Previous studies suggested that the first step in utilization of starch by Bacteroides thetaiotaomicron was binding of the polysaccharide to the cell surface, followed by translocation of the polysaccharide across the outer membrane into the periplasm. In this study, we report the molecular characterization of a gene that encodes an outer membrane protein that is essential for utilization of both maltooligosaccharides and starch. The gene, susC, encoded a protein of 115.3 kDa. Antibodies were raised against SusC, and the outer membrane location of SusC could be confirmed by Western blot (immunoblot) analysis. SusC had a possible signal sequence of between 20 and 39 amino acids, depending on which N-terminal methionine initiates the start of the protein. It also had some features typical of well-characterized outer membrane proteins from members of the family Enterobacteriaceae, such as a terminal phenylalanine residue and a region in the amino portion of the protein thought to be involved in stabilizing the protein in the outer membrane. The amino acid sequence, together with results of gene disruption experiments, suggested that SusC was not an amylolytic enzyme. Transcriptional fusion experiments, using beta-glucuronidase as a reporter group, showed that expression of susC was maltose regulated at the transcriptional level. This is the first molecular characterization of a B. thetaiotaomicron outer membrane protein involved in maltooligosaccharide and starch utilization.

Bacteroides thetaiotaomicron, a gram-negative colonic anaerobe, can utilize three forms of starch: amylose, amylopectin, and pullulan. Previously, a neopullulanase, a pullulanase, and an ␣-glucosidase from B. thetaiotaomicron had been purified and characterized biochemically. The neopullulanase and ␣-glucosidase appeared to be the main enzymes involved in the breakdown of starch, because they were responsible for most of the starch-degrading activity detected in B. thetaiotaomicron cell extracts. To determine the importance of these enzymes in the starch utilization pathway, we cloned the genes encoding the neopullulanase and ␣-glucosidase. The gene encoding the neopullulanase (susA) was located upstream of the gene encoding the ␣-glucosidase (susB). Both genes were closely linked to another starch utilization gene, susC, which encodes a 115-kDa outer membrane protein that is essential for growth on starch. The gene encoding the pullulanase, pulI, was not located in this region in the chromosome. Disruption of the neopullulanase gene, susA, reduced the rate of growth on starch by about 30%. Elimination of susA in this strain allowed us to detect a low residual level of enzyme activity, which was localized to the membrane fraction. Previously, we had shown that a disruption in the pulI gene did not affect the rate of growth on pullulan. We have now shown that a double mutant, with a disruption in susA and in the pullulanase gene, pulI, was also able to grow on pullulan. Thus, there is at least one other starch-degrading enzyme besides the neopullulanase and the pullulanase. Disruption of the ␣-glucosidase gene, susB, reduced the rate of growth on starch only slightly. No residual ␣-glucosidase activity was detectable in extracts from this strain. Since this strain could still grow on maltose, maltotriose, and starch, there must be at least one other enzyme capable of degrading the small oligomers produced by the starchdegrading enzymes. Our results show that the starch utilization system of B. thetaiotaomicron is quite complex and contains a number of apparently redundant degradative enzymes.

Bacteroides thetaiotaomicron, a gram-negative obligate anaerobe, appears to utilize starch by first binding the polymer to its surface and then translocating it into the periplasmic space. Several genes that encode enzymes or outer membrane proteins involved in starch utilization have been identified. These have been called sus genes, for starch utilization system. Previous studies have shown that sus structural genes are regulated at the transcriptional level and their expression is induced by maltose. We report here the identification and characterization of a gene, susR, which appears to be responsible for maltose-dependent regulation of the sus structural genes. The deduced amino acid sequence of SusR protein had a helix-turn-helix motif at its carboxy-terminal end, and this region had highest sequence similarity to the corresponding regions of known transcriptional activators. A disruption in susR eliminated the expression of all known sus structural genes, as expected if susR encoded an activator of sus gene expression. The expression of susR itself was not affected by the growth substrate and was not autoregulated, suggesting that binding of SusR to maltose might be the step that activates SusR. Three susR-controlled structural genes, susA, susB, and susC, are located immediately upstream of susR. These genes are organized into two transcriptional units, one containing susA and another containing susB and susC. susA was expressed at a lower level than susBC, and susA expression was more sensitive to the gene dosage of susR than was that of the susBC operon. An unexpected finding was that increasing the number of copies of susR in B. thetaiotaomicron increased the rate of growth on starch. This effect could be due to higher levels of susA expression. Whatever the explanation, the level of SusR in the cell appears to be a limiting factor for growth on starch.

The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5'-end of pyr mRNA. PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes. The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay. Titration indicated that PyrR binds RNA in an equimolar ratio. PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site. PyrR bound BL2 RNA 4-5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator. The minimal RNA that bound tightly to PyrR was 28 nt long. Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity. Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding. Conserved elements of RNA secondary structure were also required for tight binding. PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments. PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure.

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