Michael Adams and his team at the University of Georgia have purified four tungsten-containing enzymes used by P. Adams says that the genome sequence of P. Enzymes are protein molecules that act as catalysts in biochemical reactions, spurring the reactions to work faster and more efficiently.
Adams says two of the tungstoenzymes of P. One of the tungstoenzymes is involved in carbohydrate metabolism, but is unlike molybdoenzymes because the reaction it catalyses is different.
The function of the fourth purified tungstoenzyme is still unclear. While tungsten and molybdenum make up part of their respective enzymes in a similar fashion, the tungstoenzymes in P. Adams says this difference suggests that tungstoenzymes and molybdoenzymes probably evolved independently. Heme-containing enzymes, also known as hemoproteins, are a class of enzymes found in all mammalian cells hemoglobin is one of the better known hemoproteins.
Similarly, the molybdenum- and tungsten-containing families evolved separately, even though they contain a common cofactor. Generally, these proteins show unique features consistent with their origins, such as extreme thermostability and resistance against chemical denaturants, organic solvents, high salinity, and extremes of pH Fujinami and Fujisawa, ; Wende et al.
Many industrial enzymes isolated from hyper thermophiles, such as DNA polymerase, amylases, cellulases, esterases, and proteases, are now used in life sciences, food, chemical, and pharmaceutical industries and environmental biotechnology Lu and Erickson, ; Harris et al. As the first isolated Pyrococcus species, Pyrococcus furiosus P. Because of the advantage of thermostability several common industrial enzymes have been characterized and used in biotechnology Harris et al.
Structural genomics, along with the genomics, focuses on determining protein structures on the scale of the whole genome Adams et al.
Until now, more than two hundred protein structures from P. These structures are from proteins involved in almost each cellular metabolism pathway, especially in the genetic information process Nakamura et al. Restriction enzyme cloning plays an important role in preparing recombinant proteins for biochemical characterization and structural studies. However, it is not suitable for investigating protein's function and structure in high throughout. To clone genes in high throughout format, several cloning methods have been developed, such as ligation-independent cloning LIC Aslanidis and de Jong, ; Nisson et al.
These high throughout cloning techniques are independent of restriction endonuclease and are based on universal treatment of insert and vector DNA.
We constructed a P. Cloning of P. This P. Escherichia coli E. All other reagents were of analytical grade. The phosphorylated primers were used directly without any purification. All experiments, including PCR, recombinant reaction, and transformation and screening positive clones, were performed in well plate. Each P. Symbols of — denote the phosphorothioate modification. The complementary sequences between gene-specific and common primer are underlined.
The linear prokaryotic expression vector pDEST17 about 4. The PCR products of P. Transformation of E. To check the induced expression of P. The overnight cultures were diluted into fresh media in a volume ratio of The schematic presentation of P. The nick in the circular recombinant plasmid resulting from combining the two modified DNAs can be repaired by bacterial DNA repair system after transforming into E.
Figure 1. Schematic representation for constructing of a P. A Amplification of P. Finally, the annealed plasmid was repaired in vivo by E. Considering that protease K can be used to degrade DNA polymerase, we tested the effect of protease K on cloning efficiency.
Consistent with the analysis, protease K remarkably increased the number of recombinant clones Figure 2A. If the digestion time was longer than 10 min, the clone number decreased in the absence of protease K. Moreover, the purification of PCR fragments did not result in a distinct increase of clone numbers Figure 2 , indicating that the other elements of PCR mixtures were not harmful to the cloning efficiency. Figure 2.
Promotion of cloning efficiency by protease K. The abbreviations of S and PK indicate phosphorothioate and protease K. Before starting to clone genes in well plates, we first determined the cloning efficiency of various length P. Considering that the insert was excessive to vector, the effect of ratio of insert to vector was less significant.
In general the number of recombinant clones was inversely in proportion to the length of cloned genes Table 1.
For the inserts shorter than bp, the number of recombinants was about 50— For DNA inserts longer than bp, the number of recombinants sharply decreased.
The longest P. To compare the cloning efficiency of different length DNA fragments, the molar ratios of insert to vector is calculated for each gene Table 1. Interestingly, for the insert PF with the highest ratio of insert to vector, the clone number was not the highest.
In summary, the cloning efficiency of our HTP cloning method was suitable for constructing a P. Table 1. Number of recombinant clones of P. According to the described protocol, ORFs were amplified from P. To be most efficient in amplifying genes by PCR, all P. Agarose-gel electrophoresis images of other gene groups are available on request. Colony PCR results of the 17th group genes are shown in Figure 4. Size bars are nm, each. The genome of P. Over the years, we have identified at least 2 strains differing from the original P.
The same isolate was repeatedly regrown for ca. The three strains of P. Figure 4. History of the P. Strains P. We therefore asked if the newly discovered flagellin gene flaB0 is conserved not only in the type strain but also in the two lab derivates.
Hence, genomic DNA was isolated and primers f and r were used to amplify the region around flaB0. For all three strains a 2. As the primer numbers refer to the binding position in the public genome of P. Genomic sequencing of the flaB0 region confirmed the sequence we determined earlier for the missing bp segment in all three strains data not shown.
Figure 5. Detection of the flaB0 gene in three different P. Genomic DNA was isolated from the three strains P. Very clearly a ca. We asked if all flagella-related genes of P. A negative control without addition of cDNA proved that in all cases only transcripts from mRNA were analyzed data not shown.
We detected different length cotranscripts for each of the genes of the flagellar operon with exception of flaJ were only the single gene transcript was found Figure 6A. The original data using the different primers are shown exemplarily for flaB0 in Figures 6B,C , all other data are given in Supplementary Figure S1. Several transcripts including flaB0 were found whereof the largest with ca. Besides, various transcripts for the genes flaF-flaI were detected.
Interestingly, we found a transcript containing hth and fam whereas flaJ and PF were never part of a cotranscript. Analyses of RNA of cells from different growth phases showed that the transcripts changed over time; the original data are shown exemplarily in Figure 7. In early exponential phase, only few short transcripts were present compared to late exponential and stationary phase indicating that the flagellar operon is transcribed only to a limited degree in early exponential growth phase.
Figure 6. Transcripts observed for the P. A Flagellar operon of Pyrococcus furiosus with neighboring genes in the upper part. D Northern blot experiments using a flaB0 probe. RNA was isolated from late exponentially growing cells lane 1 and cells in stationary phase lane 2 and separated in a denaturing agarose gel. The gel migration behavior of an RNA standard is indicated to the left. Figure 7. Transcriptional analyses of the P. Results are shown exemplarily for two different potential cotranscripts.
A Analysis of the potential cotranscript flaB0-flaD. B Analysis of the potential cotranscript flaF-flaH. Northern blot experiments using RNA isolated from late exponentially growing cells showed the existence of a prominent ca.
In addition a much less prominent smear above ca. A general problem with this approach is the fact that some genes are difficult to clone or might be even toxic for the host, normally Escherichia coli. In our studies, we found that flaB0 could not be cloned into E. Cloning in a vector system with expression under the strong T7 polymerase promoter as used e.
We furthermore experienced problems with subcloning parts of P. Only the middle part of flaB0 could be cloned, but not the N- and C-terminal regions. Hence, we suggest that these problems in cloning might also have happened during the original genome sequencing. The only way we could obtain the flaB0 sequence was to sequence directly from genomic DNA via primer walking. The here newly described gene flaB0 codes for the major flagellin of P.
This statement is supported by the fact that one major glycoprotein of ca. There was no heterogeneity at position 2 and the presence of FlaB1 and FlaB2 was proven only by use of specific antibodies. In further experiments we were able to show that antisera raised against the least conserved part of the flagellins FlaB1 and FlaB2 labeled CsCl-gradient purified flagella mostly on their ends, whilst an antiserum raised against purified flagella reacted much stronger over the whole length of flagella.
Flagellins derived from SDS- plus heat-denatured flagella could clearly be repolymerized into smaller aggregates and fibrillar structures via simple heat treatment. The ultrastructure and diameter of such fibrils differs obviously from that of purified flagella. This, however, is not too surprising if one takes into account that for flagella assembly most likely a platform containing at least the proteins FlaC, FlaD, FlaF, and FlaG is necessary in vivo see Jarrell et al.
In addition this process is supposed to require ATP; in our hands repeated ATP addition to the in vitro repolymerization assays, however had no effect. Transcription of genes pf to pf is from the negatively oriented DNA strand, whilst the neighboring genes are transcribed form the positively oriented strand. Therefore, we analyzed this part of the genome for transcription including the flagellar operon neighboring genes pf and pf Both, our RT-PCR experiments and Northern Blot analyses show that there is not a single cotranscript detectable for the genes inside the flagellar operon of P.
In addition, the final acetate concentration in the medium was analyzed after 55 h incubation right axis. C Documentation of the incubated bottles after x h incubation, P. This strong difference in the observed acetate concentrations clearly indicates that the genetically engineered P.
Chitin is highly acetylated and we therefore assume that acetate is released from the chitin during growth of P. In comparison, in an experiment with similar cell density and incubation time grown on cellobiose as substrate, an acetate concentration of about 4 mM was observed [ 26 ].
Due to the insolubility of the chitin and the fact that many cells stick on the chitin it was very difficult to report reliable OD values, but the visual inspection of the incubated bottles of the wild type and the mutant strain clearly demonstrated chitin degradation Figure 4 C.
The bottle containing the wild type after an incubation of 55 h still exhibited a milky turbidity due to the insolubility of the chitin. In contrast, the amount of insoluble chitin in the bottle with the genetically engineered P.
Figure 4 D shows a phase contrast microscopic picture of the mutant strain. It can be clearly seen that the cells are attached to the chitin particles.
The experiments presented so far clearly indicate that the P. In this context it is interesting to note the overall high sequence identity of the two chitinases between P. This indicates that this mutation occurred very recently or there is still some kind of selection pressure for a functional enzyme. It is possible that P. This situation was recently described for the expression of the fucA1 gene in Sulfolobus acidocaldarius. To exclude or to confirm the idea of programmed frameshifting additional experiments will be necessary.
It is not possible to use available proteomics data of P. Furthermore, first attempts to use purified enzyme for a detailed mass spectrometry-based analysis were complicated by the finding that the enzyme is most likely the target of intensive proteolytic cleavage activity data not shown.
To analyze if we could further stimulate the growth of P. Oku and Ishikawa also demonstrated in in vitro experiments that a reduced recombinant version of the chitinase consisting of the chitin binding and the catalytic domain B had a higher activity as the whole enzyme [ 16 ]. To analyze if this in vitro result with heterologous expressed proteins could be confirmed in vivo , the additional plasmid pMUR50 was created Figure 5.
It contained the resistance cassette, the catalytic and the chitin binding domain of PF, the signal peptide sequence of PF and the corresponding upstream sequence for homologous recombination by single cross-over.
After sequence verification the plasmid was used to create the marker-less P. Schematic drawing for the construction of strain MUR24Pf. Plasmid pMUR50 possesses a modified chitinase construct together with the resistance cassette as used for the construction of plasmid pMUR The negative selection with 6-methylpurine resulted in strain MUR24Pf with a modified version of the chitinase.
We also performed growth experiments using similar conditions as outlined above with the strain containing the ChiB domain Figure 6. For comparison, the data of the mutant strain MUR23Pf with the redesigned chitinase as a single enzyme were included. Furthermore, the stability of the cells in the stationary phase was lower and the acetate concentration in the medium was reduced by one third in comparison to the strain with the redesigned complete chitinase Figure 6. Taken together, the data clearly indicate that the P.
This result did not confirm the in vitro data from Oku and Ishikawa and is therefore a nice example that sometimes in vitro results do not match with in vivo results [ 16 ]. Our data demonstrate that wild type P. In contrast, a genetically engineered strain with the deleted nucleotide is able to grow on chitin.
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