FlrA Represses Transcription of the Biofilm-Associated bpfA Operon in Shewanella putrefaciens.

Manipulation of biofilm formation in Shewanella is beneficial for application to industrial and environmental biotechnology. BpfA is an adhesin largely responsible for biofilm formation in many Shewanella species. However, the mechanism underlying BpfA production and the resulting biofilm remains vaguely understood. We previously described the finding that BpfA expression is enhanced by DosD, an oxygen-stimulated diguanylate cyclase, under aerobic growth. In the present work, we identify FlrA as a critical transcription regulator of the bpfA operon in Shewanella putrefaciens CN32 by transposon mutagenesis. FlrA acted as a repressor of the operon promoter by binding to two boxes overlapping the -10 and -35 sites recognized by σ70 DosD regulation of the expression of the bpfA operon was mediated by FlrA, and cyclic diguanylic acid (c-di-GMP) abolished FlrA binding to the operon promoter. We also demonstrate that FlhG, an accessory protein for flagellum synthesis, antagonized FlrA repression of the expression of the bpfA operon. Collectively, this work demonstrates that FlrA acts as a central mediator in the signaling pathway from c-di-GMP to BpfA-associated biofilm formation in S. putrefaciens CN32. IMPORTANCE: Motility and biofilm are mutually exclusive lifestyles, shifts between which are under the strict regulation of bacteria attempting to adapt to the fluctuation of diverse environmental conditions. The FlrA protein in many bacteria is known to control motility as a master regulator of flagellum synthesis. This work elucidates its effect on biofilm formation by controlling the expression of the adhesin BpfA in S. putrefaciens CN32 in response to c-di-GMP. Therefore, FlrA plays a dual role in controlling motility and biofilm formation in S. putrefaciens CN32. The cooccurrence of flrA, bpfA, and the FlrA box in the promoter region of the bpfA operon in diverse Shewanella strains suggests that bpfA is a common mechanism that controls biofilm formation in this bacterial species.

A new marine bacterial laccase with chloride-enhancing, alkaline-dependent activity and dye decolorization ability.

A bacterial laccase gene designated as lac21 was screened from a marine microbial metagenomic library of the South China Sea based on sequence screening strategy. The protein encoded by lac21 shared less than 40% sequence identities with all of the laccases found. Lac21, which was recombinantly expressed in Escherichia coli, showed high activity toward syringaldazine at an optimum pH of 7.5 and temperature of 45°C. Lac21 was stable at pH values ranging from 5.5 to 9.0 and temperatures lower than 40°C. Interestingly, chloride enhanced the laccase activity, with concomitant increase in substrate affinity. Furthermore, Lac21 has high decolorization capability toward azo dyes in the absence of redox mediators, with 80% of Reactive Deep Blue M-2GE (50mg/L) being decolorized by 15U/L enzyme after 24h incubation at 20°C. These unusual properties demonstrate that the new bacterial laccase Lac21 has potentials in specific industrial or environmental applications.

[Cloning and heterologous expression of the gene of laccase C from Trametes sp. 420 and potential of recombinant laccase in dye decolorization].

A new laccase gene (lacC) was cloned from the genomic DNA isolated from Trametes sp. 420, a new laccase-producing fungus, using the degenerate primers based on the conserved copper-binding regions in fungal laccases. Long distance-inverse PCR (LD-IPCR) was used to amplify the flanking sequences of the gene. The lacC DNA sequence obtained was 3640 base pairs (bp), including the entire open reading frame (2263bp) and the 5'-and 3'-noncoding regions. The lacC cDNA sequence is 1560bp, encoding a 519 amino acid protein. The deduced peptide sequence of LacC contains ten putative N-glycosylation sites and four conserved copper-binding regions. The lacC cDNA without its signal sequence was cloned into the expression vector pPIC9K through the pPIC9 plasmid and transformed into the Pichia pastoris strain GS115.The positive transformant was cultured at 20 degrees C in BMM medium containing 0.3mmol/L CuSO4 and 0.8% alanine, with the yield of the recombinant laccase rLacC being 1.62 x 10(4) U/L after a 9-day cell growth. Furthermore, the crude enzyme was used to decolorize several synthetic dyes at a final concentration of 50mg/L. The results showed that rLacC (6U/L) possessed the valuable ability to decolorize dyes of triarylmethane and azo types tested. The presence of low molecular weight redox mediators of ABTS and HBT increased the efficiency and velocity of dye decolorization significantly.

Cloning of a laccase gene from a novel basidiomycete Trametes sp. 420 and its heterologous expression in Pichia pastoris.

The laccase gene lacD, cloned from a novel laccase-producing basidiomycete Trametes sp. 420, contained 2,052 base pairs (bp) interrupted by 8 introns. lacD displayed a relatively high homology with laccase genes from other white rot fungi, whereas the homology between lacD and laccase genes from plants, insects, or bacteria was less than 25%. A 498-amino acid peptide encoded by the lacD cDNA was heterologously expressed in the Pichia pastoris strain GS115, resulting in the highest yield of laccase (8.3 x 10(4) U/l) as determined with ABTS (2,2'-azinobis [3-ethylbenzothia-zoline-6-sulfonic acid]) as the substrate. Additionally, the enzyme activity of recombinant laccase on decolorization of some industrial dyes was assessed.

[High output of a Trametes laccase in Pichia pastoris and characterization of recombinant enzymes].

A laccase gene (lacD) from the basidiomycete Trametes sp. 420 was heterologously expressed in Pichia pastoris in two ways, resulting in two recombinant enzymes of rLacDx with native N-terminus and rLacDe with eight additional amino acid residues at N-terminus. The yields of rLacDx and rLacDe in shaken-flask cultures after an 18-day growth were 1.21 x 10(5) u/L and 7.38 x 10(4) u/L, respectively, as determined with 2,2'-azinobis(3-ethylbenzothia-zoline- 6-sulfonic acid) (ABTS) as substrate. The yield of rLacDx was further increased to 2.39 x 10(5) u/L under high-density fermentation while the production process was decreased to 7.5 days. In addition, rLacDx and rLacDe exhibited similar enzymatic characters in oxidizing substrate guaiacol, and were stable at 50 degrees C and at a pH range from 3 to 10. However, the specific activity of rLacDx (1761 u/mg) for ABTS was higher than that of rLacDe (1122 u/mg), and the apparent Km value of rLacDx (427 microM) was less than that of rLacDe (604 microM).

[Inducing synthesis of LacA from Trametes sp. AH28-2 and cloning & analysis of 5'-terminal sequence of transcription control of the gene].

Copper ion was necessary for the transcription of all laccase isozyme genes from Trametes sp. AH28-2, with higher concentrations of Cu2+ (1-2 mmol/L) being more favorable to the synthesis of laccase. In the glucose media containing 0.5 mmol/L Cu2+, the laccase activity of the supernate was rather low (44.3 u/L) and had an increase of 60.3% (71.0 u/L) when 4.0 mmol/L o-toluidine was added. Moreover, the activity reached up to 2584 u/L as the glucose was replaced by cellobiose. And Native-PAGE showed that LacA was the main laccase component if fungus was induced by o-toluidine or copper ions. It had been demonstrated by quantitative RT-PCR that the regulation of lacA expression, induced by o-toluidine, occurred at the transcriptional level, with the accumulation of mRNA transcripts being accompanied by the increase of laccase activity of the culture fluid. In addition, the structural gene of lacA interrupted by 10 introns was 2110 bp in length and the corresponding cDNA sequence was 1560 bp encoding a 520 aa protein, which had high similarities with other laccases from basidiomycetes. Furthermore, a length of 1881 bp of 5'-terminal sequence of transcription control of lacA, amplified by the improved inverse PCR, contained a TATA box, seven CAAT boxes as well as a number of putative cis-acting elements important for its expression, including five MREs, nine CreA-binding sites, four XREs, two STREs and seven nitrogen factor binding sites. The existence of these elements was well in agreement with the data obtained from Trametes sp. AH28-2 shaken-flask cultures.

Degradation of lignin in pulp mill wastewaters by white-rot fungi on biofilm.

An investigation was conducted to explore the lignin-degrading capacity of attached-growth white-rot fungi. Five white-rot fungi, Phanerochaete chrysosporium, Pleurotus ostreatus, Lentinus edodes, Trametes versicolor and S22, grown on a porous plastic media, were individually used to treat black liquor from a pulp and paper mill. Over 71% of lignin and 48% of chemical oxygen demand (COD) were removed from the wastewater. Several factors, including pH, concentrations of carbon, nitrogen and trace elements in wastewater, all had significant effects on the degradation of lignin and the removal of COD. Three white-rot fungi, P. chrysosporium, P. ostreatus and S22, showed high capacity for lignin degradation at pH 9.0-11.0. The addition of 1 g l-1 glucose and 0.2 g l-1 ammonium tartrate was beneficial for the degradation of lignin by the white-rot fungi studied.

[Studies on production, purification and partial characteristics of the extracellular laccase from Armilliria mellea].

The production conditions of extracellular laccase from Armilliria mellea and the characteristics of the enzyme were studied. The experiment proved that initial pH5.5 of the culture medium and temperature at 25 degrees C were favorable for laccase synthesis. As carbon resources, cellobiose and raffinose were better in terms of productivity than maltose, sorbose and galactose. Organic nitrogen source was more suitable for Armilliria mellea to synthesize laccase than inorganic nitrogen source. Peat extract (PE) enhanced notably the yield of laccase; the maximal yield was 7 times as much as that of the control when PE concentration was 50%. Three isozymes were detected in culture supernatant named A, B and C respectively after their mobility on PAGE. After concentrated by (NH4)2SO4 precipitation, laccase A was further purified to homogeneity by preparative native PAGE and anion exchange column chromatography. The native enzyme was a single polypeptide with a molecular mass of approximately 59 kD estimated by SDS-PAGE, while 58 kD by gel filtration chromatography under non-denaturing conditions. Determined by IEF its isoelectric point was 4.0. The optimal pH value and temperature were 5.6 and 60 degrees C respectively in catalytic reaction of oxidizing guaiacol. At 60 degrees C and 65 degrees C, half-lives of laccase A were 45 min and 36.8 min, respectively. Enzyme activity was inhibited with 100 mmol/L Cl-, but was activated with 1 mmol/L SO4(2-). However, if the concentration of NaN3 was only 1 mmol/L, laccase A lost its activity completely. 10 mmol/L EDTA had no effect on laccase A, while 1 mmol/L Cu2+ could enhance its activity. Laccase A showed a good stability when the pH of the buffer varied from 5.2 to 7.2. Using guaiacol as the substrate, the Km was 1.026 mmol/L and the Vmax was 5 mumol/(min.mg); using ABTS instead, the Km was 0.22 mmol/L and Vmax was 69 mumol/(min.mg).

Effects of Site-specific Mutagenesis of K253 and N184V on the Thermostability of D-Glucose Isomerase.

In order to enhance the thermostability of D-Glucose isomerase (GI), its mutants, GIK253R and GIN184V, were obtained with the double primer method of site-directed mutagenesis. Then their biochemical properties were assayed and compared with wild-type GI. The results showed that: (1) The mutant K253R was less stable than the wild-type GI at 70 degrees and 80 degrees. But in 1 M L-Rhaminose at 70 degrees, they had similar rate of heat inactivation. Furthermore, the mutant K253R has higher specific activity than the wild-type GI. (2) The mutant N184V had much less thermostability and specific activity as compared with the wild-type GI. These results were explained by their kinetic parameters and crystal structure model.