Susanne Behrens

The Periplasmic Chaperone SurA Exploits Two Features Characteristic of Integral Outer Membrane Proteins for Selective Substrate Recognition

The Escherichia coli periplasmic chaperone and peptidyl-prolyl isomerase (PPIase) SurA facilitates the maturation of outer membrane porins. Although the PPIase activity exhibited by one of its two parvulin-like domains is dispensable for this function, the chaperone activity residing in the non-PPIase regions of SurA, a sizable N-terminal domain and a short C-terminal tail, is essential. Unlike most cytoplasmic chaperones SurA is selective for particular substrates and recognizes outer membrane porins synthesized in vitro much more efficiently than other proteins. Thus, SurA may be specialized for the maturation of outer membrane proteins. We have characterized the substrate specificity of SurA based on its natural, biologically relevant substrates by screening cellulose-bound peptide libraries representing outer membrane proteins. We show that two features are critical for peptide binding by SurA: specific patterns of aromatic residues and the orientation of their side chains, which are found more frequently in integral outer membrane proteins than in other proteins. For the first time this sufficiently explains the capability of SurA to discriminate between outer membrane protein and non-outer membrane protein folding intermediates. Furthermore, peptide binding by SurA requires neither an active PPIase domain nor the presence of proline, indicating that the observed substrate specificity relates to the chaperone function of SurA. Finally, we show that SurA is capable of associating with the outer membrane. Together, our data support a model in which SurA is specialized to interact with non-native periplasmic outer membrane protein folding intermediates and to assist in their maturation from early to late outer membrane-associated steps.

Is there a link between infant botulism and sudden infant death? Bacteriological results obtained in Central Germany

Abstract. Despite the fact that botulism was described in Germany for the first time by Kerner in 1820, the disease is almost forgotten in this country. Only about 10–20 cases of classical botulism (intoxication) are recorded every year, including 1–2 cases of clinical infant botulism. As we assumed a high incidence of botulism to be connected with cases of sudden infant death (SID), we undertook the research work presented here. From every case of unexpected infant death up to 12 months of age, standardised specimens (blood, liver and intestine) were taken at autopsy. They were tested for the presence of botulinum neurotoxin (BoNT) and/or bacterial forms of Clostridium botulinum with subsequent BoNT neutralisation tests by the international standard mouse bioassay. Age, sex, pathological findings and season were recorded. Over a 5-year period, 75 samples including 57 SID cases were tested. Free toxin was found in nine and bacterial forms were detected in six samples. Toxin neutralisation revealed the definite presence of BoNT/BoNT producing bacteria (mainly type E), whereas another 11 toxin tests were inconclusive. According to international literature, these 15 cases are to be interpreted as infant botulism. Conclusion: the results show a remarkable incidence of infant botulism without any known previous medical history, partly hidden as sudden infant death. We propose to systematically search for botulism in connection with sudden infant death

Molecular analysis of the mannitol operon of Clostridium acetobutylicum encoding a phosphotransferase system and a putative PTS-modulated regulator

Clostridium acetobutylicum DSM 792 accumulates and phosphorylates mannitol via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). PEP-dependent mannitol phosphorylation by extracts of cells grown on mannitol required both soluble and membrane fractions. Neither the soluble nor the membrane fraction could be complemented by the opposite fraction prepared from glucose-grown cells, indicating that the mannitol-specific PTS consists of both a soluble (IIA) and a membrane-bound (IICB) component. The mannitol (mtl) operon of C. acetobutylicum DSM 792 comprises four genes in the order mtlARFD. Sequence analysis of deduced protein products indicated that the mtlA and mtlF genes respectively encode the IICB and IIA components of the mannitol PTS, which is a member of the fructose-mannitol (Fru) family. The mtlD gene product is a mannitol-1-phosphate dehydrogenase, while mtlR encodes a putative transcriptional regulator. MtlR contains two PTS regulatory domains (PRDs), which have been found in a number of DNA-binding transcriptional regulators and in transcriptional antiterminators of the Escherichia coli BglG family. Also, near the C-terminus is a well-conserved signature motif characteristic of members of the IIA(Fru)/IIA(Mtl)/IIA(Ntr) PTS protein family. These regions are probably the sites of PTS-dependent phosphorylation to regulate the activity of the protein. A helix-turn-helix DNA-binding motif was not found in MtlR. Transcriptional analysis of the mtl genes by Northern blotting indicated that the genes were transcribed as a polycistronic operon, expression of which was induced by mannitol and repressed by glucose. Primer extension experiments identified a transcriptional start point 42 bp upstream of the mtlA start codon. Two catabolite-responsive elements (CREs), one of which overlapped the putative -35 region of the promoter, were located within the 100 bp upstream of the start codon. These sequences may be involved in regulation of expression of the operon.

Periplasmic Chaperones—Preservers of Subunit Folding Energy for Organelle Assembly

The periplasmic PapD-like chaperones have long been known to be necessary for the assembly of bacterial surface organelles. New structural work now suggests that they control assembly by arresting subunit folding. This step may be required to preserve energy for fiber formation.

Periplasmic Chaperones—New Structural and Functional Insights

Although chaperones exist in the periplasmic compartment of Gram-negative bacterial cells, how they function is not well understood. New intriguing functional insights are provided by the solved crystal structure of the periplasmic chaperone SurA.