Jenn Tu

Characterization and pulp refining activity of a Paenibacillus campinasensis cellulase expressed in Escherichia coli.

The Cel-BL11 gene from Paenibacillus campinasensis BL11 was cloned and expressed in Escherichia coli as a His-tag fusion protein. Zymographic analysis of the recombinant protein revealed cellulase activity corresponding to a protein with a 38-kDa molecular weight. The optimum temperature and pH for purified cellulase were 60 °C and pH 7.0, respectively. The enzyme retained more than 80% activity after 8h at 60 °C at pH 6 and 7. The cellulase has a Km of 11.25 mg/ml and a Vmax of 1250 μmol/min/mg with carboxylmethyl cellulose (CMC). Then enzyme was active on Avicel, swollen Avicel, CMC, barley β-glucan, laminarin in the presence of 100 mM acetate buffer. It was inhibited by Hg²⁺, Cu²⁺ and Zn²⁺. Significant kraft pulp refining energy saving, 10%, was exhibited by the pretreatment of this cellulase applied at 2 IU per gram of oven-dried pulp. Broad pH and temperature stability render this cellulase a convenient applicability toward current mainstream biomass conversion and other industrial processes.

An inducible transposon system to terminate the function of a selectable marker in transgenic plants

Since the maize transposon Ac can move to a new location within the genome, it has been used in removing selectable markers in transgenic plants. In this paper, we developed an inducible transposon system to truncate a selectable marker in transgenic plants. In this system, the marker gene was accompanied by the inducible transposon, but one end of the transposon was located in the intron of the marker gene. As an example of a marker gene, we isolated the rice 5-enolpyruvylshikimate-3-phosphate synthase (epsps) and modified it for glyphosate tolerance. The transposon contained Ac transposase, which fused with the promoter of the inducible gene for pathogenesis-related protein 1a (PR-1a). This construct was engineered into an expression vector pCAMBIA1300, harboring a hygromycin-resistant gene. The construct was first transformed into rice calli, and transformed plants were selected on hygromycin-containing medium. The stably transformed calli underwent determination for normal transcripts and tolerance to glyphosate. The results were applied to a rice transformation with the same construct, but using glyphosate as the selective agent. By determining the transformation efficiency, T-DNA copy patterns, we demonstrate that the modified epsps could be a suitable selectable marker to create transgenic rice. Furthermore, after obtaining stable transgenic plants and inducing transposition by salicylic acid, the transposon was excised, the marker gene became truncated, and its expression was terminated. This strategy could be applicable to yield self-stabilizing transposon by locating the transposon’s end in the transposase gene’s intron.

Complete Genome Sequence of Serratia marcescens WW4

ABSTRACT Serratia marcescens WW4 is a biofilm-forming bacterium isolated from paper machine aggregates. Under conditions of phosphate limitation, this bacterium exhibits intergeneric inhibition of Pseudomonas aeruginosa. Here, the complete genome sequence of S. marcescens WW4, which consists of one circular chromosome (5,241,455 bp) and one plasmid (pSmWW4; 3,248 bp), was determined.

The resolvase encoded by Xanthomonas campestris transposable element ISXc5 constitutes a new subfamily closely related to DNA invertases

Conservative site‐specific recombination is responsible for the resolution of cointegrates which result during the transposition of class II transposable elements. Resolution is catalysed by a transposon‐encoded recombinase, resolvase, that belongs to a large family of recombinases, including DNA invertases. Resolvases and the related invertases are likely to employ similar reaction mechanisms during recombination. There are important differences, however. Resolvases require two accessory DNA binding sites within each of the two directly repeated recombination sites. Invertases instead need a host factor, Fis, and an enhancer type DNA sequence, in addition to two inversely orientated recombination sites.

Transposable elements ofXanthomonas campestris pv.citri originating from indigenous plasmids

SummaryAfter introduction of the broad host range plasmid RP4 inXanthomonas campestris pv.citri strain XAS4501 twoXanthomonas transposable elements, ISXC4 and ISXC5, were isolated. These elements were found to be capable of transposition inEscherichia coli. Restriction analysis, DNA hybridization and heteroduplex experiments revealed that ISXC4 and ISXC5 are about 5.55 and 6.95 kb in size, respectively, possess inverted repeats about 50±18 bp in length and share DNA homology in their left (5.0 kb) and right (0.6 kb) ends. ISXC4 and ISXC5 were found to originate from plasmids pXW45N and pXW45J, which are indigenous replicons inX. campestris pv.citri strain XW45.

Phosphate limitation induces the intergeneric inhibition of Pseudomonas aeruginosa by Serratia marcescens isolated from paper machines

Phosphate is an essential nutrient for heterotrophic bacteria, affecting bacterioplankton in aquatic ecosystems and bacteria in biofilms. However, the influence of phosphate limitation on bacterial competition and biofilm development in multispecies populations has received limited attention in existing studies. To address this issue, we isolated 13 adhesive bacteria from paper machine aggregates. Intergeneric inhibition of Pseudomonas aeruginosa WW5 by Serratia marcescens WW4 was identified under phosphate-limited conditions, but not in Luria–Bertani medium or M9 minimal medium. The viable numbers of the pure S. marcescens WW4 culture decreased over 3 days in the phosphate-limited medium; however, the mortality of S. marcescens WW4 was significantly reduced when it was co-cultured with P. aeruginosa WW5, which appeared to sustain the S. marcescens WW4 biofilm. In contrast, viable P. aeruginosa WW5 cells immediately declined in the phosphate-limited co-culture. To identify the genetic/inhibitory element(s) involved in this process, we inserted a mini-Tn5 mutant of S. marcescens WW4 that lacked inhibitory effect. The results showed that an endonuclease bacteriocin was involved in this intergeneric inhibition by S. marcescens WW4 under phosphate limitation. In conclusion, this study highlights the importance of nutrient limitation in bacterial interactions and provides a strong candidate gene for future functional characterisation.

A 200-bp constructed inducible PR-1a promoter fusion to the Ac transposase gene drives higher transposition of a Ds element than the native PR-1a promoter fusion drives

Abstract An inducible transposon tagging technique in plants with a large genome was accessed. The open reading frame coding for the transposase gene of the maize transposon Activator ( Ac ) was expressed in transgenic tobacco plants under the control of the promoter of the inducible gene for pathogenesis-related protein 1a (PR-1a). Based on the native PR-1a promoter, several inducible promoters were constructed in order to induce variability of the Ac transposase expression level. Excision of a non-autonomous transposable element ( Ds ) from the chimeric β -glucuronidase gene construct was employed to analyze the induction of the Ac transposase by salicylic acid (SA). Applying the β -glucuronidase histochemical assay, Ds excision efficiency was determined in the regeneration calli after treatment of a tobacco leaf disc with SA. A 111-bp regulatory element, located between nucleotides −699 to −588 of the PR-1a promoter, was fused to the 89-bp CaMV35S core promoter to serve as the inducible promoter, PRΔD. When the PRΔD promoter was fused with the Ac transposase gene and induced by SA, it triggered a 26-fold higher Ds excision efficiency than the native PR-1a promoter fusion. Furthermore, Ds excision events occurred mainly in the regeneration calli at day 8 after induction with SA. Because Ds excision events could be induced in such a short period, an alternative gene tagging strategy is suggested.