Regulation of expression of the fructan hydrolase gene of Streptococcus mutans GS-5 by induction and carbon catabolite repression

Abstract

The polymers of fructose, levan and inulin, as well as sucrose and raffinose, are substrates for the product of the fruA gene of Streptococcus mutans GS-5. The purpose of this study was to characterize the DNA immediately flanking fruA, to explore the regulation of expression of fruA by the carbohydrate source, and to begin to elucidate the molecular basis for differential expression of the gene. Located 3' to fruA was an open reading frame (ORF) with similarity to beta-fructosidases which was cotranscribed with fruA. A transcriptional initiation site, located an appropriate distance from an extended -10-like promoter, was mapped at 165 bp 5' to the fruA structural gene. By the use of computer algorithms, two overlapping, stable stem-loop sequences with the potential to function as rho-independent terminators were found in the 5' untranslated region. Catabolite response elements (CREs), which have been shown to govern carbon catabolite repression (CCR) by functioning as negative cis elements in gram-positive bacteria, were located close to the promoter. The levels of production of fruA mRNA and FruA were elevated in cells growing on levan, inulin, or sucrose as the sole carbohydrate source, and repression was observed when cells were grown on readily metabolizable hexoses. Deletion derivatives containing fusions of fruA promoter regions, lacking sequences 5' or 3' to the promoter, and a promoterless chloramphenicol acetyltransferase gene were used (i) to demonstrate the functionality of the promoter mapped by primer extension, (ii) to demonstrate that CCR of the fru operon requires the CRE that is located 3' to the promoter region, and (iii) to provide preliminary evidence that supports the involvement of an antitermination mechanism in fruA induction.

FIG. 1

Relevant nucleotide sequences and features of the fruAB gene cluster. The nucleotide sequences of the 5′ region of fruA, the fruAB intergenic region, and the 3′ end of fruB are shown. The sequences of the structural genes have been omitted to save space. Sequence numbering corresponds to the original GenBank submission of the fruA sequence (accession no. L03358 and AF093758). The fruA structural gene begins at position 685 and ends at position 4953. The putative RBS of fruB begins at position 5016. The fruB start codon is at position 5027, and the structural gene extends to position 6599. Underlined and in boldface is the extended −10-like promoter element that drives fru transcription. Boxed and labeled as CRE-W (weaker) and CRE-S (strong) are the CREs described in the text. The inverted-repeat structures with characteristics of rho-independent terminators predicted by the Terminator program of the University of Wisconsin GCG software (indicated by opposing dashed arrows above the sequence) are in the 5′ leader mRNA of fruA (SL1, positions 599 to 626; SL2, positions 613 to 633) and after the fruB gene (SL4, positions 6618 to 6644). These three inverted repeats are followed by a T (U)-rich 9-nt sequence. There is also a stable, previously identified inverted repeat (SL3) at the end of fruA (9) indicated by dashed arrows above the sequence, but this is not recognized by computer algorithms as a rho-independent terminator. The sequence with partial similarity to RAT-like sequences (RAT; positions 595 to 619), underlined with a boldfaced dashed line, overlaps with both stem-loop structures in the leader region. The start and stop codons of fruA and fruB are in boldface, and the stop codons are labeled with asterisks. −35 and −10 hrcA indicate the promoter for the dnaK operon (31). TIS, the transcriptional initiation site mapped by primer extension.

FIG. 2

GAP alignment of S. mutans FruB and B. subtilis YveB. The deduced amino acid sequences of the fruB (top) and yveB genes were aligned by using the GAP program of the University of Wisconsin GCG package. Vertical lines indicate identity, and single and double dots indicate lower and high degrees of amino acid similarity, respectively. The aspartic acid (Asp 47) believed to correspond to that in the yeast invertase which was shown to be involved in catalysis is in boldface, as is a highly conserved cysteine residue (position 230) found in many fructosidases. Overlined with dashed lines are regions in FruB that exhibit some homology to conserved regions in fructosidases or which have some similarity to regions conserved between S. mutans FruA and B. subtilis SacC, which are known levanases. Asterisks indicate a sequence which is very highly conserved among FruB, YveB, and the known levanase of B. stearothermophilus, SurC.

FIG. 3

RT PCR. Shown are ethidium bromide-stained agarose gels of products obtained by RT PCR with primers spanning the fruA-fruB intergenic region, using RNA from cells grown on inulin (A) or glucose (B). RT PCRs were performed with RNA from cells grown on different carbohydrates to determine whether the carbohydrate source affected transcriptional readthrough and because RT PCR of fructan-grown cells always produced a band that was slightly smeared, perhaps from carryover of polysaccharide. The sense-strand primer corresponded to nt 4794 to 4830, and the antisense primer corresponded to nt 5140 to 5118 for the 0.65-kbp product or to nt 5415 to 5398 for the 0.32-kbp product (nucleotide positions are in reference to those of GenBank accession no. AF093758). (A) Lanes 1 and 8 are DNA size standards (LTI). Products from the PCRs were derived as follows: cDNA prepared from S. mutans RNA with RT (lanes 2 and 5), from S. mutans RNA without RT (lanes 3 and 6), and from GS-5 chromosomal DNA (lanes 4 and 7). (B) Lanes 1 and 6 contain the 1-kbp ladder (LTI). Products from the PCRs were derived as follows: from GS-5 chromosomal DNA (lane 2), from cDNA prepared from S. mutans RNA with RT (lane 3), and from S. mutans RNA without RT (lane 4), Lane 5 contains a no DNA-no RNA-no RT control.

FIG. 4

Slot blot of total RNA from S. mutans grown under induced and repressed conditions. S. mutans GS-5 was grown in TV medium supplemented with inulin, glucose, or both at concentrations of 0.5%. Total RNA (1 μg) was isolated from exponentially growing S. mutans cells, treated with RNase-free DNase, and transferred to a membrane as detailed in the text. Hybridizations were performed with an internal fragment of either fruA (A) or fruB (B) as indicated in Materials and Methods. Inulin plus RNase samples were treated with RNase prior to application to the membrane as a control to show that the RNAs were free of DNA contamination.

FIG. 5

Mapping the fruA promoter. This figure shows the results of primer extension reactions used to map the transcriptional initiation site for fruA. RNA was isolated from S. mutans cells growing exponentially with fructose (F) or levan (L) as the carbohydrate source. Reactions were performed as detailed in the text. Adjacent to the primer extensions is a sequencing reaction performed on pFRU1 (13) with the same primer used in the primer extension.

FIG. 6

Promoter fusions and deletion derivatives. Shown are schematic diagrams of the derivatives of the 5′ region of fruA which were generated by PCRs and fused to an E. coli cat gene. The fruA promoter (P_fru) is indicated with a labeled box. The two CREs are indicated as shaded boxes overlapping the promoter (CRE-W) or beginning at position +2 (CRE-S) in the mRNA. SD_s (for Shine-Dalgarno–Streptococcus) indicates the cognate RBS of fruA, and SD_E (for Shine-Dalgarno–E. coli) indicates the cognate RBS of cat. (See Materials and Methods for details.) The lollipop-like markings indicate the approximate position of the stem-loop structures in the fruA leader mRNA. The 5′ ends of WHFRU, FCAT, Δ18, and Δ12 promoter fusions described in the text correspond to positions 121, 439, 552, and 581, respectively. The 3′ end point of WHFRU, Δ12, and Δ18 is at position 786, and the 3′ end of FCAT constructs is at position 684 (Fig. 1). All integrated constructs were introduced into wild-type S. mutans GS-5, and all plasmid-borne derivatives were analyzed in US100, a recA derivative of GS-5 constructed for this study. TIS, transcriptional initiation site.