Here we examined the significance of Avt6, a vacuolar exporter of glutamate and aspartate suggested by the in vitro membrane vesicle experiment, in vacuolar compartmentalization of amino acids in Saccharomyces cerevisiae cells. Fluorescent microscopic observation of GFP-fused Avt6 revealed it to be exclusively localized to the vacuolar membrane, with the amount of Myc-tagged Avt6 significantly increased under nitrogen starvation. Glutamate uptake by cells was enhanced by deletion of the AVT6 gene, indicating indirect involvement of Avt6 in cellular glutamate accumulation. Differences in acidic amino acid content of both total and vacuolar fractions were insignificant between the parent and avt6Δ cells when cultured in nutrient-rich conditions. However, in nitrogen-starved conditions, the amount of glutamate and aspartate in the vacuolar fraction was notably increased in the avt6Δ cells. Avt6 is thus involved in vacuolar amino acid compartmentalization in S. cerevisiae cells, especially under conditions of nitrogen starvation.
A pair of primers targeting the hlyA gene for Vibrio cholerae which could distinguish the classical from El Tor biotypes was designed and combined with other specific primers for ompW, rfb complex, and virulence genes such as ctxA, toxR, and tcpI in a multiplex PCR (m-PCR) assay. This m-PCR correctly identified 39 V. cholerae from clinical, water and seafood samples. The efficiency of this multiplex PCR (m-PCR) was compared with conventional biochemical and serogrouping methods. One O139 and 25 O1 V. cholerae strains including 10 environmental strains harbored all virulence-associated genes except 1 clinical strain which only had toxR and hlyA genes. Thirteen environmental strains were classified as non-O1/non-O139 and had the toxR and hlyA genes only. The detection limit of m-PCR was 7 × 104 cfu/ml. The m-PCR test was reliable and rapid and reduced the identification time to 4 h.
The imino amino acid, proline, has roles in both cellular nutrition and response to stress. Proline uptake in Saccharomyces cerevisiae is largely mediated by a high affinity, specific permease, Put4p, and a low affinity general amino acid permease, Gap1p. Both are subject to nitrogen catabolite repression (NCR) and nitrogen catabolite inactivation (NCI). In order for proline to be fully exploited, its transport must be derepressed, as occurs upon depletion of preferred nitrogen sources, and molecular oxygen must be present to allow the first step of catabolism via proline oxidase. This study focuses on the isolation of variants of Put4p, which are insensitive to repression by a preferred nitrogen source (ammonia) and their subsequent effect on proline transport and stress tolerance. Specific amino acid residues in the carboxy-terminal region of Put4p were targeted by site-directed mutagenesis. Substitution at Serine605, a potential phosphorylation target, led to the amelioration of ammonia-induced down-regulation of Put4p. When combined with a promoter mutation (−160), the S605A mutation resulted in increased proline uptake and accumulation. This increase in proline accumulation was associated with increased cell viability in conditions of high temperature and osmotic stress raising possible benefits in industrial fermentation applications.
Nitrate-positive strains of anamorphic yeasts representing the genus Cryptococcus were isolated on fern infusion agar from Athyrium filix-femina. Phylogenetic analyses of the D1/D2 domain of the LSU rRNA and of the ITS region placed them in Cylindricus clade in the Filobasidiales. Morphological and physiological characteristics, as well as mycocinotyping and molecular analysis, show a close affinity between strains. Subsequent comparative studies of new isolates revealed that they were not conspecific with any other known Cryptococcus species, and the cultures represented an undescribed species with two varieties, for which the names Cryptococcus filicatus var. filicatus (type strain VKM Y-2954 = CBS 10874) and Cryptococcus filicatus var. pelliculosus (type strain VKM Y-2955 = CBS 10875) are proposed.
Laguna (L.) Negra and L. Verde are high altitude Andean lakes located at the 4,400 m altitude in the Andean desert (Puna) in the Argentine northwest. Both lakes are exposed to extreme weather conditions but differ in salinity contents (salinity 6.7% for L. Negra and 0.27% for L. Verde). The aim of this work was to isolate ultraviolet B fraction (UV-B) resistant bacteria under UV-stress in order to determine, a possible connection, between resistance to UV-B and tolerance to salinity. DNA damage was determined by measuring CPDs accumulation. Connection among pigmentation production and UV resistance was also studied. Water samples were exposed to artificial UV-B radiation for 24 h. Water aliquots were plated along the exposition on different media, with different salinity and carbon source content (Lake medium (LM) done with the lake water plus agar and LB). CFU were counted and DNA damage accumulation was determined. Isolated bacteria were identified by 16S rDNA sequence. Their salinity tolerance, were measured at 1, 5 and 10% NaCl and their pigment production in both media was determined. In general it was found that UV resistance and pigment production were the optimum in Lake Medium done with lake water which maintained similar salinity. The most resistant bacteria in L. Negra were different strains of Exiguobacterium sp. and, in L. Verde, Staphylococcus sp. and Stenotrophomonas maltophilia. These bacteria showed the production and increase of UV-Vis absorbing compounds under UV stress and in LM. Bacterial communities from both lakes were well adapted to high UV-B exposure under the experimental conditions, and in many cases UV-B even stimulated growth. The idea that resistance to UV-B could be related to adaptation to high salinity is still an open question that has to be answered with future experiments.
Thirteen poly (L-lactide)-degrading microorganisms were isolated and selected based on their ability of clear zone formation on an emulsified PLA agar plate and the enzyme activity in culture broth. According to phenotypic properties and 16S rRNA gene sequence, these strains were classified to various families such as Thermomonosporaceae, Micromonosporaceae, Streptosporangiaceae, Bacillaceae and Thermoactinomycetaceae. Strain T16-1, identified as Actinomadura sp., demonstrated the highest PLA-degrading activity in the liquid culture using PLA film as a carbon source. A PLA-degrading enzyme produced by the strain was purified to homogeneity shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with specific activity of 38.3 unit/mg protein. The optimum pH and temperature were 10.0 and 70ºC, respectively, which are higher than previously reported among PLA-degrading enzyme. The enzyme was stable at pH 11-12. However, the enzyme activity remained at 70% when kept at 70ºC for 1 h. The molecular weight of purified PLA-degrading enzyme from the strain T16-1 was 30 kDa. The purified enzyme was inhibited by 5 mM EDTA and 5 mM phenylmethylsulfonyl fluoride and diisopropyl fluorophosphates, strongly hydrolyzed Suc-(Ala)3-pNA, gelatin and PLA, but showed low activity on casein. The results indicated the PLA-degrading enzyme produced by the strain Actinomadura sp. T16-1 should be classified as serine protease.
The intrageneric structure of the genus Rhodopseudomonas was evaluated by studying sequence information on 16S rRNA genes, 16S-23S rRNA gene internal transcribed spacer (ITS) regions, and puf genes using 33 test strains. The topology of phylogenetic trees based on these sequences was similar to those of every other independent method for tree construction. These phylogenetic data indicated that the test strains were grouped into at least 7 clusters possibly at the species level. This was supported by genomic DNA-DNA similarities among 12 representative test strains selected from these clusters. Our molecular data confirmed that the currently available strains of Rhodopseudomonas (Rps.) palustris are genetically quite heterogeneous within the genus. For example, Rps. palustris strains DSM 123T and ATCC 17001T are different from each other at the species level despite their status as the type strain of the species. Rps. palustris strain ATCC 17005 and the full genome-sequenced strains BisA53, BisB18, BisB5, and HaA2 should be re-classified into different species from Rps. palustris or as novel species of the genus Rhodopseudomonas.
Characterization of three bacterial symbionts (BC1, BC2 and BC3) of fruit fly Bactrocera tau including morphological, biochemical and 16S rDNA (rrs gene) analysis was done to determine their taxonomic position. Morphological and biochemical characterization placed two bacteria (BC1, Klebsiella oxytoca and BC2, Pantoea agglomerans) into family Enterobacteriaceae and the third one (BC3, Staphylococcus sp.) into family Staphylococcaceae. 16S rDNA gene sequence comparison with the available NCBI database sequences further confirmed the characterizations of bacterial symbionts. Molecular phylogeny of Klebsiella oxytoca and Pantoea agglomerans closely related to the other free living enterobacterial members with 74 to 93% sequence homology (genetic distance 0.000 to 0.085); however, they showed only 74 to 87% similarity with other insect symbionts (genetic distance 0.090 to 0.121). Staphylococcus sp. showed 94% sequence homology with other members of family Staphylococcaceae with the genetic distance of 0.013. Population of these symbionts in adult fruit flies increased exponentially up to the 10th day of adult emergence and thereafter it became almost constant.
Post-translational modifications (PTMs) are important for cellular functions. The regulation of histone acetyltransferases (HATs) and histone deacetylases (HDACs) is one of important PTMs for epigenetic control, protein activity and protein stability. The regulation of acetylation of the N-terminal histone tails of core histone affects gene expression. Two class I HDAC genes and two class II HDAC genes have been identified in the Magnaporthe oryzae genome. Treatment with Rpd3/Hda1 family (classical) HDAC inhibitor inhibited the appressorium differentiation of M. oryzae. Treatment with trichostatin A, a classical HDAC inhibitor, also decreased pathogenesis. Furthermore, analyses of HDAC mutants indicated that MoHda1 and MoHos2 were required for vegetative growth and conidiation, and MoHos2 was required for appressorium formation. Disruption MoRPD3 was unsuccessful, as in the case with Aspergillus nidulans RpdA. These data indicated that HDACs have important roles in the asexual differentiation of M. oryzae.