Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

ATP-dependent fructose uptake system in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

The bacterial phosphoenolpyruvate (PEP)-dependent group translocation system (PTS) requires the presence of both membrane-bound and cytoplasmic components to phosphorylate and translocate sugar. Deinococcus radiodurans has a functional fruA gene coding for the membrane-bound components of the fructose-specific PTS. However, fruB gene coding for the fructose-specific cytosolic components of PTS is a pseudogene. Yet, this bacterium metabolized fructose readily. In vitro studies showed that both cell membranes and cytoplasmic fractions of the cells were needed for fructose phosphorylation. Further studies showed that fructose phosphorylation required ATP, not PEP, as the phosphate donor. Unlike most PEP-dependent PTS systems, fructose phosphorylation is sensitive to sodium fluoride, a kinase inhibitor. Fructose phosphorylation was also inhibited in the presence of antiserum against a kinase phosphorylation site. Rhodobacter capsulatus has a functional fruA–fruB system. Complementation assays by reconstituting the membrane fraction of D. radiodurans to the cytoplasmic fraction of R. capsulatus resulted in a PEP-dependent fructose phosphorylation, whereas mixing the membranes of R. capsulatus and the deinococcal cytosol in vitro resulted in an ATP-dependent fructose phosphorylation.     

The single-stranded DNA-binding protein of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Background

Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored.

Results

A segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF). The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSB_Dr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer.

Conclusions

The correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer. The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions. The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.

     

Analysis of RNA cleavage by RNA polymerases from <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

RNA polymerase can both synthesize and cleave RNA. Both reactions occur at the same catalytic center containing two magnesium ions bound to three aspartic acid residues of the absolutely conserved NADFDGD motif of the RNA polymerase beta subunit. We have demonstrated that RNA polymerase from Deinococcus radiodurans possesses much higher rate of intrinsic RNA cleavage than RNA polymerase from Escherichia coli (the difference in the rates is about 15-fold at 20 degrees C). However, these RNA polymerases do not differ in the rates of RNA synthesis. Comparison of the RNA polymerase sequences adjacent to the NADFDGD motif reveals the only amino acid substitution in this region (Glu751 in D. radiodurans vs. Ala455 in E. coli), which is localized in the secondary enzyme channel and can potentially affect the rate of RNA cleavage. Introduction of the corresponding substitution in the E. coli RNA polymerase leads to a slight (about 2-3-fold) increase in the cleavage rate, but does not affect RNA synthesis. Thus, the difference in the RNA cleavage rates between E. coli and D. radiodurans RNA polymerases is likely determined by multiple amino acid substitutions, which do not affect the rate of RNA synthesis and are localized in several regions of the active center.     

High cell density production of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> under optimized conditions

Deinococcus radiodurans is a bacterium being investigated for mechanisms of extreme radiation resistance and for bioremediation of environmental radioactive waste sites. In both fundamental and applied research settings, methods for large-scale production of D. radiodurans are needed. In this study, a systematic investigation was carried out to optimize D. radiodurans production at the 20-L fermentor scale. In defined medium, the phosphate buffer typically used was found to be inhibitory to D. radiodurans growth, and caused cell aggregation. Substitution of HEPES and MOPS buffers for phosphate buffer improved D. radiodurans growth characteristics. Several antifoaming agents were investigated to support large-scale production with submerged aeration, and the defoamer KFO 673 was chosen based on its ability to prevent foaming without affecting D. radiodurans growth. The conventional undefined rich medium tryptone/glucose/yeast extract (TGY) maximally supported D. radiodurans growth to an OD₆₀₀ of 10. Using a ‘design of experiments’ approach, we found glucose, Mg and Mn to be critical in supporting high-density growth of D. radiodurans. The optimal pH and temperature for D. radiodurans growth in large-scale preparations were 7.0 and 37°C, respectively. Growth was carried out in a 20-L fermentor using the newly developed media under the optimal conditions. With addition of 10 g/L glucose, 0.5 g/L MgSO₄ · 7H₂O, 5 μM MnCl₂ into TGY media, an OD₆₀₀ of 40 was achieved.     

<Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> RecX and <Emphasis Type="Italic">Escherichia coli</Emphasis> RecX proteins are capable to replace each other in vivo and in vitro

A plasmid carrying the Deinococcus radiodurans recX gene under the control of a lactose promoter decreases the Escherichia coli cell resistance to UV irradiation and γ irradiation and also influences the conjugational recombination process. The D. radiodurans RecX protein functions in the Escherichia coli cells similarly to the E. coli RecX protein. Isolated and purified D. radiodurans RecX and E. coli RecX proteins are able to replace each other interacting with the E. coli RecA and D. radiodurans RecA proteins in vitro. Data obtained demonstrated that regulatory interaction of RecA and RecX proteins preserves a high degree of conservatism despite all the differences in the recombination reparation system between E. coli and D. radiodurans.     

Comparative genomics of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> and <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>: divergent routes of adaptation to thermophily and radiation resistance

Background  

Thermus thermophilus and Deinococcus radiodurans belong to a distinct bacterial clade but have remarkably different phenotypes. T. thermophilus is a thermophile, which is relatively sensitive to ionizing radiation and desiccation, whereas D. radiodurans is a mesophile, which is highly radiation- and desiccation-resistant. Here we present an in-depth comparison of the genomes of these two related but differently adapted bacteria.     

Three <Emphasis Type="Italic">nth</Emphasis> homologs are all required for efficient repair of spontaneous DNA damage in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans is a bacterium that can survive extreme DNA damage. To understand the role of endonuclease III (Nth) in oxidative repair and mutagenesis, we constructed nth single, double and triple mutants. The nth mutants showed no significant difference with wild type in both IR resistance and H₂O₂ resistance. We characterized these strains with regard to mutation rates and mutation spectrum using the rpoB/Rif^r system. The Rif^r frequency of mutant MK1 (△dr0289) was twofold higher than that of wild type. The triple mutant of nth (ME3)generated a mutation frequency 34.4-fold, and a mutation rate 13.8-fold higher than the wild type. All strains demonstrated specific mutational hotspots. Each single mutant had higher spontaneous mutation frequency than wild type at base substitution (G:C → A:T). The mutational response was further increased in the double and triple mutants. The higher mutation rate and mutational response in ME3 suggested that the three nth homologs had non-overlapped and overlapped substrate spectrum in endogenous oxidative DNA repair.     

<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">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.     

<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.     

Desiccation tolerance in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Staphylococcus aureus is a multidrug-resistant pathogen that not only causes a diverse array of human diseases, but also is able to survive in potentially dry and stressful environments, such as the human nose, on skin and on inanimate surfaces such as clothing and surfaces. This study investigated parameters governing desiccation tolerance of S. aureus and identified several components involved in the process. Initially, the role of environmental parameters such as temperature, growth phase, cell density, desiccation time and protectants in desiccation tolerance were determined. This established a robust model of desiccation tolerance in which S. aureus has the ability to survive on dry plastic surfaces for more than 1,097 days. Using a combination of a random screen and defined mutants, clpX, sigB and yjbH were identified as being required for desiccation tolerance. ClpX is a part of the ATP-dependent ClpXP protease, important for protein turnover, and YjbH has a proposed linked function. SigB is an accessory sigma factor with a role in generalized stress resistance. Understanding the molecular mechanisms that govern desiccation tolerance may determine the break points to be exploited to prevent the spread of this dangerous pathogen in hospitals and communities.     

Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Background  

Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of γ-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair.     

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.     

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.