Primary structure and functional analysis of the lysis genes of Lactobacillus gasseri bacteriophage phi adh

Abstract

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...

Expression of Mycobacteriophage Ms6 Lysis Genes Is Driven by Two σ70-Like Promoters and Is Dependent on a Transcription Termination Signal Present in the Leader RNA

“…However, permeabilization of the plasma membrane by chloroform upon expression of the lysA gene resulted in immediate lysis. These observations are consistent with other studies, in which the overexpression of phage lysins in the absence of the respective holin functions does not inhibit E. coli growth unless chloroform has been added (3,15). These results suggest that ORF2 encodes a lysin with mureinolytic activity that is functional in E. coli.…”

“…The holin is a hydrophobic membrane protein that forms pores or lesions in the cell membrane through which the murein hydrolase is released to the periplasm and gains access to the peptidoglycan substrate. Such a dual-component lysis system has recently been discovered in many bacteriophages of both gram-negative and grampositive bacteria (15,17,22,25,27,32). The synthesis of the bacteriophage holin (protein S) is tightly controlled at the transcriptional, translational, and posttranslational levels because it determines the time of lysis.…”

The lytic cassette of mycobacteriophage Ms6 encodes an enzyme with lipolytic activity

“…Growth and viability of the recombinant strain E. coli JM109 (pMP302) was not affected, even after the addition of 2 % CHCl 3 (data not shown). It has been shown for other phages that expression of a bacteriophage lysin gene in E. coli causes cellular lysis after permeabilization of the plasma membrane with chloroform (Chandry et al, 1997;Henrich et al, 1995); this effect is also observed with the lysin LysA of mycobacteriophage Ms6 (Garcia et al, 2002). The result indicates that LysB is not an additional endolysin.…”

Sequence analysis of the lactococcal bacteriophage bIL170: insights into structural proteins and HNH endonucleases in dairy phages The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are bIL170, AF009630; bIL120, AY054975; bIL15, AY054976; bIL191, AY054977; bIL77, AY054978.

“…They are detailed in the remaining paragraphs according to their functional category. Structural genes Structural proteins (Chung et al, 1991 ;Kim & Batt, 1991a) (Lubbers et al, 1995), origin of replication (Waterfield et al, 1996), lysogeny (Nauta et al, 1996) (Johnsen et al, 1996 ;Johnsen et al, 1995 ;Pedersen et al, 2000), replication (Ostergaard et al, 2001), lysogeny (Breuner et al, 1999 ;Christiansen et al, 1996) (Kakikawa et al, 1996), lysis (Oki et al, , 1996 phi adh Lactobacillus gasseri AJ131519* Lysis (Henrich et al, 1995), structural proteins and origin of replication (Altermann et al, 1999) …”