Regulation of the alpha-galactosidase activity in Streptococcus pneumoniae: characterization of the raffinose utilization system - PubMed (original) (raw)

Regulation of the alpha-galactosidase activity in Streptococcus pneumoniae: characterization of the raffinose utilization system

C Rosenow et al. Genome Res. 1999 Dec.

Abstract

A 10.2-kb gene region was identified in the Streptococcus pneumoniae genome sequence that contains eight genes involved in regulation and metabolism of raffinose. The genes rafR and rafS are transcribed as one operon, and their gene products regulate the raffinose-dependent stimulation of a divergently transcribed second promoter (P(A)) directing the expression of aga, the structural gene for alpha-galactosidase. Raffinose-mediated transcription from P(A) results in a 500-fold increase in alpha-galactosidase activity in the cell. A third promoter within the cluster is responsible for the transcription of the remaining five genes (rafE, rafF, rafG, gtfA, and rafX), whose gene products might be involved in transport and metabolism of raffinose. The presence of additional internal promoters cannot be excluded. The aga promoter P(A) is negatively regulated by the presence of sucrose in the growth medium. Consistent with catabolite repression (CR), a DNA sequence with high homology to the CRE (cis-active element) was identified upstream of the aga promoter. Sucrose-mediated CR depends on the phosphoenolpyruvate: sucrose phosphotransferase system (PTS) but is unaffected by a mutation in a gene encoding a homolog of the CRE regulatory protein CcpA.

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Figures

Figure 1

Figure 1

Map of the raf genes in the S. pneumoniae contig. Arrows indicate operons and the orientation of transcription. The number of base pairs (bp), amino acids (aa), and the molecular mass (_M_r) of the corresponding proteins are indicated.

Figure 2

Figure 2

Sequence analysis of the intergenic region between rafR and aga (A) and aga and rafE (B). Potential ribosome binding sites (RBS), −10 and −35 promoter elements (italics), and the direct repeats (arrows) are indicated. A 15-bp sequence with homology to the CRE consensus sequence is underlined.

Figure 3

Figure 3

α-Galactosidase activities in the cell lysates of S. pneumoniae strain R6x grown in the presence of 0.2% glucose and different raffinose concentrations in the growth medium. The activities are expressed in units (nmoles of _p_-nitrophenol produced per min of incubation at room temperature) per mg protein with their

s.d.

.

Figure 4

Figure 4

Amplified cDNA from total RNA from the S. pneumoniae strain R6x grown in C+Y supplemented with 0.2% raffinose and 0.2% glucose. In each reaction (lanes 1_–_10) one primer pair was used, as indicated by the lines. The expected size for each fragment is given in kb. Lane 11 is a negative control (−) without RT and the primer pair from lane 3 in the reaction mixture; Lane 12 is a positive control (+) with total RNA and primers provided with the Access RT–PCR introductory System. The arrows indicate the identified potential promoters.

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