Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612, USA1
Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL 32611, USA2
Author for correspondence: Allen L. Honeyman. Tel: +1 813 974 2362. Fax: +1 813 974 4151. e-mail: ahoneyma{at}com1.med.usf.edu
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ABSTRACT |
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Keywords: Streptococcus mutans, ß-glucosides, aesculin, phosphoenolpyruvate-dependent phosphotransferase system
Abbreviations: CTAB, cetyltrimethylammonium bromide; EII, Enzyme II; PTS, phosphoenolpyruvate-dependent phosphotransferase system
The GenBank accession number for the sequence reported in this paper is AF206272.
a Present address: Dental Research Institute, University of Toronto School of Dentistry, Toronto, Ontario, Canada.
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INTRODUCTION |
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ß-Glucoside utilization, facilitated by the PTS, has been best characterized in Escherichia coli (Fox & Wilson, 1968 ; Schaefler, 1967
). The E. coli PTS ß-glucoside operon (the bglGFB operon) contains all of the genetic elements for the uptake and utilization of ß-glucosides, but is cryptic in nature (Schnetz et al., 1987
). A genetic mutation is required for E. coli to utilize ß-glucosides (Schnetz & Rak, 1988
). The majority of the mutations in E. coli leading to the activation of the bgl operon are the result of the transposition of an insertion sequence into the region of the operon that is upstream of bglG (Schnetz & Rak, 1988
). Once activated, the bglGFB operon is subject to regulation via the antiterminator protein BglG. The bglF gene encodes the EII of the PTS, while bglB encodes a phospho-ß-glucosidase (Amster-Choder et al., 1989
; Chen et al., 1997
). Both the BglF and the BglG proteins are components of a sensory system that induces the bgl genes in the presence of ß-glucosides (Chen & Amster-Choder, 1998
).
Erwinia chrysanthemi has a system for the metabolism of ß-glucosides very similar to that encoded by the bglGFB operon of Escherichia coli. The arb genes in Er. chrysanthemi are similar in both structure and function to the bglGFB operon of E. coli, but are not cryptic (el Hassouni et al., 1990 ). In Er. chrysanthemi, the arbF gene encodes a ß-glucoside-specific EII permease and the arbB gene encodes a phospho-ß-glucosidase. In both Er. chrysanthemi and E. coli, the PTS EIIs that are encoded by the arbF and bglF genes, respectively, are specific for the aromatic ß-glucosides arbutin and salicin. While neither of these organisms can utilize aesculin as a sole carbon source, the proteins encoded by the E. coli bglGFB genes and the arbGFB genes in Er. chrysanthemi allow for the hydrolysis of this glucoside (el Hassouni et al., 1990
).
Gram-positive bacteria have been shown to contain ß-glucoside systems similar to those of the Gram-negative bacteria. In Bacillus subtilis, the bglPH operon is responsible for the production of a PTS EII (bglP) and a phospho-ß-glucosidase (bglH) (Kruger et al., 1996 ). In this organism, the synthesis of the BglP protein is induced by the presence of either arbutin or salicin. A second ß-glucoside system has also been identified and characterized in B. subtilis (Tobisch et al., 1997
). This system is responsible for the transport and utilization of cellobiose and lichenan. A ß-glucoside-specific EII is encoded by the licABC operon, and a 6-phospho-ß-glucosidase is encoded by licH (Tobisch et al., 1997
).
Clostridium longisporum, a ruminal Gram-positive bacterium, also relies on the PTS for the uptake of ß-glucosides. C. longisporum utilizes an aryl-ß-glucoside uptake and utilization system that is composed of several abg genes (Brown & Thomson, 1998 ). This includes a PTS EII that is specific for salicin and arbutin, which is encoded by the abgF gene. The phospho-ß-glucosidase in C. longisporum is encoded by the abgA gene.
In both Gram-negative and Gram-positive organisms, the operons associated with ß-glucoside utilization are repressed in the presence of a more readily metabolized carbon source, such as glucose. It has been noted that the regulation of ß-glucoside systems is markedly similar in both Gram-negative and Gram-positive bacteria (Rutberg, 1997 ). One common feature of these PTS-mediated systems is the unusual regulation that is associated with the catabolite repression of these genes. Throughout the ß-glucosidase operons discussed, antitermination is the main regulatory mechanism (Bardowski et al., 1994
; Kruger & Hecker, 1995
; Schnetz & Rak, 1988
; Schnetz et al., 1996
). Thus, the study of the ß-glucoside-specific PTS operon from S. mutans is of importance because of the potential regulatory mechanism for these genes.
The work presented here describes a ß-glucoside-specific PTS regulon isolated from S. mutans. This regulon contains genes encoding a ß-glucoside-specific permease (bglP) and a phospho-ß-glucosidase (bglA). The region of DNA between these genes contains three additional ORFs, one of which is transcribed in the opposite direction. Two of these ORFs are hypothesized to encode functional proteins.
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METHODS |
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Large-scale plasmid DNA preparations were done as described by Sambrook et al. (1989) , but with omission of the PEG precipitation step. Mini-plasmid preparations were performed using QIAprep Spin Miniprep Kits and QIAprep 8 Miniprep Kits (Qiagen). All DNA manipulations and enzyme procedures were done according to the manufacturers instructions and under NIH recombinant DNA guidelines.
Recombinant plasmid constructs were electroporated into E. coli (Dower, 1990 ). Prior to electroporation, ligation mixtures were butanol precipitated. Briefly, 500 µl butanol was added to a 50 µl ligation reaction. The mixture was vortexed briefly and microcentrifuged for 15 min. The butanol was removed and the pellet was washed with 500 µl 100% ethanol. The pellet was air-dried and resuspended in 12 µl water. Three microlitres of the resuspended ligation was used for electroporation. S. mutans was transformed using the method described by Murchison et al. (1986)
. All chemicals and reagents were purchased from Sigma unless otherwise noted.
Isolation of the S. mutans ß-glucoside-specific regulon.
A 780 bp PCR fragment was obtained from the chromosome of S. mutans NG8 using degenerate primers designed for the glutamine synthetase gene. The PCR fragment was cloned and its nucleotide sequence determined. Sequence analysis revealed that this PCR fragment would encode a protein that had similarity with the ß-glucoside-specific PTS EIIs from various bacteria rather than the glutamine synthetase protein. The cloned PCR fragment was then moved into the E. coliS. mutans shuttle vector pVA891 (Macrina et al., 1983 ), resulting in the construction of pALH103. This non-replicating shuttle vector was transformed into S. mutans NG8 and transformants were selected for by erythromycin resistance. One such transformant, ALH96, which contained pALH103 integrated into the chromosome, was used as the source of DNA for the isolation of the bgl regulon.
To isolate the bgl genes surrounding the integration site of pALH103, chromosomal DNA was isolated from S. mutans ALH96 and was digested with various restriction endonucleases that cut outside the origin of replication and the antibiotic-resistance marker of the integrated vector. The restriction digests were phenol/chloroform extracted and the DNA was self-ligated overnight at low DNA concentrations. This resulted in the self-ligation of part of the integrated shuttle vector to the adjacent chromosomal DNA that flanked the original integration site. The ligation mixtures were butanol precipitated prior to electroporation into E. coli. Chloramphenicol resistance was used to select for cells containing plasmids carrying the chromosomal DNA that flanks the integration site. This procedure resulted in a 13·9 kb plasmid when the restriction endonuclease BamHI was used. This construct, pALH104, contained approximately 8 kb of chromosomal DNA located 3' from the original integration site. Repeating the described procedure with the chromosomal DNA isolated from ALH96 using the restriction enzyme XbaI resulted in a 7·5 kb plasmid. This construct, pALH105, contained approximately 2 kb of chromosomal DNA located 5' from the original integration site. The original integration into the chromosome and the subsequent clones that contained flanking DNA are shown in Fig. 1.
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PCR amplification.
S. mutans chromosomal DNA was used as the template in PCR reactions. The polymerase KlenTaq-LA (Clonetech) was used, according to the manufacturers recommendations. The primers used to isolate the native chromosome surrounding the integration site of pALH103 into the bgl genes were Bgl-5' (GTCTCCCATGTTGGTGGC) and Bgl-3' (GGAGCAATTTGCCAACCCC).
Construction of deletion strains and plasmids.
Table 1 and Figs 1
and 2
show the various strains and plasmids used in this report. To create a S. mutans strain containing an insertional mutation of the bglP gene, the E. coliS. mutans shuttle construct pALH103 was integrated into the chromosome. This was accomplished via homologous recombination between the cloned PCR fragment and the chromosome of S. mutans, resulting in strain ALH96. Strain ALH168, containing an insertional mutation of the bglA gene, was created by the insertion of an
-Kan cassette (Perez-Casal et al., 1991
) into an EcoRV site within the cloned bglA gene. The construct containing the desired mutation of bglA was linearized with XbaI and integrated into the chromosome of S. mutans NG8 via a double homologous recombination.
Strain ALH167, containing a nearly complete deletion of the entire regulon was created by cloning the 2·0 kb XbaIBamHI fragment from pALH105 (Fig. 1) and the 1·2 kb PstIEcoRI fragment from pALH104 into the same plasmid vector. This construct, pALH168, has these two restriction fragments in the same orientation as they are found in the native S. mutans chromosome. An
-Kan cassette was inserted into a unique SmaI site located between these two subcloned restriction fragments to create pALH169. This plasmid was linearized within the vector sequence and the deletion integrated into the chromosome by selection for the
-Kan marker. This produced the strain ALH167.
Subcloned fragments from this ß-glucoside-specific regulon were used to create plasmids for expression of these S. mutans genes in E. coli. The bglP deletion construct pALH164 was created by cloning a 5·3 kb EcoRI fragment from the isolated regulon into pACKS30. This fragment was located downstream from the bglP gene, but contains the bglC and bglA genes. The bglA deletion construct pALH165 contains the XhoIEcoRV fragment from the 5' end of the regulon. This region contains the bglP and the bglB genes and deletes all of the bglA gene. The construct pALH163, which contains the entire regulon described here, was generated from several smaller subclones (Fig. 2).
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RESULTS AND DISCUSSION |
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bglP.
The first ORF spans 1935 nucleotides (base-pairs 4202354 in the submitted sequence) and would encode a deduced protein of 645 amino acids. A putative ribosome-binding site, with the sequence AAGGAG, is located 6 bases upstream from the start of this ORF. This sequence resembles binding sites in other Gram-positive bacteria (De Vos, 1987 ; Moran et al., 1982
). The predicted protein has extensive homology with the ß-glucoside-specific PTS EIIs from other organisms (Table 2a
). This protein is likely responsible for the translocation of the carbohydrate into the bacterial cell. The PTS EIIs can have three domains that are present on one or more proteins. The ß-glucoside-specific PTS EII encoded by this ORF is predicted to contain all three domains (EIIB, EIIC and EIIA) on a single protein (EIIBCA). The similarity observed between this deduced protein and the PTS EIIs is significant throughout the full length of the deduced protein. Due to the similarity between the EII gene products, which act as permeases, and the deduced protein, the first ORF has been named bglP.
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bglC.
The third ORF of this region is transcribed from the opposite strand of DNA. It consists of 906 nucleotides (base-pairs 42545159) and would encode a protein of 302 amino acids. There is no apparent ribosome-binding site located immediately 5' to this ORF. This deduced protein has similarity with several proteins in the XylS/AraC family of transcriptional regulators, which are involved with the regulation of various operons in Listeria monocytogenes, B. subtilis, E. coli and Haemophilus influenzae (Table 2b). These proteins contain helixturnhelix DNA-binding domains. Therefore, this ORF would encode a putative transcriptional regulator and has been named bglC. No function has been assigned to this ORF at this time.
ORF 4.
The fourth ORF is 423 nucleotides in length (base-pairs 5344 to 5766) and the predicted protein has very low similarity with the TraG and TrsG regulatory proteins from Staphylococcus aureus. There is no apparent ribosome-binding site adjacent to this ORF and it is not known if the ORF encodes a functional protein. Therefore, this ORF has not been named at this time.
bglA.
The fifth ORF is 1440 nucleotides long (base-pairs 59747413) and would encode a protein of 480 amino acids. A putative ribosome-binding site, AAGGA, is positioned 6 bases from the start of this gene. This deduced protein would have high similarity to the phospho-ß-glucosidases from other organisms, including C. longisporum, B. subtilis, E. coli, Klebsiella oxytoca and Er. chrysanthemi (Table 2c). As with the S. mutans bglP gene product, this similarity is apparent throughout the entire deduced protein. This ORF has been named bglA because of the similarities between the deduced BglA protein and the phospho-ß-glucosidase from C. longisporum. It is the bglA gene product that is responsible for the hydrolysis of ß-glucosides.
Phenotypes of various bgl strains
Various deletion and interposon insertion strains were constructed in an attempt to assign phenotypes to the genes located in this region of DNA. Strain ALH167 has a partial deletion of both the bglP and bglA genes and the complete deletion of the bglB, bglC and ORF4 genes (see Methods). This deletion strain was tested on media containing various ß-glucosides as the carbon source in order to detect a phenotype and any potential functions of the proteins encoded by this locus. Strain ALH167 retained the ability to breakdown arbutin, salicin and cellobiose when assayed on Purple Broth plates containing these ß-glucosides as carbon sources (data not shown). The phenotype of this strain was also assayed on aesculin plates, which only detect the breakdown of aesculin and are not indicative of glucose fermentation. This strain was able to break down aesculin when grown on aesculin plates without glucose, but not when glucose was added to the aesculin plates (not shown). The wild-type S. mutans NG8 consistently hydrolysed aesculin in either the presence or the absence of glucose. These results, summarized in Table 3, indicate that this region is responsible, at least in part, for the utilization of the ß-glucoside aesculin, supporting the assignment of the bgl gene nomenclature.
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Additional knockout strains were constructed with a specific insertion into either the bglP or the bglA gene in order to determine the function of the individual gene products. The bglP knockout strain, ALH96, which would not produce the putative PTS EII responsible for ß-glucosidase translocation, was unable to break down aesculin in the presence of glucose; in the absence of glucose, it was able to break down the aesculin nearly as efficiently as the wild-type strain (Table 3). These phenotypic data indicate that bglP is necessary for the translocation of aesculin in the presence of glucose.
A knockout strain was also constructed that contained an insertional mutation in the bglA gene. This strain, ALH168, would not produce the putative phospho-ß-glucosidase. The bglA knockout strain and the wild-type strain were able to hydrolyse aesculin in both the presence and absence of glucose. However, aesculin hydrolysis was less pronounced in the presence of glucose. This indicates that one of the mechanisms for breaking down ß-glucosides is repressed in the presence of glucose, while another is not repressed by glucose. These data also indicate that the S. mutans bglA gene knockout is complemented by another ß-glucosidase encoded elsewhere on the chromosome.
The phenotypic data collected from various strains of S. mutans (summarized in Table 3) allow for several interesting inferences into the mechanisms that are used to utilize ß-glucosides in S. mutans. The bglP gene, isolated and discussed in this report, is not repressed in the presence of glucose. A second permease, which is strongly repressed in the presence of glucose, must exist that can also translocate aesculin into the bacterial cell. These data also indicate the presence of at least one additional mechanism that can breakdown ß-glucosides in S. mutans. Another gene, encoding a second catabolic enzyme, that is responsible for breaking down aesculin in the absence of the BglA protein, is also repressed by glucose. This second ß-glucosidase is not repressed by glucose to the extent that the second permease is. The bglA gene product is not necessary for aesculin utilization but does play a role in ß-glucoside breakdown in the presence of glucose (Table 3
).
Other bacteria have been shown to have multiple operons or regulons in order to utilize various ß-glucosides (el Hassouni et al., 1990 ; Kruger & Hecker, 1995
; Tobisch et al., 1997
). A recurring characteristic of these organisms is the ablility to utilize different proteins which are specific for different groups of ß-glucosides. Some species have genes that are involved in the utilization of the aromatic ß-glucosides arbutin and salicin. These same organisms will also have a completely different set of genes involved in the metabolism of the ß-glucoside cellobiose. This phenomenon is exemplified by the bglPH and lic operons in B. subtilis (Kruger et al., 1996
; Tobisch et al., 1997
). In view of this carbohydrate specificity of the ß-glucoside systems, it is likely that S. mutans has other PTS systems involved in the utilization of other ß-glucosides as carbon sources.
ß-Glucoside-specific activity was assayed in E. coli CC118 containing the plasmid pALH163, which harbours the entire bgl region. E. coli CC118(pALH163) was able to break down arbutin, while the host strain CC118 was not. E. coli CC118 was also transformed with pALH164, to generate a strain containing the ß-glucoside regulon with a deletion of the bglP gene. Strain CC118(pALH164) was assayed for arbutin hydrolysis on minimal medium plates containing this ß-glucoside as the sole carbon source. It was unable to break down the arbutin. While not definitively due to the lack of the bglP gene, this result tends to indicate that the bglP gene product is necessary for arbutin utilization in E. coli. A third E. coli strain was created that lacked the bglA gene from S. mutans. This strain, E. coli CC118(pALH165), retained the ability to breakdown arbutin (Table 4). From these E. coli complementation studies, it appears that the EII encoded by bglP is necessary for arbutin hydrolysis in E. coli CC118, but that the putative phospho-ß-glucosidase encoded by the bglA gene is not.
While all of the elements required for ß-glucoside utilization are present in the bgl regulon of S. mutans, the arrangement of the genes is different from that in previously characterized operons. Generally, ß-glucoside PTS operons consist of an EII gene that is directly followed by the phospho-ß-glucosidase gene. However, in S. mutans, there are three ORFs between these genes (bglP and bglA) (Fig. 2). The idea that the bgl regulon of S. mutans is structurally different from those previously described is exemplified by the fact that the bglC gene is transcribed in the opposite direction from bglP, bglB and bglA.
In this report, we have described the isolation and characterization of a ß-glucoside-specific PTS from S. mutans. The bglP gene encodes a PTS EII component that is not repressed by glucose. The protein encoded by the S. mutans bglA gene is similar to the phospho-ß-glucosidases isolated from other bacterial PTSs. The bglC gene is thought to encode a transcriptional regulator and the potential functions of the proteins encoded by the bglB gene and ORF4 are still unknown. Further studies are being initiated to examine the possible functions of these proteins, as well as to understand the regulation of this region. This regulon will be used to further examine the unusual regulation normally associated with ß-glucoside utilization, as well as to further characterize the mechanisms responsible for catabolite repression in S. mutans.
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ACKNOWLEDGEMENTS |
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Received 3 December 1999;
revised 17 March 2000;
accepted 27 March 2000.