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.

Regulation and Physiologic Significance of the Agmatine Deiminase System of Streptococcus mutans UA159

“…4), although AgD activity was slightly higher in the ccpA and ccpB strains following growth in either glucose or galactose, supplemented with agmatine. This observation is consistent with studies of other metabolic pathways in S. mutans that showed that even though cre sequences were tightly linked to the regulatory regions, CcpA (RegM) and CcpB are not primary factors controlling CCR in this organism (40).…”

“…AgD activity was measured in S. mutans UA159 and in otherwise isogenic mutants of this strain lacking the ccpA or ccpB genes that were constructed previously in our laboratory (40). The strains were grown in TV broth containing 25 mM glucose or the nonrepressing sugar, galactose, with or without 10 mM agmatine, to mid-exponential phase prior to measurement of enzyme activity.…”

Characterization of cis- Acting Sites Controlling Arginine Deiminase Gene Expression in Streptococcus gordonii

“…CRE-S resides in close proximity to the Ϫ35 sequence, and CRE-W overlaps partly with the ArcR binding site; therefore, sequence alterations in either CRE could profoundly affect the transcriptional activity of the arc operon. A similar challenge was noted previously in our laboratory, with transcriptional analysis of two putative CREs in the promoter region of the fruA gene of S. mutans, when mutation of a CRE that overlapped with the extended Ϫ10 promoter greatly reduced fruA promoter activity (30). Notably, differences between changes in ADS expression in response to CCR in these two mutants were small and possibly not biologically significant, at least under the conditions tested.…”

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…In S. xylosus, deletion of the cre located in front of the malRA operon (for maltose/maltotriose utilization) causes a complete relief from CCR for ␣-glucosidase (204). Similarly, deletion of a cre located right behind the transcription initiation site of the fructan hydrolase-encoding fruA gene of Streptococcus mutans causes a relief from glucose repression (93,946). CCR of the ␤-amylase-encoding bamM gene from B. megaterium is mediated by a cre located in the DNA region encoding the signal peptide of this extracellular enzyme (457).…”