Characterization of the bacteriophage φ29‐encoded protein p16.7: a membrane protein involved in phage DNA replication

Abstract

SummaryAn early expressed operon, located at the right end of the linear bacteriophage f29 genome, contains open reading frame (ORF)16.7, whose deduced protein sequence of 130 amino acids is conserved in f29-related phages. Here, we show that this ORF actually encodes a protein, p16.7, which is abundantly and early expressed after infection. p16.7 is a membrane protein, and the N-terminally located transmembrane-spanning domain is required for its membrane localization. The variant p16.7A, in which the N-terminal membrane anchor was replaced by a histidine-tag, was purified and characterized. Purified p16.7A was shown to form dimers in solution. To study the in vivo role of p16.7, a f29 mutant containing a suppressible mutation in gene 16.7 was constructed. In vivo phage DNA replication was affected in the absence of p16.7, especially at early infection times. Based on the results, the putative role of p16.7 in in vivo f29 DNA replication is discussed.

Dynamic localization of membrane proteins in Bacillus subtilis

“…All the proteins appeared to have a similar heterogeneous pattern of localization that was strikingly different from the distribution of total membrane when observed with the membrane stain FM4-64. The localization pattern for p16.7 has previously been reported and described as being similar to that of FM4-64 (Meijer et al, 2001). Indeed, in this work we used the same strain, 110WA, to observe p16.7 localization.…”

“…In the absence of other phage proteins, p16.7 is simply a small integral membrane protein with no known role in B. subtilis physiology. The localization of a p16.7-GFP fusion has been previously reported and shown to be confined to the cytoplasmic membrane in a pattern very similar to FM4-64 (Meijer et al, 2001). However, we found that the p16.7-GFP localization pattern was indistinguishable from that of either SdhA-GFP or AtpA-GFP (Fig.…”

“…2D), suggesting that this punctate distribution pattern was most likely a general feature for the localization of integral membrane proteins. An explanation for the apparently different localization pattern for p16.7 found by us and by Meijer et al (2001) is given in the Discussion.…”

“…However, we do not feel that our results contradict those previously reported; rather, our analysis and interpretation has been more focused on the signal heterogeneity. In addition, our images were processed to remove out-of-focus light (see Methods) whereas Meijer et al (2001) used unprocessed images. Both sets of results indicate that p16.7 is distributed within the cytoplasmic membrane, and it is possible to observe signal heterogeneity in the insert box of Fig.…”

“…Penicillinbinding proteins involved in cell wall synthesis have also been shown to localize in several patterns, including to division septa and discrete foci along the long axis of the cell, reflecting the role of these proteins in specific stages of cell wall synthesis (Scheffers et al, 2004). However, analysis of the localization of the E. coli Sec protein secretion machinery, BglF sugar sensor and B. subtilis phage w29 DNA replication protein p16.7 indicates that these proteins are homogeneously distributed around the cytoplasmic membrane, with no reported preference for cell poles or other observable domains (Brandon et al, 2003;Lopian et al, 2003;Meijer et al, 2001). Certainly, the images presented in these papers indicate that the localization pattern of these proteins creates a clear outline of the cytoplasmic membrane.…”