Extracellular expression of keratinase Ker P from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

Keratinase from Pseudomonas aeruginosa KS-1 was expressed constitutively as an extracellular protein in Escherichia coli with high specific activity of 3.7 kU/mg. It was purified fourfold as a 33 kDa monomeric protein by Q-Sepharose ion exchange chromatography with a recovery of 95%. It is a serine protease with optimal activity at pH 9 and 50°C. It was stable from pH 4 to 12 for 1 h with a t_1/2 of 12 min at 70°C. It hydrolyzed haemoglobin > fibrin > feather keratin > azo-casein > casein > meat protein > gelatin. Among synthetic substrates, it efficiently hydrolyzed N-Suc-ala-ala-pro-phe-pNA, N-Suc-ala-ala-ala-pNA, N-Suc-ala-ala-pro-leu-pNA and also plasmin substrate, d-Val-Leu-Lys-pNA     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> as a polymicrobial infection model for <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.     

Biofilm dispersion in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

In recent decades, many researchers have written numerous articles about microbial biofilms. Biofilm is a complex community of microorganisms and an example of bacterial group behavior. Biofilm is usually considered a sessile mode of life derived from the attached growth of microbes to surfaces, and most biofilms are embedded in self-produced extracellular matrix composed of extracellular polymeric substances (EPSs), such as polysaccharides, extracellular DNAs (eDNA), and proteins. Dispersal, a mode of biofilm detachment indicates active mechanisms that cause individual cells to separate from the biofilm and return to planktonic life. Since biofilm cells are cemented and surrounded by EPSs, dispersal is not simple to do and many researchers are now paying more attention to this active detachment process. Unlike other modes of biofilm detachment such as erosion or sloughing, which are generally considered passive processes, dispersal occurs as a result of complex spatial differentiation and molecular events in biofilm cells in response to various environmental cues, and there are many biological reasons that force bacterial cells to disperse from the biofilms. In this review, we mainly focus on the spatial differentiation of biofilm that is a prerequisite for dispersal, as well as environmental cues and molecular events related to the biofilm dispersal. More specifically, we discuss the dispersal-related phenomena and mechanisms observed in Pseudomonas aeruginosa, an important opportunistic human pathogen and representative model organism for biofilm study.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

Urovirulence of<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>: Planktonic cells<Emphasis Type="Italic">vs.</Emphasis> biofilm cells

Planktonic and biofilm cells of a clinical urinary isolate of P. aeruginosa were compared in vitro for their ability to adhere to uroepithelial cells, interaction with macrophages, and for production of virulence factors like extracellular proteinase, elastase, hemolysin, phospholipase C and pyochelin. Biofilm cells showed increased adherence to UECs, which was coupled with reduced uptake and intracellular killing by macrophages. Overall there was a decrease in production of extracellular products by biofilm cells. Comparing the two cell forms for their ability to establish infection in an ascending model of acute pyelonephritis, significant enhancement of renal bacterial load, as well as more pronounced renal pathology developed with biofilm cells.     

Transferable Carbenicillin Resistance in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

IN 1969, after carbenicillin had been in use for three years in this unit, highly resistant strains of Pseudomonas aeruginosa were isolated for the first time¹. Because these resistant strains included, from their first appearance, representatives of two unrelated types, it seemed likely that the resistance was transferable; this hypothesis was supported by experiments showing the transfer of carbenicillin resistance between Ps. aeruginosa and Escherichia coli K12 in vitro and in vivo^2–4;. The resistant Ps. aeruginosa produced a penicillinase (β lactamase) similar to that normally produced by some strains of Enterobacteria and different from that normally produced by Ps. aeruginosa^2,3, so it seemed likely that the Ps. aeruginosa had initially acquired resistance by the transfer of an R factor from a carbenicillin-resistant member of the Enterobacteriaceae colonizing the same burn. This hypothesis is now supported by a study on strains of Enterobacteria and Ps. aeruginosa isolated in a number of hospitals. We have also found evidence suggesting that Ps. aeruginosa which has acquired this R factor may not show resistance until it has been exposed repeatedly to carbenicillin.     

Ecophysiological Variabilities in Ectohydrolytic Enzyme Activities of Some <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> Species, <Emphasis Type="Italic">P. citrea,P. issachenkonii</Emphasis>, and <Emphasis Type="Italic">P. nigrifaciens</Emphasis>

The ecophysiological variabilities in the ectohydrolytic enzyme profiles of the three species of Pseudoalteromonas, P. citrea, P. issachenkonii, and P. nigrifaciens, have been investigated. Forty-one bacteria isolated from several invertebrates, macroalgae, sea grass, and the surrounding water exhibited different patterns of hydrolytic enzyme activities measured as the hydrolysis of either native biopolymers or fluorogenic substrates. The activities of the following enzymes were assayed: proteinase, tyrosinase, lipase, amylase, chitinase, agarase, fucoidan hydrolase, laminaranase, alginase, pustulanase, cellulase, β-glucosidase, α- and β-galactosidases, β-N-acetylglucosaminidase, β-glucosaminidase, β-xylosidase, and α-mannosidase. The occurrence and cell-specific activities of all enzymes varied over a broad range (from 0 to 44 μmol EU per hour) and depended not only on taxonomic affiliation of the strain, but also on the source/place of its isolation. This suggests ‘specialization’ of different species for different types of polymeric substrates as, for example, all strains of P. citrea and P. issachenkonii hydrolyzed alginate and laminaran, while strains of P. nigrifaciens were lacking the ability to hydrolyze most of the algal polysaccharides. The incidence of certain enzymes such as fucoidan hydrolases, alginate lyases, agarases, and α-galactosidases might be strain specific and reflect its particular ecological habitat. Received: 15 February 2002 / Accepted: 27 March 2002     

Ectomycorrhizae of <Emphasis Type="Italic">Tuber huidongense</Emphasis> and <Emphasis Type="Italic">T. liyuanum</Emphasis> with <Emphasis Type="Italic">Castanea mollissima</Emphasis> and <Emphasis Type="Italic">Pinus armandii</Emphasis>

Tuber huidongense and T. liyuanum are common commercial white truffles in China that belong to the Rufum and Puberulum groups of the genus Tuber, respectively. Their mycorrhizae were successfully synthesized with two native trees—Castanea mollissima and Pinus armandii—under greenhouse conditions. The identities of the mycorrhizae were confirmed through internal transcribed spacer (ITS) sequence analyses, and their morphological characteristics were described. All of the obtained mycorrhizae have an interlocking pseudoparenchymatous mantle, which is a typical feature of truffle mycorrhizae. The mycorrhizae of T. huidongense on the two trees have hyaline branched emanating hyphae, similar to the documented mycorrhizae of the Rufum group. The unramified, spiky, and hyaline cystidia on the mycorrhizae of T. liyuanum with both C. mollissima and P. armandii further confirmed that this characteristic is constant for the mycorrhizae of the Puberulum group. The successful mycorrhizal syntheses on the two nut-producing trees will be of economic importance in the cultivation of the two truffles.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.     

Rhizosphere competent <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in the management of <Emphasis Type="Italic">Heterodera cajani</Emphasis> on sesame

Biological control of the cyst forming nematode Heterodera cajani was studied on sesame using plant growth promoting rhizobacteria (PGPR) Pseudomonas aeruginosa LPT3 and LPT5. Based on plant growth promoting attributes, two fluorescent pseudomonads, LPT3 and LPT5 were evaluated for their efficacy against cyst forming nematode Heterodera cajani that parasitize Sesamum indicum. Pseudomonas aeruginosa LPT5 produced IAA, HCN, chitinase, glucanase and siderophore, and also solubilized inorganic phosphate in vitro. Moreover, LPT5 resulted in mortality of second stage juveniles of H. cajani, which was 13% higher as compared to P. aeruginosa LPT3. Interestingly, when both strains were inoculated together for the management of H. cajani on Sesamum indicum the population of H. cajani was reduced significantly, in field trial. Approximately 60% reduction in cyst and juveniles population was recorded with LPT5 coated seeds, while LPT3 resulted in 49% reduction in cyst and juvenile population as compared to control. Plants grown with seeds bacterized with LPT5 and reduced doses of urea, diammonium phosphate (DAP), muriate of potash (K) and gypsum gave maximum increase in yield, in comparison to that of plants raised under the influence of recommended or full doses of the chemical fertilizers. Pseudomonas aeruginosa LPT5 also showed excellent root colonization.