These phage proteins assemble stable, nonspecific pores in the bacterial envelope, allowing phage-encoded lysins (endolysins) to access their substrate (peptidoglycan) (Young & Bläsi, 1995; Wang et al., 2000). Several holin-like proteins are encoded in bacterial genomes including Gram-positive such as Staphylococcus aureus and Bacillus spp. (Loessner et al., 1999; Real et al.,
2005; Anthony et al., 2010), which display a regulatory role in the activity of murein hydrolases, autolysis and spore morphogenesis (Rice & Bayles, 2003). In the Gram-negative bacteria Borrelia burgdorferi, BlyA exhibits a holin-like function promoting the endolysin-dependent lysis and enhancing haemolytic phenotype in animal erythrocytes (Guina & Oliver, 1997; Damman et al., 2000). In addition, Escherichia coli and Salmonella spp. genomes contain Selleck Crizotinib holin-like genes, but little is known about their function.
Selumetinib In this work, we performed a combination of bioinformatic, genetic and biochemical experiments in order to characterize the STY1365 small ORF of S. Typhi. Bacterial strains and plasmids used in this study are listed in Table 1. Cells were routinely grown in 2 mL Luria–Bertani (LB) broth at 37 °C with shaking. When required, media were supplemented with ampicillin (100 μg mL−1), chloramphenicol (20 μg mL−1), kanamycin (50 μg mL−1) and l-arabinose (2 μg μL−1). Solid media were prepared by addition of 1.5 g w/v agar. The nucleotide sequence from S. Typhi CT18 genome (AL513382) was accessed via the National Center for Biotechnology Information (NCBI) Genome database (http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome) Methocarbamol and was used to compare STY1365 and both flanking regions with S. Typhimurium DT104
prophage-like element (AB104436, Saitoh et al., 2005). The STY1365 coding sequence of S. Typhi STH2370 strain was sequenced previously and it was shown to be identical to the corresponding genomic region of S. Typhi CT18 (Rodas et al., 2010). Transmembrane domains of STY1365 were analyzed using tmhmm server v2.0 program (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Analysis of STY1365 predicted amino acid sequence (NC_003198.1) was performed using psi-blast program (http://www.ncbi.nlm.nih.gov/BLAST/). Multiple sequence alignments of STY1365 amino acid sequences and EcolTa2 holin of E. coli TA271 (ZP_07522128.1), ESCE_1669 holin of E. coli SE11 (YP_002292944.1), ECDG_01257 holin of E. coli B185 (ZP_06657343.1) and holin 1 of phage ΦP27 (NP_543080.1) were constructed using vector nt suite v.8 software (Invitrogen). For the chromosomal deletion of STY1365, the ‘one step inactivation’ method described by Datsenko & Wanner (2000) was used. Following mutagenesis, the aph resistance cassette was removed by FLP-mediated recombination. The FRT site generated by excision of antibiotic resistance cassette was used to integrate plasmid pCE36, generating a transcriptional lacZY fusion (Ellermeier et al., 2002).