Gene organization in a central DNA fragment of Oenococcus oeni bacteriophage fOg44 encoding lytic, integrative and non-essential functions

Parreira, Ricardo; São-José, Carlos; Isidro, Anabela; Domingues, Susana; Vieira, G.; Santos, Mário A.

doi:10.1016/s0378-1119(98)00554-x

Cited by 23 publications

(13 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We have recently described the sequences of the lysin and holin genes from the Oenococcus oeni bacteriophage fOg44 and noted that the N-terminal region of its putative lysin (Lys44) was highly hydrophobic (23). A similar observation was made earlier concerning a related enzyme from the lactococcal phage Tuc2009 (2).…”

supporting

confidence: 64%

“…6, lanes 8 and 9). Lys⌬C lacks the C-terminal 103 residues of Lys44 (Table 2), encompassing the pair of 48-aa repeats which were previously identified in its sequence (23).…”

Section: Resultsmentioning

confidence: 99%

“…The active, presumably exported form of Lys44 was first detected in O. oeni-infected cells at 80 min postinfection, a time matching the appearance of a 1.8-kb transcript specifically hybridizing with probes internal to the fOg44 lysis genes (23). Considering that under the conditions used here, the fOg44 latent period extends for about 150 min (28), it would appear that the lysin is targeted to the cell wall compartment as it is being synthesized, rather than at the end of phage maturation, as observed in and related systems.…”

Section: Figmentioning

confidence: 89%

“…Totalprotein extracts were prepared by resuspending the pellet from 1-ml culture samples in 0.1 ml of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (19) followed by treatment at 100°C for 5 min. Proteins (5 l per lane, unless otherwise indicated) were analyzed by electrophoresis on SDS-11% PAGE gels and visualized by Coomassie blue staining or blotted onto nitrocellulose membranes (Bio-Rad) as previously described (23). Lysin polypeptides were immunodetected using rabbit polyclonal antibodies raised against the 51-kDa His-Lys44 fusion protein (anti-Lys antibodies).…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

The N-Terminal Region of the Oenococcus oeni Bacteriophage fOg44 Lysin Behaves as a Bona Fide Signal Peptide in Escherichia coli and as a cis -Inhibitory Element, Preventing Lytic Activity on Oenococcal Cells

et al. 2000

Self Cite

View full text Add to dashboard Cite

The function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an export signal was investigated. We observed that when induced in Escherichia coli, Lys44 was cleaved between residues 27 and 28 in a SecA-dependent manner. Lys44 processing could be blocked by a specific signal peptidase inhibitor and was severely reduced by modification of the cleavage site. The lethal effect of Lys44 expression observed in E. coli was ascribed to the presence of its N-terminal 27-residue sequence, as its deletion resulted in the production of a nontoxic, albeit active, product. We have further established that lytic activity in oenococcal cells was dependent on Lys44 processing. An active protein with the molecular mass expected for the cleaved enzyme was detected in extracts from O. oeni-infected cells. The temporal pattern of its appearance suggests that synthesis and export of Lys44 in the infected host progress along with phage maturation. Overall, these results provide, for the first time, experimental evidence for the presence of a signal peptide in a bacteriophage lysin. Database searches and alignment of protein sequences support the prediction that other known O. oeni and Lactococcus lactis phages also encode secretory lysins. The evolutionary significance of a putative phage lysis mechanism relying on secretory lytic enzymes is tentatively discussed, on the basis of host cell wall structure and autolytic capacity.All tailed bacteriophages with double-stranded DNA genomes appear to accomplish lysis of the host cell by the concerted action of a peptidoglycan hydrolase (referred to as endolysin or lysin) and a small hydrophobic protein (holin) presumed to form nonspecific lesions upon oligomerization in the membrane (for a review, see reference 41). The latter function seems essential to allow access of the lytic enzyme to the cell wall compartment since in the phage lysins examined so far, the presence of a signal peptide (SP) that would target them to the translocase of the general secretion pathway (GSP) has never been demonstrated.We have recently described the sequences of the lysin and holin genes from the Oenococcus oeni bacteriophage fOg44 and noted that the N-terminal region of its putative lysin (Lys44) was highly hydrophobic (23). A similar observation was made earlier concerning a related enzyme from the lactococcal phage Tuc2009 (2). In spite of its hydrophobic character, the function of the N-terminal sequence of the Tuc2009 lysin as a possible SP was not considered, presumably because the presence of a holin gene upstream of lys argued for a standard holin-dependent lysin export mechanism. This assumption was strengthened by the observation that the expression of an almost identical lysin in Escherichia coli (LysB from the Lactococcus lactis phage LC3) did not result in a decrease in culture absorbance unless the corresponding holin was simultaneously induced (3).Interestingly, however, during an attempt to overproduce Lys44 in an easily purifiable form (as a histidine-tagged fusi...

show abstract

supporting

confidence: 64%

“…6, lanes 8 and 9). Lys⌬C lacks the C-terminal 103 residues of Lys44 (Table 2), encompassing the pair of 48-aa repeats which were previously identified in its sequence (23).…”

Section: Resultsmentioning

confidence: 99%

Section: Figmentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The N-Terminal Region of the Oenococcus oeni Bacteriophage fOg44 Lysin Behaves as a Bona Fide Signal Peptide in Escherichia coli and as a cis -Inhibitory Element, Preventing Lytic Activity on Oenococcal Cells

et al. 2000

Self Cite

View full text Add to dashboard Cite

show abstract

“…An inverted organization (lys upstream of hol) was reported for the Oenococcus oeni bacteriophage fOg44 (24), and in many cases the genes are not even linked (e.g., T4) (29). In many phages of Streptococcus thermophilus (30) and in phage Av-1, which infects the Gram-positive bacteria Actinomyces naeslundii (3), two putative holin genes precede the endolysin gene.…”

Section: Discussionmentioning

confidence: 99%

Functional Analysis of the Holin-Like Proteins of Mycobacteriophage Ms6

et al. 2011

View full text Add to dashboard Cite

The mycobacteriophage Ms6 is a temperate double-stranded DNA (dsDNA) bacteriophage which, in addition to the predicted endolysin (LysA)-holin (Gp4) lysis system, encodes three additional proteins within its lysis module: Gp1, LysB, and Gp5. Ms6 Gp4 was previously described as a class II holin-like protein. By analysis of the amino acid sequence of Gp4, an N-terminal signal-arrest-release (SAR) domain was identified, followed by a typical transmembrane domain (TMD), features which have previously been observed for pinholins. A second putative holin gene (gp5) encoding a protein with a predicted single TMD at the N-terminal region was identified at the end of the Ms6 lytic operon. Neither the putative class II holin nor the single TMD polypeptide could trigger lysis in pairwise combinations with the endolysin LysA in Escherichia coli. One-step growth curves and single-burst-size experiments of different Ms6 derivatives with deletions in different regions of the lysis operon demonstrated that the gene products of gp4 and gp5, although nonessential for phage viability, appear to play a role in controlling the timing of lysis: an Ms6 mutant with a deletion of gp4 (Ms6 ⌬gp4 ) caused slightly accelerated lysis, whereas an Ms6 ⌬gp5 deletion mutant delayed lysis, which is consistent with holin function. Additionally, cross-linking experiments showed that Ms6 Gp4 and Gp5 oligomerize and that both proteins interact. Our results suggest that in Ms6 infection, the correct and programmed timing of lysis is achieved by the combined action of Gp4 and Gp5.

show abstract