Analysis of cis- and trans-acting factors involved in regulation of the Streptococcus mutans fructanase gene (fruA)

Abstract

There are two primary levels of control of the expression of the fructanase gene (fruA) of Streptococcus mutans: induction by levan, inulin, or sucrose and repression in the presence of glucose and other readily metabolized sugars. The goals of this study were to assess the functionality of putative cis-acting regulatory elements and to begin to identify the trans-acting factors involved in induction and catabolite repression of fruA. The fruA promoter and its derivatives generated by deletions and/or site-directed mutagenesis were fused to a promoterless chloramphenicol acetyltransferase (CAT) gene as a reporter, and strains carrying the transcriptional fusions were then analyzed for CAT activities in response to growth on various carbon sources. A dyadic sequence, ATGACA(TC)TGTCAT, located at -72 to -59 relative to the transcription initiation site was shown to be essential for expression of fruA. Inactivation of the genes that encode fructose-specific enzymes II resulted in elevated expression from the fruA promoter, suggesting negative regulation of fruA expression by the fructose phosphotransferase system. Mutagenesis of a terminator-like structure located in the 165-base 5' untranslated region of the fruA mRNA or insertional inactivation of antiterminator genes revealed that antitermination was not a mechanism controlling induction or repression of fruA, although the untranslated leader mRNA may play a role in optimal expression of fructanase. Deletion or mutation of a consensus catabolite response element alleviated glucose repression of fruA, but interestingly, inactivation of the ccpA gene had no discernible effect on catabolite repression of fruA. Accumulating data suggest that expression of fruA is regulated by a mechanism that has several unique features that distinguish it from archetypical polysaccharide catabolic operons of other gram-positive bacteria.

FIG. 1.

Relevant nucleotide sequences and features of the 5′ region of fruA (positions −80 to 152 relative to the TIS). In boldface is the extended −10-like promoter that directs transcription of fruA. Boxed and labeled are two CREs, CRE-S (positions 2 to 15 relative to the TIS) and CRE-W (−26 to −14). Two inverted repeats, SL1 (positions 80 to 106) and SL2 (94 to 116), with potential to function as Rho-independent transcriptional terminators, are indicated with dashed arrows. Overlapping SL1 and SL2 and underlined is a sequence with 50% similarity to a RAT sequence (positions 76 to 104). A dyadic sequence (DS) located at −72 to −59 relative to the TIS is a putative binding site of a positive regulatory protein. The start codon of fruA is indicated with an arrow.

FIG. 2.

The fruA promoter (PfruA) and its derivatives (left panel) and CAT activities expressed from the respective fusions after integration into the chromosome and growth of cells with the indicated carbohydrates(s) (right panel). Bar PfruA shows the intact fruA promoter and the putative cis elements SL1 (positions 80 to 106 relative to the TIS), SL2 (94 to 116), CRE-S (2 to 15), CRE-W (−26 to −14), extended −10-like promoter, and DS (−72 to −59) (see Fig. 1 for more detail). All constructs, PfruA and its derivatives generated from deletions and/or site-directed mutagenesis, were fused with a promoterless cat gene, which had its own ribosome binding site. The transcriptional fusions were then integrated into the S. mutans UA159 chromosome. PfruA-I, intact fruA promoter (−80 to 152) fusion integrated in the chromosome; DC-I, PfruA with deletions of CRE-S and the UTR 3′ to the promoter; DT-I, PfruA with deletions of SL1 and SL2 and their 3′UTR; MP-I, PfruA with base substitutions of SL1; MM-I, PfruA with mutations of both CRE-S and SL1; DP-I, PfruA with internal deletions of SL1 and SL2 (positions 78 to 116); UP1-I, PfruA with deletions of DS and its 5′ region; UP2-I, PfruA with deletions of both DS and CRE-S and its 3′UTR; and 18-I, base substitutions of DS. Nucleotides (in boldface) underneath each element indicate the substitutions made. Expression of the fusions were examined by CAT assays of cells grown on TV medium with the indicated sugar(s) as the sole carbohydrate source (see text for more details). CAT activity is expressed as nanomoles minute⁻¹ milligram of protein⁻¹, and the values presented here represent means ± standard deviations from no fewer than four separate experiments. n.d., not detectable.