Thermococcus kodakarensis Genetics: TK1827-Encoded β-Glycosidase, New Positive-Selection Protocol, and Targeted and Repetitive Deletion Technology

ABSTRACT Inactivation of TK1761, the reporter gene established for Thermococcus kodakarensis, revealed the presence of a second β-glycosidase that we have identified as the product of TK1827. This enzyme (pTK1827) has been purified and shown to hydrolyze glucopyranoside but not mannopyranoside, have optimal activity at 95°C and from pH 8 to 9.5, and have a functional half-life of ∼7 min at 100°C. To generate a strain with both TK1761 and TK1827 deleted, a new selection/counterselection protocol has been developed, and the levels of β-glycosidase activity in T. kodakarensis strains with TK1761 and/or TK1827 deleted and with these genes expressed from heterologous promoters are described. Genetic tools and strains have been developed that extend the use of this selection/counterselection procedure to delete any nonessential gene from the T. kodakarensis chromosome. Using this technology, TK0149 was deleted to obtain an agmatine auxotroph that grows on nutrient-rich medium only when agmatine is added. Transformants can therefore be selected rapidly, and replicating plasmids can be maintained in this strain growing in rich medium by complementation of the TK0149 deletion.

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Quantitative analysis of intermediary metabolism in Tetrahymena. Cells grown in proteose-peptone and resuspended in a defined nutrient-rich medium.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)86720-0 ◽

1979 ◽

Vol 254 (20) ◽

pp. 10385-10395

Author(s):

R.B. Stein ◽

J.J. Blum

Keyword(s):

Quantitative Analysis ◽

Intermediary Metabolism ◽

Rich Medium ◽

Proteose Peptone ◽

Nutrient Rich Medium

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Leporid immunoglobulin G shows evidence of strong selective pressure on the hinge and CH3 domains

Open Biology ◽

10.1098/rsob.140088 ◽

2014 ◽

Vol 4 (9) ◽

pp. 140088 ◽

Cited By ~ 15

Author(s):

Ana Pinheiro ◽

Jenny M. Woof ◽

Tereza Almeida ◽

Joana Abrantes ◽

Paulo C. Alves ◽

...

Keyword(s):

Positive Selection ◽

Immunoglobulin G ◽

Half Life ◽

Selective Pressure ◽

Hinge Region ◽

European Rabbit ◽

Rabbit Igg ◽

Immunological Research ◽

Constant Domains ◽

Positively Selected Sites

Immunoglobulin G (IgG) is the predominant serum immunoglobulin and has the longest serum half-life of all the antibody classes. The European rabbit IgG has been of significant importance in immunological research, and is therefore well characterized. However, the IgG of other leporids has been disregarded. To evaluate the evolution of this gene in leporids, we sequenced the complete IGHG for six other genera: Bunolagus , Brachylagus , Lepus , Pentalagus , Romerolagus and Sylvilagus . The newly sequenced leporid IGHG gene has an organization and structure similar to that of the European rabbit IgG. A gradient in leporid IgG constant domain diversity was observed, with the CH1 being the most conserved and the CH3 the most variable domain. Positive selection was found to be acting on all constant domains, but with a greater incidence in the CH3 domain, where a cluster of three positively selected sites was identified. In the hinge region, only three polymorphic positions were observed. The same hinge length was observed for all leporids. Unlike the variation observed for the European rabbit, all 11 Lepus species studied share exactly the same hinge motif, suggesting its maintenance as a result of an advantageous structure or conformation.

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Improved gfp and inaZ Broad-Host-Range Promoter-Probe Vectors

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.11.1243 ◽

2000 ◽

Vol 13 (11) ◽

pp. 1243-1250 ◽

Cited By ~ 383

Author(s):

William G. Miller ◽

Johan H. J. Leveau ◽

Steven E. Lindow

Keyword(s):

Host Range ◽

Reporter Gene ◽

Half Life ◽

The Other ◽

Broad Host Range ◽

Promoter Probe ◽

Restriction Sites ◽

Unique Restriction ◽

Selection For ◽

Derivatives Of

A new set of broad-host-range promoter-probe vectors has been constructed. One subset contains the pVS1 and p15a replicons and confers resistance to either gentamicin or kanamycin. The other set contains the broad-host-range replicon from pBBR1 and confers resistance to kanamycin, tetracycline, ampicillin, or spectinomycin/streptomycin. Both plasmid sets are highly stable and are maintained without selection for more than 30 generations in several bacterial taxa. Each plasmid contains a promoter-probe cassette that consists of a multicloning site, containing several unique restriction sites, and gfp or inaZ as a reporter gene. The cassette is bound by transcriptional terminators to permit the insertion of strong promoters and to insulate the cassette from external transcription enabling the detection of weak or moderate promoters. The vector suite was augmented with derivatives of the kanamycin-resistant gfp promoter-probe plasmids that encode Gfp variants with different half-life times.

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Electrochemical Corrosion Behavior of Cu-15Ni-8Sn Alloy in Marine Microbial Medium

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.79-82.1099 ◽

2009 ◽

Vol 79-82 ◽

pp. 1099-1102

Author(s):

Yan Hua Lei ◽

Yan Sheng Yin ◽

Chao Hong Liu ◽

Xue Ting Chang ◽

Yan Chen ◽

...

Keyword(s):

Electron Microscopy ◽

Corrosion Behavior ◽

Electrochemical Corrosion ◽

Rich Medium ◽

Sem Images ◽

Electrochemical Corrosion Behavior ◽

Nutrient Rich Medium ◽

Simulated Seawater ◽

Control Samples ◽

Scanning Electron

A comparative study of the corrosion behavior of the copper-nickel-tin alloy in a nutrient–rich simulated seawater-based nutrient-rich medium in the presence and the absence of the marine bacteria was carried out by electrochemical experiments, microscopic methods. Comparing to the corresponding control samples, the electrochemical data demonstrated that the presence of the bacteria accelerated the corrosion of the alloy. Scanning electron microscopy (SEM) images revealed the occurrence of micro-pitting and intergranular corrosion underneath the biofilm on the alloy surface.

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MEIOSIS CAN INDUCE RECOMBINATION IN rad52 MUTANTS OF SACCHAROMYCES CEREVISIAE

Genetics ◽

10.1093/genetics/113.3.531 ◽

1986 ◽

Vol 113 (3) ◽

pp. 531-550

Author(s):

Michael A Resnick ◽

John Nitiss ◽

Charles Edwards ◽

Robert E Malone

Keyword(s):

Saccharomyces Cerevisiae ◽

Meiotic Recombination ◽

Selective Medium ◽

Complete Loss ◽

Double Strand Breaks ◽

Rich Medium ◽

Strand Breaks ◽

Selective Media ◽

Single Strand Breaks ◽

Nutrient Rich Medium

ABSTRACT The RAD52 and RAD50 genes have previously been shown to be required for normal meiotic recombination and for various types of recombination occurring in mitotic cells. Recent evidence suggests that rad52 mutants might be defective in an intermediate recombination step; we therefore examined recombination during meiosis in several rad52 mutants at several different loci and in genetic backgrounds that yield efficient sporulation and synchronous meiosis. Similar to previous reports, spores from rad52 diploids are inviable and meiotic recombination is greatly reduced by rad52 mutations. However, intragenic recombinants were detected when cells were plated on selective media during meiosis; rad52 mutants experience induction of recombination between homologues under these special conditions. The frequencies of recombination at four loci were considerably greater than the mitotic controls; however, they were still at least 20 times lower than corresponding Rad+ strains. The prototrophs induced by meiosis in rad52 mutants were not typical meiotic recombinants because incubation in nutrient-rich medium before plating to selective medium resulted in the complete loss of recombinants. We propose that previously observed single-strand breaks that accumulate in rad52 mutants may be associated with recombinational intermediates that are resolved when cells are returned to selective mitotic media and that the meiosis-induced recombination in rad52 cells does not involve double-strand breaks.

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Probing Activated Sludge with Fluorescently Labeled rRNA Targeted Oligonucleotides

Water Science & Technology ◽

10.2166/wst.1994.0294 ◽

1994 ◽

Vol 29 (7) ◽

pp. 15-23 ◽

Cited By ~ 22

Author(s):

M. Wagner ◽

R. Amann ◽

H. Lemmer ◽

W. Manz ◽

K. H. Schleifer

Keyword(s):

Activated Sludge ◽

Ribosomal Rna ◽

Phosphate Removal ◽

Municipal Sewage ◽

Correct Identification ◽

Rich Medium ◽

Phylogenetic Groups ◽

In Situ Investigation ◽

Nutrient Rich Medium

Activated sludge samples from municipal sewage treatment plants were characterized using 16S and 23S ribosomal RNA targeted oligonucleotide probes specific for defined phylogenetic groups of bacteria. Comparison of in situ community structures as determined by molecular biological methods with the composition of the heterotrophic saprophyte flora isolated on nutrient rich medium revealed large discrepancies. These are caused by the selectivity of media and culture conditions. The most significant effect of cultivation on nutrient rich medium is an underestimation of bacteria belonging to the beta-subclass of Proteobacteria and an overestimation of bacteria belonging to the gamma-subclass of Proteobacteria. Therefore, culture dependent enumerations of the gamma-subclass bacteria of the genus Acinetobacter in plants with enhanced biological phosphate removal (EBPR) resulted in significant overestimations. In situ identification by fluorescent oligonucleotide probing revealed that Acinetobacter numbers were below 8% of the active bacterial cells in the examined EBPR-plants. In situ hybridization techniques also bear the potential for the early and correct identification of filamentous bacteria as indicators for sludge bulking and foaming. A 16S ribosomal RNA targeted oligonucleotide probe specific for Sphaerotilus spec, was developed and successfully applied for in situ investigation of this filamentous bacterium.

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Novel Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase in Wautersia eutropha H16

Journal of Bacteriology ◽

10.1128/jb.187.15.5129-5135.2005 ◽

2005 ◽

Vol 187 (15) ◽

pp. 5129-5135 ◽

Cited By ~ 45

Author(s):

Teruyuki Kobayashi ◽

Keiichi Uchino ◽

Tomoko Abe ◽

Yuya Yamazaki ◽

Terumi Saito

Keyword(s):

Inclusion Bodies ◽

Ralstonia Eutropha ◽

Specific Activity ◽

High Specific Activity ◽

Immunoblot Analysis ◽

Polyclonal Antiserum ◽

Logarithmic Phase ◽

Rich Medium ◽

Wautersia Eutropha ◽

Nutrient Rich Medium

ABSTRACT Wautersia eutropha H16 (formerly Ralstonia eutropha) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (PHB) with intracellular poly(3-hydroxybutyrate) depolymerases. In this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZc) gene was cloned and overexpressed in Escherichia coli. Then PhaZc was purified and characterized. Immunoblot analysis with polyclonal antiserum against PhaZc revealed that most PhaZc is present in the cytosolic fraction and a small amount is present in the poly(3-hydroxybutyrate) inclusion bodies of W. eutropha. PhaZc degraded various 3-hydroxybutyrate oligomers at a high specific activity and artificial amorphous poly(3-hydroxybutyrate) at a lower specific activity. Native PHB granules and semicrystalline PHB were not degraded by PhaZc. A PhaZ deletion mutation enhanced the deposition of PHB in the logarithmic phase in nutrient-rich medium. PhaZc differs from the hydrolases of W. eutropha previously reported and is a novel type of intracellular 3-hydroxybutyrate-oligomer hydrolase, and it participates in the mobilization of PHB along with other hydrolases.

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