Clostridium difficile, a leading cause of hospital-acquired infection, possesses a dense surface layer (S-layer) that mediates host-pathogen interactions. The key structural components of the S-layer result from proteolytic cleavage of a precursor protein, SlpA, into high-and low-molecularweight components. Here we report the discovery and optimization of the first inhibitors of this process in live bacteria, and their application for probing S-layer processing. We also describe the design and in vivo application of activity-based probes that identify the protein Cwp84 as the cysteine protease that mediates SlpA cleavage. This work provides novel chemical tools for the analysis of Slayer biogenesis, and for the potential identification of novel drug targets within clostridia and related bacterial pathogens.

Growth experiments showed that adenine and hypoxanthine can be used as nitrogen sources by several strains of K. pneumoniae under aerobic conditions. The assimilation of all nitrogens from these purines indicates that the catabolic pathway is complete and proceeds past allantoin. Here we identify the genetic system responsible for the oxidation of hypoxanthine to allantoin in K. pneumoniae. The hpx cluster consists of seven genes, for which an organization in four transcriptional units, hpxDE, hpxR, hpxO, and hpxPQT, is proposed. The proteins involved in the oxidation of hypoxanthine (HpxDE) or uric acid (HpxO) did not display any similarity to other reported enzymes known to catalyze these reactions but instead are similar to oxygenases acting on aromatic compounds. Expression of the hpx system is activated by nitrogen limitation and by the presence of specific substrates, with hpxDE and hpxPQT controlled by both signals. Nitrogen control of hpxPQT transcription, which depends on 54 , is mediated by the Ntr system. In contrast, neither NtrC nor the nitrogen assimilation control protein is involved in the nitrogen control of hpxDE, which is dependent on 70 for transcription. Activation of these operons by the specific substrates is also mediated by different effectors and regulatory proteins. Induction of hpxPQT requires uric acid formation, whereas expression of hpxDE is induced by the presence of hypoxanthine through the regulatory protein HpxR. This LysR-type regulator binds to a TCTGC-N 4 -GCAAA site in the intergenic hpxD-hpxR region. When bound to this site for hpxDE activation, HpxR negatively controls its own transcription.

Clostridium difficile expresses a number of cell wall proteins, including the abundant high-molecular-weight and low-molecular-weight S-layer proteins (SLPs). These proteins are generated by posttranslational cleavage of the precursor SlpA by the cysteine protease Cwp84. We compared the phenotypes of C. difficile strains containing insertional mutations in either cwp84 or its paralog cwp13 and complemented with plasmids expressing wild-type or mutant forms of their genes. We show that the presence of uncleaved SlpA in the cell wall of the cwp84 mutant results in aberrant retention of other cell wall proteins at the cell surface, as demonstrated by secretion of the proteins Cwp66 and Cwp2 into the growth medium. These phenotypes are restored by complementation with a plasmid expressing wild-type Cwp84 enzyme but not with one encoding a Cys116Ala substitution in the active site. The cwp13 mutant cleaved the SlpA precursor normally and had a wild-type-like colony phenotype. Both Cwp84 and Cwp13 are produced as proenzymes which are processed by cleavage to produce mature enzymes. In the case of Cwp84, this cleavage does not appear to be autocatalytic, whereas in Cwp13 autocatalysis was demonstrated as a Cys109Ala mutant did not undergo processing. Cwp13 appears to have a role in processing of Cwp84 but is not essential for Cwp84 activity. Cwp13 cleaves SlpA in the HMW SLP domain, which we suggest may reflect a role in cleavage and degradation of misfolded proteins at the cell surface.

The capacity to both ferment and oxidize L-ascorbate has been widely documented for a number of enteric bacteria. Here we present evidence that all the strains of Klebsiella pneumoniae tested in this study ferment L-ascorbate using the ula regulon-encoded proteins. Under aerobic conditions, several phenotypes were observed for the strains. Our results showed that the yiaK-S system is required for this aerobic metabolic process. Gel shift experiments performed with UlaR and YiaJ and probes corresponding to the specific promoters indicated that L-ascorbate-6-phosphate is the effector molecule recognized by both regulators, since binding of the repressors to their recognition sites was impaired by the presence of this compound. We demonstrated that in K. pneumoniae cells L-ascorbate-6-phosphate is formed only by the action of the UlaABC phosphotransferase system. This finding explains why strains that lack the ula genetic system and therefore are unable to form the inducer intracellularly cannot efficiently use this vitamin as a carbon source under either anaerobic or aerobic conditions. Thus, efficient aerobic metabolism of L-ascorbate in K. pneumoniae is dependent on the presence of both the yiaK-S and ula systems. The expression of the yiaK-S operon, but not the expression of the ula regulon, is controlled by oxygen availability. Both systems are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and by IHF.Several studies have described the ability of a number of enteric bacteria to both ferment and oxidize L-ascorbate (7,31,33). Dissimilation of L-ascorbate by Escherichia coli under anaerobic conditions has been extensively documented (4,5,32,34) and has been shown to be carried out by proteins encoded by the ula regulon (Fig. 1D). The ula system of E. coli is formed by two divergently transcribed operons (5): the ulaG operon, which is thought to encode the L-ascorbate-6-phosphate lactonase (32), and the ulaA-F operon, which encodes the three components of the L-ascorbate phosphotransferase transport system (UlaABC) (34), as well as three catabolic enzymes (UlaDEF) (32). The UlaA, UlaB, and UlaC gene products (formerly designated SgaT, SgaB, and SgaA, respectively) are involved in the uptake and phosphorylation of L-ascorbate (34). Intracellular L-ascorbate-6-phosphate may be transformed by L-ascorbate-6-phosphate lactonase to 3-keto-L-gulonate-6-phosphate. It has been proposed that this compound is decarboxylated by UlaD to L-xylulose-5-phosphate, which is then converted to D-xylulose-5-phosphate by the sequential action of UlaE (having 3-epimerase activity) and UlaF (having 4-epimerase activity) (32). Thus, the functions of the gene products of the ula system are transport of L-ascorbate and transformation of this compound into D-xylulose-5-phosphate ( Fig. 1E) (32), which is subsequently metabolized by the pentose phosphate pathway. The ula regulon is under the control of the UlaR repressor (4, 5), which belongs to the DeoR repressor family (http://us.expasy.org/uniprot/P0A9W0; http: //p...