No abstract

FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of about a dozen division proteins that localize to the septal ring. FtsI comprises a short amino-terminal cytoplasmic domain, a single transmembrane helix (TMH), and a large periplasmic domain that encodes the catalytic (transpeptidase) activity. We show here that a 26-amino-acid fragment of FtsI is sufficient to direct green fluorescent protein to the septal ring in cells depleted of wild-type FtsI. This fragment extends from W22 to V47 and corresponds to the TMH. This is a remarkable finding because it is usual for a TMH to target a protein to a site more specific than the membrane. Alanine-scanning mutagenesis of the TMH identified several residues important for septal localization. These residues cluster on one side of an alpha-helix, which we propose interacts directly with another division protein to recruit FtsI to the septal ring.The use of fluorescence microscopy to visualize proteins in bacteria has revealed that many proteins are not distributed randomly but instead localize to specific subcellular sites, such as the midcell or pole(s) (22, 37). Moreover, proteins that are targeted to specific sites often fail to function properly if they are mislocalized. Despite the importance of proper localization, little is known about how targeting information is encoded in the amino acid sequences of bacterial proteins. In this report, we describe a small peptide from a bacterial cell division protein, FtsI, that is sufficient to target green fluorescent protein (GFP) to the division site in Escherichia coli. Interestingly, this peptide is a transmembrane helix (TMH). These findings help to clarify how targeting information is encoded in FtsI's primary sequence and demonstrate that a bacterial TMH can serve as a targeting signal.FtsI, also known as penicillin-binding protein 3 (PBP3), is a transpeptidase needed for cross-linking septal peptidoglycan (1,3,38). Previous studies from a number of laboratories have shown that FtsI is one of over a dozen proteins that localize to the division site, where they form a structure called the septal ring (for recent reviews, see references 12 and 43). As division proceeds, the ring constricts so as to remain at the leading edge of the developing septum. The septal ring is thought to be a multiprotein complex that mediates cell division. Studies of septal ring assembly in various mutant backgrounds have revealed that, at least in E. coli, the division proteins are recruited to the ring in a sequential fashion. In this hierarchy, FtsI is one of the last proteins to join the ring; localization of FtsI appears to depend upon the prior localization of FtsZ, FtsA, ZipA, FtsEX (though this is a leaky requirement), FtsK, FtsQ, FtsBL, and FtsW. This scheme suggests that FtsI is recruited to the septal ring by a cascade of protein-protein interactions involved in the assembly of a multiprotein complex. Moreover, FtsI might localize by binding to ...

Phage lambda DNA packaging is accompanied by prohead expansion, due to structural changes in gpE, the major capsid protein. Rearrangement of the gpE lattice creates binding sites for trimers of gpD, the head stabilization protein. lambda-Like phage 21's shp gene is homologous to lambda's D gene. gpD and gpShp share 49% amino acid identity. To ask whether gpShp could stabilize the lambda head shell, we replaced lambda's D gene with shp, creating lambda shp. Unlike lambda or 21, lambda shp was strictly dependent on the presence of 10(-2) M Mg2+, and lambda shp virions were very sensitive to chelating agents. Density gradient studies indicated that the lambda gpE lattice was underpopulated with gpShp. gpD's N-terminus has been proposed to contact gpE, and we found that lambda D/shp, which produces a chimeric protein with the N-terminus of gpD and the C-terminus of gpShp, was Mg2+-independent and more stable than lambda shp.

Sepsis caused by multidrug-resistant bacterial infections in critically injured patients has become a major clinical problem. Recently, Acinetobacter baumannii (AB) wound infections, especially in our critically injured soldiers fighting in Iraq and Afghanistan, is posing a major clinical problem and an economic burden. ConjuGon, Inc., has developed a novel antibacterial therapeutic technology using bacterial conjugation. The donor cells are attenuated Escherichia coli carrying a conjugative plasmid. The expression of bactericidal genes cloned on the plasmid is tightly repressed in the donor cells but becomes de-repressed once mobilized into a pathogen and disrupts protein synthesis. Here, we tested the efficacy of this novel conjugation technology to control and eradicate a drug-resistant clinical isolate of AB wound infection both in vitro and in a murine burn sepsis model. C57Blk/6J mice were divided into burn (B) and burn sepsis (BS) groups. All animals received a 12% TBSA dorsal scald full-thickness burn. The BS group was inoculated with multidrug-resistant AB (1 x 10(5) colony-forming units [CFU]) at the burn wound site. BS animals were either untreated or treated with increasing concentrations (10(3) - 19(10) CFU) of attenuated donor E. coli encoding bactericidal proteins. The survival rate was monitored for 10 days. The ability of donor cells to significantly diminish AB levels in the burn wound 24 hours after injury was determined by quantitative cultures. Donor cells were highly effective in killing AB in vitro. In the burn sepsis model, 90% B group animals survived, and 40% to 50% BS animals survived with no treatment in 5 to 6 days. Treatment with donor cells at 10(10) to 10(6) provided significant survival advantage (P < .05). Quantitative cultures of burn wounds revealed that AB numbers increased from 3 x 10(4) CFU to 7.8 +/- 4.4 x 10(9) CFU in 24 hours in the untreated group. Single treatment with donor cells (10(10) CFU) significantly reduced AB in the burn wound to less than the levels seeded into the wound (1.23 +/- 0.5 x 10(4) CFU; P < .05). Taken together, these results indicate that this novel technology is an efficient method to control drug-resistant AB burn wound infections and prevent their systemic spread.