationships of the se- lected microorganisms. The phylogenetic relationships were obtained from 16S ribosomal DNA alignment. All proteobac- teria appeared distant from the lactic acid bacteria, and the Clostridium species formed a well defined cluster between Thermotoga maritima and the bacillales (Fig. 5A). For the fructosidases, all enzymes obtained from the LAB sucrose operons clustered extremely well together at the left end of the tree, whereas there was apparent shuffling of the other three groups (Fig. 5B). The paralogs of those fructosidases in S. mutans, S. pneumoniae, and L. acidophilus clustered at the opposite end of the tree. Interestingly, the L. acidophilus fruc- tosidase was distant from the LAB sucrose hydrolases cluster and showed strong homology to enzymes experimentally asso- ciated with oligosaccharide hydrolysis, in organisms such as T. maritima, Microbacterium laevaniformans, and B. subtilis. Each component of the ABC transport system clustered together (Fig. 5C), namelyMsmE,MsmF,MsmG, andMsmK for substrate-binding membrane-spanning proteins and nucleotide- binding unit, respectively. For MsmE, MsmF, and MsmG, three consistent subclusters were obtained: (i) the two Bacillus species; (ii) L. acidophilus, S. mutans, and S. pneumoniae from the Fig. 2. Sugar induction and repression. (A) Transcriptional induction of the msmE and bfrA genes, monitored by RT-PCR (Upper) and RNA slot blots (Lower). Cells were grown on glucose (Glc), fructose (Fru), sucrose (Suc), FOS GFn, and FOS Fn. Chromosomal DNA was used as a positive control for the probe. (B) Transcriptional repression analysis of msmE and bfrA by variable levels of Glc and Fru: 0.1% (5.5 mM), 0.5% (28 mM), and 1.0% (55 mM), in the presence of 1% Fn. Cells were grown in the presence of Fn until OD600 approximated 0.5–0.6, glucose was added, and cells were propagated for an additional 30 min. Fig. 3. Growth curves. The two mutants, bfrA (Upper) and msmE (Lower), were grown on semisynthetic medium supplemented with 0.5% wt�vol car- bohydrate: fructose (F), GFn (E), Fn (�), after one passage on Fn (■). The lacZ mutant grown on Fn was used as control (ƒ). Barrangou et al. PNAS � July 22, 2003 � vol. 100 � no. 15 � 8959 M IC RO BI O LO G Y operons bearing a galactosidase; and (iii) L. acidophilus and S. pneumoniae from the operons bearing a fructosidase. For the PTS transporters, the clustering did not proceed according to phylogeny, especially for lactic acid bacteria, which formed two separate clusters (Fig. 5D). The two distant trans- porters at the bottom of the tree are non-PTS sucrose trans- porters of the major facilitator family of transporters, as sug- gested by their initial annotation. All regulators were repressors, with the exception of those regulators of L. acidophilus, S. pneumoniae, and S. mutans clustering at the bottom of the tree (Fig. 5E), which activate transcription of operons bearing an ABC transport system associated with a galactosidase (20). In contrast, the msm regulators for both S. pneumoniae and L. acidophilus seemed to be repressors similar to that of the sucrose operon (5E). The helix-turn-helix DNA-binding motif of the regulator was very well conserved among selected regulator