To orchestrate a complex life style in changing environments, the filamentous cyanobacterium Nostoc punctiforme facilitates communication between neighboring cells through septal junction complexes. This is achieved by nanopores that perforate the peptidoglycan (PGN) layer and traverse the cell septa. The N‐acetylmuramoyl‐l‐alanine amidase AmiC2 (Npun_F1846; http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/5/1/28.html) in N. punctiforme generates arrays of such nanopores in the septal PGN, in contrast to homologous amidases that mediate daughter cell separation after cell division in unicellular bacteria. Nanopore formation is therefore a novel property of AmiC homologs. Immunofluorescence shows that native AmiC2 localizes to the maturing septum. The high‐resolution crystal structure (1.12 Å) of its catalytic domain (AmiC2‐cat) differs significantly from known structures of cell splitting and PGN recycling amidases. A wide and shallow binding cavity allows easy access of the substrate to the active site, which harbors an essential zinc ion. AmiC2‐cat exhibits strong hydrolytic activity in vitro. A single point mutation of a conserved glutamate near the zinc ion results in total loss of activity, whereas zinc removal leads to instability of AmiC2‐cat. An inhibitory α‐helix, as found in the Escherichia coli AmiCE. coli structure, is absent. Taken together, our data provide insight into the cell‐biological, biochemical and structural properties of an unusual cell wall lytic enzyme that generates nanopores for cell–cell communication in multicellular cyanobacteria. The novel structural features of the catalytic domain and the unique biological function of AmiC2 hint at mechanisms of action and regulation that are distinct from other amidases. Database The AmiC2‐cat structure has been deposited in the Protein Data Bank under accession number http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5EMI.

SummaryThe genetic switch region of temperate Streptococcus thermophilus phage TP-J34 contains two divergently oriented promoters and several predicted operator sites. It separates lytic cycle-promoting genes from those promoting lysogeny. A polycistronic transcript comprises the genes coding for repressor Crh, metalloproteinase-motif protein Rir and superinfection exclusion lipoprotein Ltp. Weak promoters effecting monocistronic transcripts were localized for ltp and int (encoding integrase) by Northern blot and 5′-RACE-PCR. These transcripts appeared in lysogenic as well as lytic state. A polycistronic transcript comprising genes coh (encoding Cro homolog), ant (encoding putative antirepressor), orf7, orf8 and orf9 was only detected in the lytic state. Four operator sites, of which three were located in the intergenic regions between crh and coh, and one between coh and ant, were identified by competition electromobility shift assays. Cooperative binding of Crh to two operator sites immediately upstream of coh could be demonstrated. Coh was shown to bind to the operator closest to crh only. Oligomerization was proven by cross-linking Crh by glutaraldehyde. Knock-out of rir revealed a key role in prophage induction. Rir and Crh were shown to form a complex in solution and Rir prevented binding of Crh to its operator sites.

The human intestinal microbiota plays an important role in human health. While adhesion to gastrointestinal mucosa is a prerequisite for colonisation, inhibition of adhesion is a property which may prevent or reduce infections by food borne pathogens. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus represent the two lactic bacteria constituting the yoghurt culture. These starter cultures have been claimed to be probiotic. In our study we compared two S. thermophilus strains (i.e. lysogenic strain J34 and corresponding non-lysogenic [prophage-cured] strain J34-6), with respect to (1) their in vitro adhesion properties to HT29 cells and (2) their cell surface hydrophobicities. Effects of the two strains on inhibition of adhesion of the pathogens Listeria monocytogenes Scott A, Staphylococcus aureus 6732 and Salmonella enteritidis S489 were studied in vitro with HT29 cell cultures. Lysogenic strain J34 was shown to be considerably more effective than the non-lysogenic derivative strain J34-6.

No abstract