The integral membrane protein p16.7 organizes in vivo φ29 DNA replication through interaction with both the terminal protein and ssDNA

Remarkably little is known about the in vivo organization of membrane-associated prokaryotic DNA replication or the proteins involved. We have studied this fundamental process using the Bacillus subtilis phage φ29 as a model system. Previously, we demonstrated that the φ29-encoded dimeric integral membrane protein p16.7 binds to ssDNA and is involved in the organization of membrane-associated φ29 DNA replication. Here we demonstrate that p16.7 forms multimers, both in vitro and in vivo, and interacts with the φ29 terminal protein. In addition, we show that in vitro multimerization is enhanced in the presence of ssDNA and that the C-terminal region of p16.7 is required for multimerization but not for ssDNA binding or interaction with the terminal protein. Moreover, we provide evidence that the ability of p16.7 to form multimers is crucial for its ssDNA-binding mode. These and previous results indicate that p16.7 encompasses four distinct modules. An integrated model of the structural and functional domains of p16.7 in relation to the organization of in vivo φ29 DNA replication is presented

Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from Gram-positive bacteria

Most small plasmids of Gram-positive bacteria use the rolling-circle mechanism of replication and several of these have been studied in considerable detail at the DNA level and for the function of their genes. Although most of the common laboratory Bacillus subtilis 168 strains do not contain plasmids, several industrial strains and natural soil isolates do contain rolling-circle replicating (RCR) plasmids. So far, knowledge about these plasmids was mainly limited to: (i) a classification into seven groups, based on size and restriction patterns; and (ii) DNA sequences of the replication region of a limited number of them. To increase the knowledge, also with respect to other functions specified by these plasmids, we have determined the complete DNA sequence of four plasmids, representing different groups, and performed computer-assisted and experimental analyses on the possible function of their genes. The plasmids analyzed are pTA1015 (5.8 kbp), pTA1040 (7.8 kbp), pTA1050 (8.4 kbp), and pTA1060 (8.7 kbp). These plasmids have a structural organization similar to most other known RCR plasmids. They contain highly related replication functions, both for leading and lagging strand synthesis. pTA1015 and pTA1060 contain a mobilization gene enabling their conjugative transfer. Strikingly, in addition to the conserved replication modules, these plasmids contain unique module(s) with genes which are not present on known RCR plasmids of other Gram-positive bacteria. Examples are genes encoding a type I signal peptidase and genes encoding proteins belonging to the family of response regulator aspartate phosphatases. The latter are likely to be involved in the regulation of post-exponential phase processes. The presence of these modules on plasmids may reflect an adaptation to the special conditions to which the host cells were exposed.

Type I signal peptidases of Bacillus subtilis

Bacillus subtilis contains at least three chromosomally-encoded type I signal peptidases (SPases; SipS, SipT, and SipU), which remove signal peptides from secretory proteins. In addition, certain B. subtilis (natto) strains contain plasmid-encoded type I SPases (SipP). The known type I SPases from B. subtilis show a high degree of similarity to SPases from related bacilli and Staphylococcus aureus and, to a lesser extent, to SPases from other organisms. In addition, the putative active site region of the Bacillus SPases shows similarity to the corresponding region of LexA-like proteases, suggesting that the type I SPases employ a serine-lysine catalytic dyed. Unlike the type I SPase of Escherichia coli, Sips, SipT and SipU are neither essential for protein secretion, nor for viability of the cell. Although non-essential, Sips is an important factor for efficient protein secretion. SipS is transcribed in a growth phase-and medium-dependent manner. Under some conditions, transcription of sipS is controlled by the DegS-DegU two-component regulatory system, indicating that expression of sipS is determined by the same factors that control the expression of most genes for secreted degradative enzymes. These observations suggest thats. subtilis can modulate its capacity and specificity for protein secretion through controlled expression of sipS.</p

A Conserved Class II Type Thioester Domain-Containing Adhesin Is Required for Efficient Conjugation in Bacillus subtilis

Conjugation, the process by which a DNA element is transferred from a donor to a recipient cell, is the main horizontal gene transfer route responsible for the spread of antibiotic resistance and virulence genes. Contact between a donor and a recipient cell is a prerequisite for conjugation, because conjugative DNA is transferred into the recipient via a channel connecting the two cells. Conjugative elements encode proteins dedicated to facilitating the recognition and attachment to recipient cells, also known as mating pair formation. A subgroup of the conjugative elements is able to mediate efficient conjugation during planktonic growth, and mechanisms facilitating mating pair formation will be particularly important in these cases. Conjugative elements of Gram-negative bacteria encode conjugative pili, also known as sex pili, some of which are retractile. Far less is known about mechanisms that promote mating pair formation in Gram-positive bacteria. The conjugative plasmid pLS20 of the Gram-positive bacterium Bacillus subtilis allows efficient conjugation in liquid medium. Here, we report the identification of an adhesin gene in the pLS20 conjugation operon. The N-terminal region of the adhesin contains a class II type thioester domain (TED) that is essential for efficient conjugation, particularly in liquid medium. We show that TED-containing adhesins are widely conserved in Gram-positive bacteria, including pathogens where they often play crucial roles in pathogenesis. Our study is the first to demonstrate the involvement of a class II type TED-containing adhesin in conjugationPeer reviewe

Type I signal peptidases of Bacillus subtilis

Bacillus subtilis contains at least three chromosomally-encoded type I signal peptidases (SPases; SipS, SipT, and SipU), which remove signal peptides from secretory proteins. In addition, certain B. subtilis (natto) strains contain plasmid-encoded type I SPases (SipP). The known type I SPases from B. subtilis show a high degree of similarity to SPases from related bacilli and Staphylococcus aureus and, to a lesser extent, to SPases from other organisms. In addition, the putative active site region of the Bacillus SPases shows similarity to the corresponding region of LexA-like proteases, suggesting that the type I SPases employ a serine-lysine catalytic dyed. Unlike the type I SPase of Escherichia coli, Sips, SipT and SipU are neither essential for protein secretion, nor for viability of the cell. Although non-essential, Sips is an important factor for efficient protein secretion. SipS is transcribed in a growth phase-and medium-dependent manner. Under some conditions, transcription of sipS is controlled by the DegS-DegU two-component regulatory system, indicating that expression of sipS is determined by the same factors that control the expression of most genes for secreted degradative enzymes. These observations suggest thats. subtilis can modulate its capacity and specificity for protein secretion through controlled expression of sipS

Dynamic relocalization of phage φ29 DNA during replication and the role of the viral protein p16.7

We have examined the localization of DNA replication of the Bacillus subtilis phage φ29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7