Establishment of gene transfer systems for and construction of the genetic map of a marine Vibrio strain.

Two gene transfer systems were established for a marine bacterium, Vibrio sp. strain 60. One was generalized transduction with a newly isolated bacteriophage, As3, and the other was conjugal gene transfer by the use of newly constructed transposon-facilitated recombination (Tfr) donors. As3 transduced various chromosomal markers at frequencies of 10(-4) to 10(-6). Tfr donors, which were constructed by introducing transposon Tn10 into both plasmid RP4 and the chromosome, mediated the polarized transfer of chromosomal genes from the sites of Tn10 insertion on the chromosome. By means of these gene transfer systems, a genetic map of the vibrio chromosome was constructed.     

Genetic analysis of the Rhizobium meliloti bacA gene: functional interchangeability with the Escherichia coli sbmA gene and phenotypes of mutants.

The Rhizobium meliloti bacA gene encodes a function that is essential for bacterial differentiation into bacteroids within plant cells in the symbiosis between R. meliloti and alfalfa. An Escherichia coli homolog of BacA, SbmA, is implicated in the uptake of microcin B17, microcin J25 (formerly microcin 25), and bleomycin. When expressed in E. coli with the lacZ promoter, the R. meliloti bacA gene was found to suppress all the known defects of E. coli sbmA mutants, namely, increased resistance to microcin B17, microcin J25, and bleomycin, demonstrating the functional similarity between the two proteins. The R. meliloti bacA386::Tn(pho)A mutant, as well as a newly constructed bacA deletion mutant, was found to show increased resistance to bleomycin. However, it also showed increased resistance to certain aminoglycosides and increased sensitivity to ethanol and detergents, suggesting that the loss of bacA function causes some defect in membrane integrity. The E. coli sbmA gene suppressed all these bacA mutant phenotypes as well as the Fix- phenotype when placed under control of the bacA promoter. Taken together, these results strongly suggest that the BacA and SbmA proteins are functionally similar and thus provide support for our previous hypothesis that BacA may be required for uptake of some compound that plays an important role in bacteroid development. However, the additional phenotypes of bacA mutants identified in this study suggest the alternative possibility that BacA may be needed for membrane integrity, which is likely to be critically important during the early stages of bacterial differentiation within plant cells.     

Actin Dynamics Regulated by the Balance of Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and Cofilin Activities Determines the Biphasic Response of Glucose-induced Insulin Secretion

Actin dynamics in pancreatic β-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic β-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic β-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic β-cells (MIN6-K8 β-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 β-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 β-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.     

An unusual new species of the genus Brachypalpus Macquart (Diptera: Syrphidae) from Eastern Asia

Brachypalpus (Brachypalpus) longifacies sp. nov. is described and illustrated from Eastern Asia (Russian Far East and Japan). It differs from other species of the nominative subgenus by several unusual characters, i.e. face in profile weakly convex dorsally and concave ventrally, antennae very elongated, and abdomen with reddish transversal maculae on 2nd tergum (male) or 2nd and 3rd terga (female). A key is given for known species of the subgenus Brachypalpus.LSID: urn:lsid:zoobank.org:pub:634CB6E2-B2C1-40EC-8811-BF3DD4BDE69A     

Genetic analysis of Rhizobium meliloti bacA-phoA fusion results in identification of degP: two loci required for symbiosis are closely linked to degP.

The function of the Rhizobium meliloti bacA gene, which is a homolog of the Escherichia coli sbmA gene, is required for an intermediate step in nodule development. A strain carrying the bacA386::TnphoA fusion was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and three mutants that had higher levels of alkaline phosphatase activity were identified. The mutations in these strains were recessive and mapped to the same genetic locus. The gene affected by these mutations was identified and sequenced and was found to be a homolog of the E. coli degP gene, which encodes a periplasmic endopeptidase. Although degP function is important for the virulence of certain intracellular pathogens of mammals, it is not required for the R. meliloti-alfalfa symbiosis. The genetic analyses involving degP were complicated by the presence of a locus immediately upstream of depP that was lethal when present in multiple copies in a DegP- background. R. meliloti derivatives carrying insertion mutations in this locus displayed an N,N,N',N'-tetramethyl-p-phenylenediamine oxidase-negative phenotype, elicited the formation of white cylindrical nodules that did not fix nitrogen, and grew slowly in rich medium, suggesting that the locus was a cyc gene encoding a protein involved in the biosynthesis of a component or components of a respiratory chain. The previously identified fix-382::TnphoA, which similarly causes the formation of white cylindrical nodules that do not fix nitrogen, was shown to affect a gene that is separate from this cyc gene but extremely closely linked to it.     

Distinct uptake of tellurate from selenate in a selenium accumulator, Indian mustard (Brassica juncea)

Tellurium (Te) is widely used in industry because of its unique chemical and physical properties, and has recently become a part of everyday life as a component of phase-change optical magnetic disks. However, the recovery of Te from the environment has not been discussed yet. In this regard, we evaluated the potential use of Indian mustard (Brassica juncea), a selenium (Se) accumulator, for the phytoremediation of Te. The Indian mustard plant was exposed to selenate and tellurate and the concentrations of Se and Te and the chemical species in the plant were determined. The Indian mustard plant accumulated less Te than Se, and the amount of Te accumulated in the plant was approximately 1/69 of that of Se. Although the incorporation of selenate was reduced by increasing sulfate concentration in the medium, the incorporation of Te was not affected by it, suggesting that this plant was able to discriminate tellurate from selenate in the roots. Three Te species were detected in the plant. The major species was tellurate. The other two species were not identical to available Te standards and thus could not be identified. Consequently, the Indian mustard plant is inappropriate for the phytoremediation of Te because it can strictly distinguish tellurate from selenate.     

Cellular responses to postsegregational killing by restriction-modification genes

Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in an Escherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC, recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted "nonself" DNA and repair restricted "self" DNA.     

Determination of Young's modulus for nanofibrillated cellulose multilayer thin films using buckling mechanics

The Young's modulus of multilayer films containing nanofibrillated cellulose (NFC) and polyethyleneimine (PEI) was determined using the strain-induced elastic buckling instability for mechanical measurements (SIEBIMM) technique. (1) Multilayer films were built up on polydimethylsiloxane substrates using electrostatic layer-by-layer assembly. At 50% relative humidity, SIEBIMM gave a constant Young's modulus of 1.5 ± 0.2 GPa for 35-75 nm thick films. Conversely, in vacuum, the Young's modulus was 10 times larger, at 17.2 ± 1.2 GPa. A slight decrease in buckling wavelength with increasing strain was observed by scanning electron microscopy with in situ compression, and above 10% strain, extensive cracking parallel to the compressive direction occurred. We conclude that whereas PEI acts as a "glue" to hold multiple layers of NFC together, it prevents full development of hydrogen bonding and specific fibril-fibril interactions, and at high humidity, its hygroscopic nature decreases the elastic modulus when compared with pure NFC films.     

Stability of EcoRI restriction-modification enzymes in vivo differentiates the EcoRI restriction-modification system from other postsegregational cell killing systems

Certain type II restriction modification gene systems can kill host cells when these gene systems are eliminated from the host cells. Such ability to cause postsegregational killing of host cells is the feature of bacterial addiction modules, each of which consists of toxin and antitoxin genes. With these addiction modules, the differential stability of toxin and antitoxin molecules in cells plays an essential role in the execution of postsegregational killing. We here examined in vivo stability of the EcoRI restriction enzyme (toxin) and modification enzyme (antitoxin), the gene system of which has previously been shown to cause postsegregational host killing in Escherichia coli. Using two different methods, namely, quantitative Western blot analysis and pulse-chase immunoprecipitation analysis, we demonstrated that both the EcoRI restriction enzyme and modification enzyme are as stable as bulk cellular proteins and that there is no marked difference in their stability. The numbers of EcoRI restriction and modification enzyme molecules present in a host cell during the steady-state growth were estimated. We monitored changes in cellular levels of the EcoRI restriction and modification enzymes during the postsegregational killing. Results from these analyses together suggest that the EcoRI gene system does not rely on differential stability between the toxin and the antitoxin molecules for execution of postsegregational cell killing. Our results provide insights into the mechanism of postsegregational killing by restriction-modification systems, which seems to be distinct from mechanisms of postsegregational killing by other bacterial addiction modules.