Secretion of nucleoside diphosphate kinase by mucoid Pseudomonas aeruginosa 8821: involvement of a carboxy-terminal motif in secretion

Nucleoside diphosphate kinase (Ndk) is a ubiquitous enzyme which functions in balancing the nucleotide pool of the cell. We have recently reported that in addition to being intracellular in both mucoid and nonmucoid Pseudomonas aeruginosa, Ndk is also secreted into the extracellular environment by mucoid P. aeruginosa cells. This secreted Ndk has biochemical activity similar to the intracellular Ndk and is 16 kDa in size. To demonstrate that Ndk is indeed secreted and to localize the secretion motif, we constructed an ndk knockout mutant, which lacks both intracellular and extracellular forms of Ndk. In this study, we report the construction of deletion derivatives made from the carboxy-terminal region of Ndk. These deletion derivatives were introduced into the ndk::Cm knockout mutant and were examined for the intracellular and extracellular presence of Ndk. It was observed that the carboxy-terminal 8-amino-acid region is required for the secretion of Ndk into the extracellular region. This region has the sequence DXXX, where X is a predominantly hydrophobic residue. Such sequences represent a conserved motif in proteins secreted by the type I secretory pathway in gram-negative microorganisms. To investigate the significance of this motif in the secretion of Ndk, we constructed a fusion protein of Ndk and the blue fluorescent protein (BFP) as well as a fusion protein of mutated Ndk (whose DTEV motif has been changed to AAAA) and the BFP. The presence of extracellular Ndk was detected only in the ndk::Cm knockout mutant harboring the wild-type BFP-Ndk protein fusion. We could not detect the presence of extracellular Ndk in the ndk::Cm knockout mutant containing the mutated BFP-Ndk protein fusion. In addition, we have also used immunofluorescence microscopy to localize the wild-type and mutated BFP-Ndk proteins in the cell. The significance of these observations is discussed.     

A new powerful antibacterial synergistic combination of trimethoprim and trimeprazine

The antihistaminic phenothiazine trimeprazine (Tz) was found to exhibit significant antibacterial activity on the basis of in vitro and in vivo tests. For the study of synergism due to a combination between Tz and trimethoprim (Tm), drug soaked filter paper discs were placed on young culture lawns of sensitive bacteria on nutrient agar plates. Calculation of the area of inhibition zones for determining the degree of synergism between Tz and Tm showed the increase to be statistically significant (p<0.01) when compared with their individual effects. By the checkerboard assessment procedure, the fractional inhibitory concentration (FIC) index was found to be 0.18, confirming synergism. The protective capacity of this combination was then assessed in Swiss white mice using S. typhimurium as the challenge bacterium, and the level of bacterial load was determined from infected autopsied animals. Statistical analysis of the data by students 't' test finally proved that a combination of Tz+Tm was highly synergistic.     

Application of bioreactor system for large-scale production of Eleutherococcus sessiliflorus somatic embryos in an air-lift bioreactor and production of eleutherosides

Embryogenic callus was induced from leaf explants of Eleutherococcus sessiliflorus cultured on Murashige and Skoog (MS) basal medium supplemented with 1 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), while no plant growth regulators were needed for embryo maturation. The addition of 1 mg l(-1) 2,4-D was needed to maintain the embryogenic culture by preventing embryo maturation. Optimal embryo germination and plantlet development was achieved on MS medium with 4 mg l(-1) gibberellic acid (GA(3)). Low-strength MS medium (1/2 and 1/3 strength) was more effective than full-strength MS for the production of normal plantlets with well-developed shoots and roots. The plants were successfully transferred to soil. Embryogenic callus was used to establish a suspension culture for subsequent production of somatic embryos in bioreactor. By inoculating 10 g of embryogenic cells (fresh weight) into a 3l balloon type bubble bioreactor (BTBB) containing 2l MS medium without plant growth regulators, 121.8 g mature somatic embryos at different developmental stages were harvested and could be separated by filtration. Cotyledonary somatic embryos were germinated, and these converted into plantlets following transfer to a 3l BTBB containing 2l MS medium with 4 mg l(-1) GA3. HPLC analysis revealed that the total eleutherosides were significantly higher in leaves of field grown plants as compared to different stages of somatic embryo. However, the content of eleutheroside B was highest in germinated embryos. Germinated embryos also had higher contents of eleutheroside E and eleutheroside E1 as compared to other developmental stages. This result indicates that an efficient protocol for the mass production of E. sessiliflorus biomass can be achieved by bioreactor culture of somatic embryos and can be used as a source of medicinal raw materials.     

'DNA' as contaminants in antibiotics and its capacity to transform bacteria to drug resistance

DNA/and deoxyribose sugars were detected in streptomycin (Sm), kanamycin, polymyxin, penicillin G, ampicillin, methicillin, cloxacillin and mitomycin C in small amounts/traces. Stained DNA could be feebly visualized directly in Sm run in agarose gel, which improved after its separation and concentration. These DNA materials were DNase sensitive, RNase and pronase resistant, and appeared to consist of fragments, c. less than or equal to 6 Mdal; this could repeatedly transform to SmR several recipient enterobacteria and vibrios; E. coli C600 and S. typhi 57, after such transformation revealed similar plasmid DNA bands that were absent in their wild-types. G + C mole% of plasmid and chromosomal DNA of recipient (57) along with that of Streptomyces griseus reference strain, suggested an extraneous origin for the plasmid DNA.     

Molecular systematics of the Awaous banana complex (River gobies; Teleostei: Oxudercidae)

Diadromous fishes can exhibit interesting evolutionary and population-level patterns given their use of freshwater and marine environments as part of their life histories. The River goby genus Awaous are prominent members of riverine ichthyofaunas and occur throughout Atlantic and Pacific slopes of the Americas from the southern United States to Ecuador and Brazil. Here we study the widespread and polymorphic Awaous banana complex to assess phylogeographic patterns and test previous hypotheses that all populations of this species in the Americas belong to the same species. Analysis of sequence data based on the mitochondrial cytochrome oxidase I gene shows multiple clades within the Atlantic and Pacific basins, which correspond to previously described species. Additionally, haplotype analysis demonstrates unique and unconnected networks between these species. Within these clades we document biogeographic patterns that are congruent with results of other co-occurring diadromous species, as well as a novel biogeographic pattern for the region. Our results support the recognition of distinct species of Awaous in the Atlantic (A. banana and A. tajasica) and Pacific (A. transandeanus) basins. These results are concordant with previously established morphological characters permitting the separation of these species.