The importance of the content of anionic phospholipids [cardiolipin (CL) and phosphatidylglycerol (PG)] in the osmotic adaptation and in the membrane structure of Bacillus subtilis cultures was investigated. Insertion mutations in the three putative cardiolipin synthase genes (ywiE, ywnE and ywjE) were obtained. Only the ywnE mutation resulted in a complete deficiency in cardiolipin and thus corresponds to a true clsA gene. The osmotolerance of a clsA mutant was impaired: although at NaCl concentrations lower than 1?2 M the growth curves were similar to those of its wild-type control, at 1?5 M NaCl (LBN medium) the lag period increased and the maximal optical density reached was lower. The membrane of the clsA mutant strain showed an increased PG content, at both exponential and stationary phase, but no trace of CL in either LB or LBN medium. As well as the deficiency in CL synthesis, the clsA mutant showed other differences in lipid and fatty acids content compared to the wild-type, suggesting a cross-regulation in membrane lipid pathways, crucial for the maintenance of membrane functionality and integrity. The biophysical characteristics of membranes and large unilamellar vesicles from the wild-type and clsA mutant strains were studied by Laurdan's steady-state fluorescence spectroscopy. At physiological temperature, the clsA mutant showed a decreased lateral lipid packing in the protein-free vesicles and isolated membranes compared with the wild-type strain. Interestingly, the lateral lipid packing of the membranes of both the wild-type and clsA mutant strains increased when they were grown in LBN. In a conditional IPTG-controlled pgsA mutant, unable to synthesize PG and CL in the absence of IPTG, the osmoresistance of the cultures correlated with their content of anionic phospholipids. The transcriptional activity of the clsA and pgsA genes was similar and increased twofold upon entry to stationary phase or under osmotic upshift. Overall, these results support the involvement of the anionic phospholipids in the growth of B. subtilis in media containing elevated NaCl concentrations.

SummaryDuring morphogenesis of mature HIV-1 cores, the viral capsid (CA) proteins assemble conical or tubular shells around the viral ribonucleoprotein complexes. This assembly step is mimicked in vitro through reactions in which CA proteins oligomerize to form long tubes, and this process can be modeled as consisting of a slow nucleation period followed by a rapid phase of tube growth. We have developed a novel fluorescence microscopy approach to monitor in vitro assembly reactions and have employed it, along with electron microscopy analysis, to characterize the assembly process. Our results indicate that temperature, salt concentration, and pH changes have differential effects on tube nucleation and growth steps. We also demonstrate that assembly can be unidirectional or bidirectional, that growth can be capped, and that proteins can assemble onto the surfaces of tubes, yielding multiwalled or nested structures. Finally, experiments show that a peptide inhibitor of in vitro assembly also can dismantle pre-existing tubes, suggesting that such reagents may possess antiviral effects against both viral assembly and uncoating. Our investigations help establish a basis for understanding the mechanism of mature HIV-1 core assembly, and avenues for antiviral inhibition.

No abstract

Burkholderia pseudomallei is a gram-negative facultative intracellular pathogen that causes melioidosis, an invasive disease of humans and animals. To address the response of this bacterium to iron-limiting conditions, we first performed a global transcriptional analysis of RNA extracted from bacteria grown under iron-limiting and iron-rich conditions by microarrays. We focused our study on those open reading frames (ORFs) induced under iron limitation, which encoded predicted proteins that could be involved in the biosynthesis and uptake of the siderophore malleobactin. We purified this siderophore and determined that it consisted of at least three compounds with different molecular weights. We demonstrated that ORFs BPSL1776 and BPSL1774, designated mbaA and mbaF, respectively, are involved in the biosynthesis of malleobactin, while BPSL1775, named fmtA, is involved in its transport. These genes are in an operon with two other ORFs (mbaJ and mbaI) whose transcription is under the control of MbaS, a protein that belongs to the extracytoplasmic function sigma factors. Interestingly, the transcription of the mbaA, fmtA, and mbaS genes is not controlled by the availability of the siderophore malleobactin.Burkholderia pseudomallei is a gram-negative facultative intracellular pathogen that is the causative agent of melioidosis. Although this invasive disease is endemic to southeast Asia and northern Australia, its worldwide availability, high rate of mortality, and aerosol infectivity resulted in its classification as a select agent (12, 65). B. pseudomallei is also a saprophytic organism that has developed high resistance to many hostile environmental conditions such as acidic environments, dehydration, prolonged nutrient starvation, and antiseptic and detergent solutions (12).Despite increasing knowledge on the epidemiology of this disease, very little is known on the molecular mechanisms of the infection; only a few virulence factors, i.e., the capsular polysaccharide, lipopolysaccharide, type IV pilus, and a type III secretion apparatus, have been described with animal models (55,64). This pathogen can invade phagocytic and nonphagocytic cells using the type III secretion apparatus to promote its escape into the cytoplasm, where the bacterium induces polar actin polymerization (56, 57). Intracellular B. pseudomallei cells can induce host cell membrane protrusions that may facilitate cell-to-cell spread of the bacteria and induce the fusion of adjacent cells forming giant multinucleated cells (24,25,54). Sequencing of the two B. pseudomallei chromosomes (22) has generated a significant amount of data expected to yield information on new virulence factors and putative targets for vaccine development.A potential virulence factor in B. pseudomallei that so far has not been studied is the ability to utilize iron, a feature that may play an important role in the pathogenesis of the disease caused by this bacterium. Iron is required for the growth of nearly all microorganisms in the environment, as well as in biologica...