The lysis genes of the Lactobacillus gasseri bacteriophage adh were isolated by complementation of a lambda Sam mutation in Escherichia coli. Nucleotide sequencing of a 1,735-bp DNA fragment revealed two adjacent coding regions of 342 bp (hol) and 951 bp (lys) in the same reading frame which appear to belong to a common transcriptional unit. Proteins corresponding to the predicted gene products, holin (12.9 kDa) and lysin (34.7 kDa), were identified by in vitro and in vivo expression of the cloned genes. The adh holin is a membrane-bound protein with structural similarity to lysis proteins of other phage, known to be required for the transit of murein hydrolases through the cytoplasmic membrane. The adh lysin shows homology with mureinolytic enzymes encoded by the Lactobacillus bulgaricus phage mv4, the Streptococcus pneumoniae phage Cp-1, Cp-7, and Cp-9, and the Lactococcus lactis phage LC3. Significant homology with the N termini of known muramidases suggests that adh lysin acts by a similar catalytic mechanism. In E. coli, the adh lysin seems to be associated with the total membrane fraction, from which it can be extracted with lauryl sarcosinate. Either one of the adh lysis proteins provoked lysis of E. coli when expressed along with holins or lysins of phage lambda or Bacillus subtilis phage 29. Concomitant expression of the combined holin and lysin functions of adh in E. coli, however, did not result in efficient cell lysis.Large Escherichia coli phage in general appear to encode at least two lysis functions, a murein hydrolase, required for destruction of the peptidoglycan, and a protein termed holin which permits access of the lytic enzyme to the periplasm (for a review, see reference 78). In the case of bacteriophage lambda, oligomerization of the holin (protein S) in the inner membrane of E. coli apparently leads to formation of a nonspecific lesion through which the lambda transglycosylase is released to the periplasm at the end of the vegetative cycle (80). The expression of the S gene and thus the kinetics of formation of the S-dependent hole in the inner membrane is tightly controlled at the transcriptional (35) and at the translational level (7, 53), as well as posttranslationally by virtue of two S-encoded polypeptides with opposing functions (6, 70). Holin functions have also been attributed to the products of P22 gene 13 (58), to phage 21 gene S (9), and recently to protein 14 of the Bacillus subtilis phage 29 (70), the first holin identified from a phage of gram-positive bacteria. Large phage therefore appear to pursue an evolutionarily conserved lysis pathway.In phage lambda, P22, 21, and 29, the genes encoding the corresponding holins and murein hydrolases are arranged identically. The holin gene in all cases precedes the gene encoding the murein hydrolase and overlaps at least with its ribosome-binding site (9,13,30,58). With the exception of the products of the lambda S and P22 13 genes, which are nearly identical (58), the known holins show no homology with each other (78). Since lambda S...
The lambda-holin protein is cytotoxic for eukaryotic cells in vitro and inhibits tumour growth in vivo suggesting potential therapeutic use in cancer gene therapy.
With the aim to extend the presently available inducible gene expression systems for lactobacilli, we have isolated a thermoinducible promoter-repressor cassette from the temperate Lactobacillus casei phage FSW-TI in Escherichia coli. The FSW-TI promoter fragment was abutted to the plasmid-borne promoterless -glucuronidase (gusA) reporter gene and shown to direct its transcription in L. casei. In addition, the functionality of the promoter-repressor system was verified in the L. casei FSW-TI lysogen by showing that the gusA reporter gene, controlled by the isolated FSW-TI promoter, was repressed at 28°C and expressed at 42°C. Moreover, a homology search revealed that the C terminus of the isolated FSW repressor shows a high similarity to the small mutS-related domain of the MutS2 protein family that is unprecedented for phageencoded repressor proteins.Given the economic importance of lactic acid bacteria, and more recently their prospective use as safe vaccines, a number of physiological traits as well as food-grade expression tools of lactobacilli and lactococci have been studied (9). Various inducible gene expression systems have been developed for Lactococcus lactis (reviewed in reference 3). In general, these transcriptional induction systems require the exogenous supply of inducers (18,19,21). In addition, a heat-inducible homologous expression system has been developed for L. lactis based on a genetically engineered thermolabile repressor of the lactococcal phage r1t (17). In lactobacilli, studies on the regulation of gene expression have focused mainly on carbon catabolism pathways, and an inducible-repressible expression system derived from the lactose operon of Lactobacillus casei has been described (5 and references therein).The temperate phage FSW-TI was isolated from L. casei strain Shirota (S1) after chemical mutagenesis of a lysogen followed by heat induction (22,24). Since the mutation responsible for the thermoinducibility was shown to reside in the phage DNA, it seemed likely that it could affect the stability of the phage repressor at the elevated temperature. In this study, we have characterized a promoter and a cognate repressor of FSW-TI with the potential to be exploited in a thermoinducible food-grade expression system for L. casei.Isolation of a promoter and a cognate heat-labile repressor of FSW-TI in Escherichia coli. The following strategy was used to isolate the thermoinducible promoter-repressor cassette of phage FSW-TI. The FSW-TI DNA was first shotgun cloned into the E. coli promoter test vector pKK232-8 (Amersham Pharmacia) carrying a promoterless cat gene. Chloramphenicol (Cm)-resistant clones were cotransformed with a FSW-TI library generated in the compatible plasmid pK194 (7). The DNA sequence encoding the protein with apparent repressor activity and its cognate promoter sequence were then obtained from clones that were sensitive and resistant to the antibiotic at 28 and 42°C, respectively.Phage FSW-TI DNA was prepared as described earlier (23). The DNA was cleaved with eithe...
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