Department of Genetics and Microbiology, Faculty of Biology1 and Department of Biochemistry and Molecular Biology B2, University of Murcia, 30100 Murcia, Spain
Author for correspondence: Antonio Sanchez-Amat. Tel: +34 968 364955. Fax: +34 968 363963. e-mail: antonio{at}um.es
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ABSTRACT |
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Keywords: Marinomonas mediterranea, marine bacterium, two-component histidine kinase, laccase, tyrosinase
Abbreviations: Amp, ampicillin; DMP, dimethoxyphenol; DMPO, DMP oxidase; DO, dopa oxidase; Gm, gentamicin; Km, kanamycin; L-dopa, 3,4-dihydroxyphenylalanine; PPO, polyphenol oxidase; RACE, rapid amplification of cDNA ends; Rif, rifampicin; SO, syringaldazine oxidase; TH, tyrosine hydroxylase
a The GenBank accession number for the sequence reported in this paper is AF398464.
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INTRODUCTION |
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Laccases are enzymes with promising biotechnological applications. They have been intensively studied in fungi, where they have been related to a variety of physiological functions (Thurston, 1994 ). Among these functions, this enzyme is used to produce dopa-melanins in the presence of diphenolic compounds in the human pathogen Cryptococcus neoformans (Williamson et al., 1998
). In another fungus, Aspergillus fumigatus, a laccase participates in conidial pigment biosynthesis (Tsai et al., 1999
).
By contrast, laccase activity has been rarely reported in prokaryotes (Givaudan et al., 1993 ; Solano et al., 1997
). In Azospirillum lipoferum this enzymic activity was described for the first time in a bacterium (Givaudan et al., 1993
). Although its physiological role is unknown, it has been related to melanin synthesis in this micro-organism (Faure et al., 1994
). Recently, there has been mounting evidence indicating that different bacterial blue multi-copper proteins also show some kind of laccase activity (Hullo et al., 2001
; Kim et al., 2001
; Solano et al., 2001
). These proteins are involved in different cellular processes such as sporulation in Bacillus (Hullo et al., 2001
) and copper resistance in Escherichia coli (Kim et al., 2001
).
The physiological relevance of the laccase activity in M. mediterranea is unknown. Strain Tn101 is a null mutant in this activity obtained by transposon mutagenesis (Solano et al., 2001 ). The preliminary characterization of this mutant has not yet shown any relevant physiological alteration under standard culture conditions. These results suggest that this enzyme is not essential for bacterial growth in these conditions and it should rather play some role in the adaptation of M. mediterranea to some specific environmental conditions. Thus some regulatory elements should be involved in the connection between environmental stimuli and expression of PPO activities and we have initiated studies to address this point.
Two-component signal transduction systems regulate the adaptive responses of prokaryotic and eukaryotic micro-organisms to changing environmental conditions (Stock et al., 1989 ). In the more typical form, they are composed of two proteins: a histidine protein kinase, also called sensor kinase, and a response regulator. The sensor kinase is a membrane protein that senses environmental stimuli through its N-terminal input domain and undergoes autophosphorylation at a highly conserved histidine residue in the C-terminal transmitter domain. Subsequently, the phosphate group is transferred to an aspartic residue sited in the N-terminal receiver domain of the response regulator. This change modulates the activity of the C-terminal output domain, generally with DNA-binding capacity, regulating gene expression in response to the stimulus.
Modified and more complex versions of the phosphoryl group-based transduction mechanism have been described (Appleby et al., 1996 ). One example is the four-step phosphorelay system, in which the phosphoryl group is transferred in the order His-Asp-His-Asp with the phosphorylated amino acids in different domains. This phosphorelay system can show different structural organizations with the four conserved domains distributed in a variable number of proteins (Appleby et al., 1996
). For instance, the process of sporulation in Bacillus subtilis was the first process regulated by the mechanism described and in this case the four phosphorylated domains are in four different proteins (Burbulys et al., 1991
). Another example of a different organization is the BvgS-BvgA system that regulates virulence factors in Bordetella pertussis (Uhl & Miller, 2001
). This phosphorelay system is only composed of two proteins: BvgS is a hybrid tripartite sensor kinase since it is fused with a phosphoaspartate receiver domain plus a second phosphotransferase domain. BvgA is its associate response regulator containing the final domain with the Asp residue.
The aim of this work was to initiate the studies on the regulation of the expression of PPO activities in M. mediterranea. A mutant affected in the regulation of these processes has been obtained by transposon mutagenesis. The sequencing of the disrupted gene in this mutant has shown that it encodes a hybrid sensor kinase with the same organization as BvgS. This sensor kinase regulates both laccase and tyrosinase activities, as well as melanin synthesis, indicating a relationship between these processes in M. mediterranea at least at the regulatory level.
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METHODS |
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Southern blotting.
Chromosomal DNA was extracted from several independent M. mediterranea rifampicin-resistant (Rifr), ampicillin-sensitive (Amps), Kmr transconjugants obtained after the E. coli S17-1 (pir) (pLOFKm)xM. mediterranea MMB-1R matings. Southern blot analysis was carried out after digesting these samples with different restriction enzymes. A digoxigenin-labelled NotI fragment from pLOFKm encompassing the kanamycin-resistance gene was used as a probe to check transposon insertion.
Cloning of the ppoS gene.
Isolated genomic DNA of M. mediterranea T103 was digested with EcoRI and ligated to pUC19 digested with the same enzyme. The ligation mixture was transformed into E. coli DH5 and transformants selected for ampicillin and kanamycin resistance. The plasmid obtained (pUCT103) was subcloned in pBKS II by using the NotI restriction sites that the transposon has close to both ends of IS10 and the EcoRI, ClaI and HindIII restriction sites in the M. mediterranea chromosome DNA. The DNA adjacent to the insertion point was sequenced using the T3 and T7 universal primers as well as some internal primers synthesized as required.
RNA extraction and Northern blot analysis.
M. mediterranea strains were inoculated in MMC, and two samples were taken, one at mid-exponential phase (OD600 around 0·3) and another at early stationary phase (OD600 around 0·9). Total RNA was extracted from M. mediterranea MMB-1R and T103 by standard protocols (Chirgwin et al., 1979 ). Ten micrograms of RNA were denatured by treatment with DMSO and glyoxal. The denatured RNA samples were electrophoresed through a 1% agarose gel in 10 mM phosphate running buffer, pH 7. Prior to blotting, certainty of equal sample loadings was verified by equivalent intensities of the 16S and 23S rRNA bands in all samples after acridine orange staining. RNA was transferred to a nylon transfer membrane Hybond-N+ (Pharmacia) by downward capillary and fixed by UV irradiation. The ppoA probe was generated by PCR using the primers DIRECT and REVTRUN (Table 1
) which encompassed a 1·2 kb fragment close to the 5' end of the gene. The ppoA probe was radioactively labelled with [
-32P]dATP by use of a random-primed DNA labelling kit (Boehringer) according to the manufacturers protocol. The labelled probe was purified by gel filtration in Sephadex G-25 columns (Pharmacia). Prehybridization and hybridization were carried out at 42 °C. The prehybridization solution contained 50% formamide, 0·9 M NaCl, 0·05 M sodium phosphate pH 8·3, 5 mM EDTA, 5x Denhardts reagent (0·1% Ficoll and 0·1% polyvinylpyrrolidone), 0·3% SDS, 0·2 mg BSA ml-1 and 0·25 mg denatured herring sperm DNA ml-1. The hybridization solution had the same composition plus 10% dextran sulfate and the labelled probe previously denatured at 100 °C for 5 min. Radioactive bands were visualized using the Molecular Imager System GS-525 (Bio-Rad) with the Molecular Analyst software (Bio-Rad).
Mapping the ppoS transcriptional start site.
To locate the transcriptional start of the ppoS gene, 5' rapid amplification of cDNA ends (RACE) experiments were carried out using a 5'/3' RACE Kit essentially as recommended by the manufacturer (Roche Molecular Biochemicals). Total RNA (2 µg) isolated from M. mediterranea at the stationary phase in MMC was reverse transcribed with the ppoS-specific primer PPOSP1 (see Table 1) which anneals 793814 bp downstream of the ppoS translational start site. A control reaction without reverse transcriptase was included to ensure that the resulting product was due to the amplification of cDNA rather than contaminating chromosomal DNA. A poly(dA) tail was appended to the 3' end of the cDNA using terminal transferase and PCR amplified using the RACE oligo(dT)-anchor primer and a nested ppoS-specific primer PPOSP2. The PCR product obtained was again PCR amplified with a kit-provided PCR anchor primer containing a ClaI site and a nested ppoS-specific primer PPOSP3 modified to introduce a BamHI restriction site. This last primer anneals 452476 bp downstream of the ppoS translational start. The resulting product was purified using a High Pure PCR product purification kit (Roche Diagnostics), cloned into the pBKSII vector at the ClaI/BamHI sites and sequenced.
Single-copy transcriptional fusion construction.
Plasmid pBFU8 containing a transcriptional fusion between ppoA and the lacZ gene was created using the auxiliary plasmid pUJ8 (de Lorenzo et al., 1990 ). The construction process is outlined in Fig. 1
. A PCR product carrying 135 bp upstream of the translational start of ppoA, and hence comprising the promoter region and extending into the coding region, was generated by amplification of M. mediterranea genomic DNA with primers ECOFUF and BAMFUR (Table 1
). This product was digested with EcoRI and BamHI cutting in the restriction sites introduced in the primers, and cloned between the corresponding sites of the plasmid pUJ8, generating a transcriptional fusion with the lacZ gene. The whole fusion was excised as an EcoRINotI fragment and inserted into the corresponding sites of the mini-Tn10 transposon present in the plasmid pBSL182 (Alexeyev & Shokolenko, 1995
), generating the plasmid pBFU8. A control plasmid named pBGAL was also created. pBGAL and pBFU8 were identical except that the former did not contain the fragment derived from the chromosome of M. mediterranea and hence the lacZ gene is promoterless. In both cases the lacZ gene is in the opposite orientation to the gentamicin (Gm)-resistance marker of the transposon (Fig. 1
). The transposons were transferred from donating E. coli into M. mediterranea MMB-1R and T103 by transposon mutagenesis as previously described (Solano et al., 2000
). Mutant strains that were Rifr, Gmr, and Amps with a single copy chromosomal insertion of the transposon were selected.
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RESULTS |
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The mutant obtained was grown in liquid MMC medium and all the PPO enzymic activities so far detected in M. mediterranea were quantitatively determined in cellular extracts (Table 2). The results obtained indicated that, consistent with the screening method, the mutant showed a significant decrease in the laccase activities (DMPO and SO) due to the multipotent PpoA, as well as in the SDS-activated tyrosinase-related activities. This pattern strongly suggested that a regulatory gene would be affected in the selected mutant.
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The strains MMB1LAC0 and T103LAC0, obtained with the control plasmid pBGAL, showed very low levels of ß-galactosidase activity (Fig. 6). This result agrees with the absence of promoter driving lacZ expression in the construct, and also indicates that its expression is not dependent on the genetic background of the strain mutagenized or the site of insertion. The opposite orientation of the gentamicin-resistance marker with respect to lacZ may contribute to the absence of reading from promoters located outside the construction.
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DISCUSSION |
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After transposon mutagenesis, we isolated a M. mediterranea mutant, strain T103, that was affected in laccase activity as well as in tyrosinase activity and melanin formation. Quantitative determinations on cellular extracts from this mutant showed a significant decrease in all the enzymic activities described in this micro-organism, that is those due to the multipotent laccase PpoA, as well as those due to the SDS-activated tyrosinase (Table 2). Both enzymes also had in common that they were growth-phase regulated in a similar fashion (Fig. 4
). These results clearly indicate that both enzymes are subjected to some coordinate regulatory mechanisms that induce their expression under specific conditions.
The gene mutated in strain T103 has been cloned and sequenced. According to BLAST homology search and sequence analysis, this gene encodes a sensor kinase belonging to a two-component regulatory system and hence it has been denominated ppoS. Two-component signal transduction systems have been described in many different prokaryotic micro-organisms and also in some eukaryotic organisms (Appleby et al., 1996 ). These systems control many different cellular processes (Stock et al., 1989
). However, this is the first example showing the involvement of these systems in the regulation of PPO activities or melanin synthesis.
As judged by the conserved domains contained in the PpoS sequence, this protein is a new member of the tripartite hybrid sensor kinase, since it shows three phosphorylable domains, H1, D1 and H2 (Table 3). This kind of sensor kinase participates in the multi-step phosphorelay, in which four phosphorylation events occur sequentially (Appleby et al., 1996
). They should work in association with another member of the two-component systems, the response regulator that contains a conserved final Asp in the fourth phosphorylated domain. This second protein should be involved in the interaction with the DNA and the transcriptional regulation of other genes. However, in spite of our attempts, the response regulator in M. mediterranea remains to be identified.
The sensor kinases are membrane proteins and in fact sequence analysis indicated that PpoS contains two transmembrane domains that must encompass a segment facing the periplasmic space. This segment should receive the environmental signal that triggers the first autophosphorylation leading to the adaptive response (Stock et al., 1989 ). An extensive search through the databases using the sequence of the periplasmic fragment of the M. mediterranea PpoS did not allow the identification of any sensor kinase showing similarity to it in that region. Similarly, the comparison between sensor kinases of B. subtilis and E. coli did not reveal similarities in the periplasmic regions, which was considered to reflect the different environments that those organisms occupy (Fabret et al., 1999
). In this regard, the marine environment from which M. mediterranea was isolated is a very different ecological niche. In addition, no information could be obtained using the whole sequence, since the proteins in the databases showing the highest identities to PpoS were GacS (LemA) of Pseudomonas syringae and BarA of E. coli, which showed identities as low as 29 and 28%, respectively.
According to the results shown in Fig. 4, there is a growth-phase regulation affecting both the multipotent laccase PpoA and the SDS-activated tyrosinase. PpoS does not seem to participate in the growth-phase regulation of PPO activities, since in the mutated strain T103 lower PPO activities are observed, but the growth-phase regulation is not suppressed. Data obtained with lacZ transcriptional fusions and Northern blots indicate that, at least for the ppoA gene, the regulation is at the transcriptional level. It is important to point out that the proposed promoter for ppoA shows a -10 region, CTAGTC (Sanchez-Amat et al., 2001
), identical to one of the putative regions in the ppoS promoter and also resembling the
38 consensus (Lee & Gralla, 2001
). These results strongly suggest that the growth-phase regulation could be controlled by an alternative sigma factor, such as
38. On the other hand, wild-type and T103 fusion strains show ß-galactosidase levels differing only by a factor of approximately two; this small difference can not justify the big difference between the two strains in the amount of PpoA RNA observed by Northern blot or in the enzymic activities. This discrepancy suggests that there is a similar transcriptional rate of the ppoA promoter in both strains and that the involvement of PpoS in the regulation of PpoA occurs at the post-transcriptional level. One likely possibility is that PpoS enhances factors stabilizing the ppoA transcript, as proposed for the regulation of BarA expression by RpoS (Mukhopadhyay et al., 2000
).
The coordinate regulation of both enzymes under stress conditions, such as reaching the stationary phase, suggests that they may form part of the adaptive response of M. mediterranea to these kinds of conditions. In this regard, melanin pigments are considered to be important factors in the resistance against stress factors such as UV radiation and oxidative damage. Under standard laboratory conditions we have shown that the important enzyme in melanin synthesis by M. mediterranea is the SDS-activated tyrosinase, which uses as substrate the tyrosine present in the culture medium (Solano et al., 1997 ; López-Serrano et al., 2002
). However, the substrate used for melanin synthesis in sea waters, the natural environment from which M. mediterranea was isolated, is unknown. It is important to bear in mind that although in marine waters it is possible to find some enriched microenvironments, such as surface biofilms, the mean amino acid concentration is in the range of 1030 nM (Lee & Bada, 1975
). In this context, the wide range of substrates that the multipotent laccase is able to oxidize may be important to facilitate melanin synthesis using a wider variety of phenolic compounds found in natural environments.
Alternatively, the redox nature of PPO enzymes implies that they can be used to either polymerize or depolymerize complex substrates such as lignin, as clearly evidenced in fungal laccases (Thurston, 1994 ). In marine waters most of the organic matter is in the form of high molecular mass compounds of phenolic nature that can even inhibit microbial metabolism (Morita, 1986
). These compounds are highly resistant to microbial degradation and hence the expression of the PPO activities might facilitate the degradation of these compounds and may give to the cell a competitive advantage when other more easily metabolizable substrates are depleted.
The regulation at the molecular level of PPO activities and melanin formation in M. mediterranea has been approached for the first time in this study. It has been shown that both PPOs are subjected to coordinate regulation. During the growth curve in complex medium, these activities are induced in stationary phase. The novel PpoS sensor histidine kinase, cloned and sequenced in this study, is involved in this regulation and allows maximal levels of enzymic activities. To go further in the characterization of the physiological relevance of PPO activities and melanin synthesis in M. mediterranea, the environmental signals sensed by PpoS and the response of M. mediterranea to stress conditions are under investigation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 4 March 2002;
revised 11 April 2002;
accepted 11 April 2002.