A predicted ABC transporter, FtsEX, is needed for cell division in Escherichia coli

FtsE and FtsX have homology to the ABC transporter superfamily of proteins and appear to be widely conserved among bacteria. Early work implicated FtsEX in cell division in Escherichia coli, but this was subsequently challenged, in part because the division defects in ftsEX mutants are often salt remedial. Strain RG60 has an ftsE::kan null mutation that is polar onto ftsX. RG60 is mildly filamentous when grown in standard Luria-Bertani medium (LB), which contains 1% NaCl, but upon shift to LB with no NaCl growth and division stop. We found that FtsN localizes to potential division sites, albeit poorly, in RG60 grown in LB with 1% NaCl. We also found that in wild-type E. coli both FtsE and FtsX localize to the division site. Localization of FtsX was studied in detail and appeared to require FtsZ, FtsA, and ZipA, but not the downstream division proteins FtsK, FtsQ, FtsL, and FtsI. Consistent with this, in media lacking salt, FtsA and ZipA localized independently of FtsEX, but the downstream proteins did not. Finally, in the absence of salt, cells depleted of FtsEX stopped dividing before any change in growth rate (mass increase) was apparent. We conclude that FtsEX participates directly in the process of cell division and is important for assembly or stability of the septal ring, especially in salt-free media.     

Nonreducing terminal modifications determine the chain length of polymannose O antigens of Escherichia coli and couple chain termination to polymer export via an ATP-binding cassette transporter

The chain length of bacterial lipopolysaccharide O antigens is regulated to give a modal distribution that is critical for pathogenesis. This paper describes the process of chain length determination in the ATP-binding cassette (ABC) transporter-dependent pathway, a pathway that is widespread among Gram-negative bacteria. Escherichia coli O8 and O9/O9a polymannans are synthesized in the cytoplasm, and an ABC transporter exports the nascent polymer across the inner membrane prior to completion of the LPS molecule. The polymannan O antigens have nonreducing terminal methyl groups. The 3-O-methyl group in serotype O8 is transferred from S-adenosylmethionine by the WbdD(O8) enzyme, and this modification terminates polymerization. Methyl groups are added to the O9a polymannan in a reaction dependent on preceding phosphorylation. The bifunctional WbdD(O9a) catalyzes both reactions, but only the kinase activity controls chain length. Chain termination occurs in a mutant lacking the ABC transporter, indicating that it precedes export. An E. coli wbdD(O9a) mutant accumulated O9a polymannan in the cytoplasm, indicating that WbdD activity coordinates polymannan chain termination with export across the inner membrane.     

Rho is involved in superoxide formation during phagocytosis of opsonized zymosans

Phagocytosis is accompanied by the production of superoxide by the NADPH oxidase complex, for which GTP-bound Rac is essential. We wanted to determine whether Rho is also involved in the production of superoxide during phagocytosis. Inhibition of Rho by Tat-C3 exoenzyme (Tat-C3) blocked superoxide formation and curtailed the phagocytosis of serum- (SOZ), C3bi- (COZ), and IgG-opsonized zymosan (IOZ) particles. Tat-C3 did not affect superoxide formation in response to phorbol myristate acetate (PMA), formyl Met-Leu-Phe (fMLP), or macrophage colony-stimulating factor (M-CSF). Superoxide formation was also reduced in J774 cells transfected with a cDNA expressing dominant-negative form of RhoA (N19RhoA). However, purified prenylated recombinant RhoA did not activate NADPH oxidase in vitro, suggesting that Rho does not interact directly with NADPH oxidase. Tat-C3 inhibited the activity of RhoA, but did not affect that of Rac in vitro or in vivo. It also inhibited the phosphorylation of p47(PHOX), one of the cytosolic components of NADPH oxidase. Taken together, these results suggest that Rho plays an important role in superoxide formation during phagocytosis of SOZ, COZ, and IOZ via phosphorylation of p47(PHOX).     

Antagonistic cross-talk between Rac and Cdc42 GTPases regulates generation of reactive oxygen species

Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b(558) and p67(phox) oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b(558) that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b(558) in vitro. Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.     

Inhibition of dengue virus serotypes 1 to 4 in vero cell cultures with morpholino oligomers

Five dengue (DEN) virus-specific R5F2R4 peptide-conjugated phosphorodiamidate morpholino oligomers (P4-PMOs) were evaluated for their ability to inhibit replication of DEN virus serotype 2 (DEN-2 virus) in mammalian cell culture. Initial growth curves of DEN-2 virus 16681 were obtained in Vero cells incubated with 20 microM P4-PMO compounds. At 6 days after infection, a P4-PMO targeting the 3'-terminal nucleotides of the DEN-2 virus genome and a random-sequence P4-PMO showed relatively little suppression of DEN-2 virus titer (0.1 and 0.9 log10, respectively). P4-PMOs targeting the AUG translation start site region of the single open reading frame and the 5' cyclization sequence region had moderate activity, generating 1.6- and 1.8-log10 reductions. Two P4-PMO compounds, 5'SL and 3'CS (targeting the 5'-terminal nucleotides and the 3' cyclization sequence region, respectively), were highly efficacious, each reducing the viral titer by greater than 5.7 log10 compared to controls at 6 days after infection with DEN-2 virus. Further experiments showed that 5'SL and 3'CS inhibited DEN-2 virus replication in a dose-dependent and sequence-specific manner. Treatment with 10 microM 3'CS reduced the titers of all four DEN virus serotypes, i.e., DEN-1 (strain 16007), DEN-2 (16681), DEN-3 (16562), and DEN-4 (1036) viruses by over 4 log10, in most cases to below detectable limits. The extent of 3'CS efficacy was affected by the timing of compound application in relation to viral infection of the cells. The 5'SL and 3'CS P4-PMOs did not suppress the replication of West Nile virus NY99 in Vero cells. These data indicate that further evaluation of the 5'SL and 3'CS compounds as potential DEN virus therapeutics is warranted.     

The size and position of heterologous insertions in a silent locus differentially affect pilin recombination in Neisseria gonorrhoeae

Gonococcal pilus antigenic and phase variation result from unidirectional, RecA-dependent recombination of DNA sequences from a silent pilin copy ( pilS  ) into the expressed pilin gene ( pilE  ). To develop a quantitative assay for pilin gene recombination that is independent of phase variation, a promoterless cat gene was inserted into pilS , and recombination of ' cat into pilE was detected by selection of chloramphenicol-resistant (Cm^R) variants expressing ' cat from the pilin promoter. Although RecA-dependent Cm^R variants occurred, none were generated by the simple transfer of ' cat into pilE . Instead, each Cm^R variant contained a new pilin locus that was a hybrid of sequences from the pilE and the pilS1 ::' cat loci in addition to the two starting loci. Therefore, this system could not be used to quantify antigenic variation. However, combined studies of these hybrid loci and of recombination products generated during additional pilS mutational analyses demonstrated that both the size and position of an insertion in pilS differentially affect pilin recombination. Also, the hybrid loci appear to be intermediates of antigenic variation. This enabled the creation of molecular models for the recombination reactions that result in pilin antigenic variation.     

Long-term trends in seasonal plankton dynamics in Lake Mead (Nevada-Arizona,USA) and implications for climate change

Where biodiversity conservation and environmental preservation are significant concerns, rapid assessment and monitoring of the colonization and spread of non-native species are essential for timely decision-making and response. We developed and evaluated the adequacy of a rapid assessment protocol (RAP) for detecting non-native fish species in 74 Eastern Brazilian lakes. The RAP consists of a single field day employing two surveyors to conduct interviews with local sport fishers, visual surveys of fish, angling with lures, and gillnetting. We compared our results with those from separate, intense, large sampling effort (LSE) field surveys. Despite requiring less than 1/100th of the field effort, the RAP was able to detect the presence of most non-native fish species that were reported in the same lakes by LSE surveys. Information from local sport fishers was invaluable, particularly for detecting species that were in low abundance, but was not available for lakes within a forest preserve area. Non-native introductions commonly lead to rapid and widespread invasion and adverse effects on native biota. Our results strongly suggest that the RAP could function as a cost-effective tool for efficiently assessing the presence of non-native fishes in lakes and monitoring their colonization and spread over large geographic areas.     

Effects of auxin concentration on induction and growth of embryogenic callus from young inflorescence explants of Old World bluestem (Bothriochloa spp.) and bermuda (Cynodon spp.) grasses

Objectives of this research were to test the effects of plant genotypes and auxin 2,4-D (2,4-dichlorophenoxyacetic acid) medium concentrations on embryogenic (E) callus production of two grass species. Two Old World bluestem,Bothriochloa ischaemum, accessions (A-8793 and A-8911c) and three bermudagrass,Cynodon dactylon (L.) Pers., accessions (A-10978b, A12164, and Brazos) supplied the explant material. Immature inflorescences 9 mm in length were placed on modified Murashige-Skoog (MS) agar medium containing 0, 1, 3, or 5 mg L^-1 of 2,4-D. Explants of all genotypes produced callus by the end of a 4-week dark incubation period at 25°C. When subcultured onto fresh media and maintained at 25°C with a 16 hr photoperiod, calli became embryogenic within 8 weeks of inoculation. Three mg L^-1 of 2,4-D in the media maximized E callus production in both bluestem genotypes and in A-10978b and A-12164 bermudagrass genotypes. Maximum E callus production from Brazos bermudagrass resulted from the 1 mg L^-1 treatment. Somatic embryos developed after subculture under light. Embryos showed scutellum-like structures and coleoptile-coleorhiza bipolar organization. Plantlets were regenerated from all genotypes except Brazos, whose embryoids failed to germinate. All callus from Brazos eventually senesced. Light and scanning electron microscopy confirmed regeneration through somatic embryogenesis.