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UV Irradiation and Desiccation Modulate the Three-dimensional Extracellular Matrix of Nostoc commune (Cyanobacteria)

2005; Elsevier BV; Volume: 280; Issue: 48 Linguagem: Inglês

10.1074/jbc.m505961200

1083-351X

Deborah J. Wright, S C Smith, Vinita Joardar, Siegfried Scherer, Jody Jervis, Andrew Warren, Richard F. Helm, Malcolm Potts,

Microbial Community Ecology and Physiology

Cyanobacterium Nostoc commune can tolerate the simultaneous stresses of desiccation, UV irradiation, and oxidation. Acidic WspA, of ∼33.6 kDa, is secreted to the three-dimensional extracellular matrix and accounts for greater than 70% of the total soluble protein. The wspA gene of N. commune strain DRH1 was cloned and found in a single genomic copy, in a monocistronic operon. Transcription of wspA and sodF (superoxide dismutase), and synthesis and secretion of WspA, were induced upon desiccation or UV-A/B irradiation of cells. Recombinant WspA binds the UV-A/B absorbing pigment scytonemin through non-covalent interactions. WspA peptide polymorphism, and heterogeneity of multiple wspA sequences within cells of a single colony, account for distinct WspA isoforms. WspA has no similarity to entries in the sequence databases and wspA, a possible xenolog, is restricted to a subset of strains in the "form species" N. commune characterized through group I intron phylogeny. We hypothesize that WspA plays a central role in the global stress response of N. commune through modulation of the structure and function of the three-dimensional extracellular matrix, particularly the transport, distribution, and/or macromolecular architecture of mycosporine and scytonemin UV-A/B absorbing pigment complexes. Cyanobacterium Nostoc commune can tolerate the simultaneous stresses of desiccation, UV irradiation, and oxidation. Acidic WspA, of ∼33.6 kDa, is secreted to the three-dimensional extracellular matrix and accounts for greater than 70% of the total soluble protein. The wspA gene of N. commune strain DRH1 was cloned and found in a single genomic copy, in a monocistronic operon. Transcription of wspA and sodF (superoxide dismutase), and synthesis and secretion of WspA, were induced upon desiccation or UV-A/B irradiation of cells. Recombinant WspA binds the UV-A/B absorbing pigment scytonemin through non-covalent interactions. WspA peptide polymorphism, and heterogeneity of multiple wspA sequences within cells of a single colony, account for distinct WspA isoforms. WspA has no similarity to entries in the sequence databases and wspA, a possible xenolog, is restricted to a subset of strains in the "form species" N. commune characterized through group I intron phylogeny. We hypothesize that WspA plays a central role in the global stress response of N. commune through modulation of the structure and function of the three-dimensional extracellular matrix, particularly the transport, distribution, and/or macromolecular architecture of mycosporine and scytonemin UV-A/B absorbing pigment complexes. Cyanobacteria make a major contribution to world photosynthesis and nitrogen fixation, but their dense and often toxic "blooms" in marine, brackish, and fresh waters are of growing concern to public health (1Whitton B.A. Potts M. Ecology of Cyanobacteria: Their Diversity in Time and Space. 1st Ed. Kluwer, Dordrecht, Netherlands2000Google Scholar). The sequences of a wide range of cyanobacterial genomes are becoming available and constitute an important resource both for the rational management and exploitation of these organisms, as well as the study of topics as diverse as carbon fixation, cell differentiation, plant and fungal symbioses, secondary product biosynthesis, and evolution. 3www.kazusa.or.jp/cyano/cyano.html and genome.ornl.gov/microbial/npun/22dec03/npun.html. 3www.kazusa.or.jp/cyano/cyano.html and genome.ornl.gov/microbial/npun/22dec03/npun.html. Why is one of the key groups of organisms in the Precambrian still one of the most important groups of phototrophs today? Almost certainly their success stems, in large part, from a capacity to withstand multiple, superimposed, environmental stresses. Those cyanobacteria with a cosmopolitan distribution are excellent model systems for molecular and biochemical studies of complex stress response pathways. A feature of some of these populations is that they are often dominated by what appears to be a single morphological form. Because such forms may be readily identified, but occur in diverse localities, the term "form species" has been used to describe such colonies. Macroscopic, readily identifiable communities of bacteria such as these, provide a unique opportunity to question how structure-function relationships contribute to our growing awareness of metagenomics (the genetic diversity in naturally occurring populations of cells; Ref. 2Venter J.C. Remington K. Heidelberg J.F. Halpern A.L. Rusch D. Eisen J.A. Wu D. Paulsen I. Nelson K.E. Nelson W. Fouts D.E. Levy S. Knap A.H. Lomas M.W. Nealson K. White O. Peterson J. Hoffman J. Parsons R. Baden-Tillson H.C.P. Rogers Y.H. Smith H.O. Science. 2004; 304: 66-74Crossref PubMed Scopus (3215) Google Scholar), and how the principles of biological chemistry can mold diversity in situ. Nostoc commune (3Wright D. Prickett T. Helm R.F. Potts M. Int. J. Syst. Evol. Microbiol. 2001; 51: 1839-1852Crossref PubMed Scopus (45) Google Scholar) is a visually conspicuous biological component of nutrient impoverished environments from the Tropics to the polar regions where it is often the principal source of fixed nitrogen (4Potts M. Whitton B.A. Potts M. Nostoc Ecology of the Cyanobacteria: Their Diversity in Time and Space. 465-504. Kluwer Academic Publishers, Dordrecht, Netherlands,2000: 465-504Google Scholar, 5Mollenhauer D. Bengtsson R. Lindstrom E.-A. Eur. J. Phycol. 1999; 34: 349-360Crossref Scopus (3) Google Scholar). In the context of the present study, it can be emphasized that as the first documented biofilm, the cyanobacterium N. commune has had recognition since Medieval Times (6Potts M. Int. J. Syst. Bacteriol. 1997; 47: 584Crossref Scopus (26) Google Scholar), yet is now an endangered species in the European Union and in neighboring countries (5Mollenhauer D. Bengtsson R. Lindstrom E.-A. Eur. J. Phycol. 1999; 34: 349-360Crossref Scopus (3) Google Scholar), as well as in China (4Potts M. Whitton B.A. Potts M. Nostoc Ecology of the Cyanobacteria: Their Diversity in Time and Space. 465-504. Kluwer Academic Publishers, Dordrecht, Netherlands,2000: 465-504Google Scholar). An understanding of the biological chemistry of N. commune is relevant in view of the simultaneous and overlapping extremes of desiccation, UV irradiation, oxidative stress, long-term metabolic arrest, and nutrient deficiency, to which this cyanobacterium is subjected (7Potts M. Microbiol. Rev. 1994; 58: 755-805Crossref PubMed Google Scholar). One feature of N. commune that warrants special emphasis is its biochemically complex extracellular matrix (8Hill D.R. Peat A. Potts M. Protoplasma. 1994; 182: 126-148Crossref Scopus (93) Google Scholar). The principal components of this matrix are a diverse collection of secreted macromolecules including water stress protein (WspA) 4The abbreviations used are: WspAwater stress protein AF-APAfluorescein 6-aminopenicillanic acidAPA6-aminopenicillanic acidORFopen reading frameBLASTPbasic local alignment search toolWwattCAPS3-(cyclohexylamino) propanesulfonic acid. 4The abbreviations used are: WspAwater stress protein AF-APAfluorescein 6-aminopenicillanic acidAPA6-aminopenicillanic acidORFopen reading frameBLASTPbasic local alignment search toolWwattCAPS3-(cyclohexylamino) propanesulfonic acid. (9Scherer S. Potts M. J. Biol. Chem. 1989; 264: 12546-12553Abstract Full Text PDF PubMed Google Scholar, 10Hill D.R. Hladun S.L. Scherer S. Potts M. J. Biol. Chem. 1994; 269: 7726-7734Abstract Full Text PDF PubMed Google Scholar), a highly stable and active superoxide dismutase (SodF) (11Shirkey B. Kovarcik D.P. Wright D.J. Wilmoth G. Prickett T.F. Helm R.F. Gregory E.M. Potts M. J. Bacteriol. 2000; 182: 189-197Crossref PubMed Scopus (129) Google Scholar), water-soluble (mycosporine amino acids) (12EhlingSchulz M. Bilger W. Scherer S. J. Bacteriol. 1997; 179: 1940-1945Crossref PubMed Google Scholar) and lipid-soluble (scytonemin) (8Hill D.R. Peat A. Potts M. Protoplasma. 1994; 182: 126-148Crossref Scopus (93) Google Scholar, 13Hunsucker S.W. Tissue B.A. Potts M. Helm R.F. Anal. Biochem. 2001; 288: 227-230Crossref PubMed Scopus (24) Google Scholar) UV absorbing pigments. All of these components are distributed within a secreted high molecular weight extracellular polysaccharide, a glycan (14Helm R.F. Huang Z. Edwards D. Leeson H. Peery W. Potts M. J. Bacteriol. 2000; 182: 974-982Crossref PubMed Scopus (123) Google Scholar), which has unusual rheological properties and constitutes the bulk of desiccated and rehydrated colonies (14Helm R.F. Huang Z. Edwards D. Leeson H. Peery W. Potts M. J. Bacteriol. 2000; 182: 974-982Crossref PubMed Scopus (123) Google Scholar, 15Shaw E. Hill D.R. Brittain N. Wright D.J. Tauber U. Marand H. Helm R.F. Potts M. Appl. Environ. Microbiol. 2003; 69: 5679-5684Crossref PubMed Scopus (48) Google Scholar). The metabolic investment in the components of the matrix is considerable and it is our hypothesis that this extracellular complex plays a central role in the response of N. commune to environmental stress. water stress protein A fluorescein 6-aminopenicillanic acid 6-aminopenicillanic acid open reading frame basic local alignment search tool watt 3-(cyclohexylamino) propanesulfonic acid. water stress protein A fluorescein 6-aminopenicillanic acid 6-aminopenicillanic acid open reading frame basic local alignment search tool watt 3-(cyclohexylamino) propanesulfonic acid. Greater than 70% of the total soluble protein in desiccated colonies of N. commune is a group of acidic polypeptides, of cryptic function, referred to as Wsp with pI values between 4.3 and 4.8 and masses of 33-39 kDa (9Scherer S. Potts M. J. Biol. Chem. 1989; 264: 12546-12553Abstract Full Text PDF PubMed Google Scholar). Previous studies demonstrated that WspAs were secreted, had similar N-terminal sequences, and co-purified with a 1,4-β-d-xylanxylanohydrolase activity (10Hill D.R. Hladun S.L. Scherer S. Potts M. J. Biol. Chem. 1994; 269: 7726-7734Abstract Full Text PDF PubMed Google Scholar). The goals of this study were to isolate and characterize the water stress protein gene wspA and its genomic locus, identify its role in the stress tolerance of N. commune, and to identify structure/function relationships within the extracellular matrix. Bacterial Strains—Desiccated colonies of the cyanobacterial form species N. commune were collected from the field (3Wright D. Prickett T. Helm R.F. Potts M. Int. J. Syst. Evol. Microbiol. 2001; 51: 1839-1852Crossref PubMed Scopus (45) Google Scholar) (TABLE ONE). These materials were kept desiccated, in the dark, prior to the analyses described here. Nostoc sp. UTEX 584 was grown as described (16Potts M. Bowman M.A. Arch. Microbiol. 1985; 141: 51-56Crossref Scopus (43) Google Scholar). Cells of N. commune DRH1 and Nostoc punctiforme ATCC 29133 were grown as shallow suspensions to optimize gas exchange and minimize attenuation of incident radiation (see below). The medium was BG110 (17Rippka R. Deruelles J. Waterbury J.B. Herdmann M. Stanier R.Y. J. Gen. Microbiol. 1979; 111: 1-61Crossref Google Scholar) diluted 1:8 with distilled water.TABLE ONECyanobacteria reference numbers of form species include a mnemonic of the place of origin and the year the sample was desiccated (see Table I in Ref.3Wright D. Prickett T. Helm R.F. Potts M. Int. J. Syst. Evol. Microbiol. 2001; 51: 1839-1852Crossref PubMed Scopus (45) Google Scholar; see also text Fig. 2D)StrainCommentRef.Form species N. communeN. commune DRH1Liquid culture derived from N. commune CHEN/1986 by Donna Rene Hill3, 8N. commune CHEN/1986Desiccated field material, China3N. commune BBC/1992Desiccated field material, Blacksburg, VA3N. commune PARA/1979Desiccated field material, Australia3Var. flagelliformeN. commune TOP/1993Desiccated field material, Topsail Island, SC3N. commune TAG/1988Desiccated field material, Tägerwilen, Switzerland3N. sphaericum SPH/1998Desiccated, purchased from health store, China3N. commune ALD8122Desiccated field material, Aldabra Atoll, Indian Ocean3Other cyanobacteriaN. punctiforme ATCC29133ATCC culture collection strain; genome sequenced agenome.ornl.gov/microbial/npun/22dec03/npun.html.N. "commune" UTEX584UTEX culture collection strain; misnamed Nostoc strain3, 4a genome.ornl.gov/microbial/npun/22dec03/npun.html. Open table in a new tab UV Irradiation of Cells—Cell suspensions of N. commune DRH1 (200 ml) were grown in flat 800-ml capacity polystyrene Nunclon® Surface cell culture bottles (Nunc A/S, Roskilde, Denmark). Suspensions were agitated on a rotary shaker at 70 rpm, at 25 °C. Irradiation was provided by Sylvania soft white Duluz® compact fluorescent 23 W bulbs (Osram Sylvania Ltd., Mississauga, Canada), a 20 W RS UV-B medical light with a spectral maximum at 310 nm (model "TL," Philips, Holland), and 15 W black lights each with a spectral maximum at 368 nm (model F15T8-BL, General Electric). Total quantum scalar irradiance was measured with a model QSL-100 meter (Biospherical Instruments Inc., San Diego, CA). The flux densities of the UV-A and UV-B components of the spectrum were measured with DIX series UV-B and 365A sensors, respectively, with a Spectroline DRC-100X digital radiometer (Spectronics Corp., Westbury, NY). In these experiments full illumination represented a continuous photon flux density in the visible range of 330 μmol of photons m-2 s-1, with UV-A and UV-B maxima of 3.8 × 106 and 0.8 × 106 μWm2, respectively. Cells were also grown under reduced illumination, and in the dark, as specified in the figure legends. All values reported were the incident fluxes within culture vessels at the immediate surface of the cell suspensions. Desiccation and Rehydration of Cells—Cell suspensions (see above) were grown in Petri dishes and, after removing the lid partially, allowed to dry overnight, at room temperature and at ∼20% relative humidity, under a stream of sterile air. The desiccated cells, with a residual water content of 0.05 g of H2O/g of cell solids, were rehydrated with distilled water containing protease inhibitors at final concentrations of: 1 mm leupeptin, 0.07 mm benzamidine, 0.1 mm dithiothreitol, 1 mm EDTA, 25 mm diisopropyl fluorophosphate, 50 mm phenylmethylsulfonyl fluoride, in 0.1 mm Tris·HCl (pH 7.2) for 5 min, before repeating the desiccation step. After subsequent rehydration (for different time periods) the cell-free supernatant fraction was recovered and subjected to ultrafiltration using a 10-kDa cut-off Centricon centrifugal filter cartridge (Amicon, Millipore, Billerica, MA). Exopolysaccharide Purification—Purified glycan of N. commune DRH1 for use in binding assays (prepared in both ionized and deionized form) was obtained as described (14Helm R.F. Huang Z. Edwards D. Leeson H. Peery W. Potts M. J. Bacteriol. 2000; 182: 974-982Crossref PubMed Scopus (123) Google Scholar). Crude samples were obtained after centrifugation of cell suspensions at 30,000 × g, for 30 min. The supernatant fraction was lyophilized and the residue (∼3.5 mg of residue/ml of supernatant fraction) was mixed with a minimal volume of distilled water and allowed to dissolve overnight at 4 °C before further analysis. Extracellular polysaccharide was stained with Alcian Blue at pH 7.0. Scytonemin Purification and Binding Assay—Scytonemin was prepared from N. punctiforme ATCC 29133 as described (13Hunsucker S.W. Tissue B.A. Potts M. Helm R.F. Anal. Biochem. 2001; 288: 227-230Crossref PubMed Scopus (24) Google Scholar). Recombinant WspA (wspA cloned in pRSET C and expressed in Escherichia coli strain BL21-AI, Invitrogen) was purified from a whole cell lysate. The band corresponding to WspA was excised from 20-cm SDS-PAGE gels. Protein was eluted using a microelectroeluter (Centrilutor, Millipore), centrifuged in a 30-kDa cut-off Centriprep spin cartridge, and dialyzed exhaustively against 50 mm, pH 7.5, Tris·HCl buffer. Scytonemin (dissolved in dimethyl formamide), glycan, and WspA were mixed in different ratios (see text), incubated at 37 °C for 2.5 h, and resolved in 12.5% Criterion pre-cast gels (Bio-Rad). Synthesis of Fluorescein 6-Aminopenicillanic Acid and Binding Assay—Fluorescein 6-aminopenicillanic acid (F-APA) was synthesized from 6-aminopenicillanic acid (F-APA; 2 mg ml-1 in distilled water) and 5(6Potts M. Int. J. Syst. Bacteriol. 1997; 47: 584Crossref Scopus (26) Google Scholar)-carboxyfluorescein N-succinimidyl ester (2 mg ml-1 in distilled water). The reagents (Fluka Chemical Co., Milwaukee, WI) were mixed and left at room temperature for 15 h. Analytical thin layer chromatography was used to assess the degree of coupling and the Rf values of the product and reactants. Aliquots of the reaction mixture (250 μl) were resolved with Whatman silica preparative TLC plates using 80:20, acetonitrile:water. F-APA was scraped from plates and resuspended in a minimal amount of 50 mm Tris·HCl (pH 7.4). The supernatant fraction was recovered following high-speed centrifugation and used immediately. Freshly prepared F-APA was used in each independent series of binding assays. Aliquots of concentrated N. commune DRH1 cell-free extracellular supernatant fractions (10 μl; 0.0528 mg ml-1) were mixed with different concentrations of F-APA and incubated for 30 min at 30 °C. Competition assays were designed with unlabeled 6-APA as described in the figure legends. The mixtures were boiled at 100 °C for 5 min to disrupt any nonspecific aggregates of protein and F-APA. The samples were then resolved through SDS-PAGE (0.25 μg of protein/lane) and transferred to Immobilon P membrane using a CAPS buffer system (18Potts M. Angeloni S.V. Ebel R.E. Bassam D. Science. 1992; 256: 1690-1692Crossref PubMed Scopus (98) Google Scholar). For further details of the assay see the supplemental materials and methods. Protein and Peptide Purification—Several methods were used to obtain protein sequence information. Desiccated colonies of N. commune CHEN/1986 (5-20 g) were frozen under liquid nitrogen and ground to a powder using a pestle and mortar. The powder was mixed with 200 to 600 ml of sterile-distilled water containing protease inhibitors (above) and the cells were allowed to rehydrate for 30 min to release soluble protein. Aliquots of the cell-free supernatant fraction were subjected to ultrafiltration (using a 10-kDa cut-off Centricon centrifugal filter cartridge; Millipore; Amicon, Billerica, MA), and analytical fast protein liquid chromatography (Amersham Biosciences) as described (10Hill D.R. Hladun S.L. Scherer S. Potts M. J. Biol. Chem. 1994; 269: 7726-7734Abstract Full Text PDF PubMed Google Scholar); using a Mono Q HR 5/5 anion exchanger, a Mono S HR 5/5 cation exchanger, a Mono P 5/20 chromatofocusing column, a phenyl-Superose HR 5/5 hydrophobic interaction column, or a Superose 12 HR 10/30 gel exclusion chromatography column. Protein sequence analysis was performed on purified fractions of WspA protein, and on partially purified fractions of WspA that were transferred to Immobilon-P membranes via Western blotting (18Potts M. Angeloni S.V. Ebel R.E. Bassam D. Science. 1992; 256: 1690-1692Crossref PubMed Scopus (98) Google Scholar). Conditions for the treatment of Wsp with Glu-C endoprotease and Lys-C endoprotease were as described (9Scherer S. Potts M. J. Biol. Chem. 1989; 264: 12546-12553Abstract Full Text PDF PubMed Google Scholar, 10Hill D.R. Hladun S.L. Scherer S. Potts M. J. Biol. Chem. 1994; 269: 7726-7734Abstract Full Text PDF PubMed Google Scholar). An aliquot of the supernatant fraction was reduced with 2-mercaptoethanol prior to alkylation of any potential cysteine residues with iodoacetamide. After dialysis the sample was concentrated and an aliquot was subjected to preparative SDS-PAGE (10% w/v gels). A polypeptide with a molecular mass of ∼35-kDa was excised from multiple lanes and recovered through electroelution in 0.1 m Hepes, 0.1% SDS buffer (w/v). N-terminal sequence analysis was performed on 7.47 mg (202 pmol) of protein. An equivalent amount of protein was taken to dryness and hydrolyzed in gas phase 6 n HCl for 20 h at 110 °C. After hydrolysis the sample was dissolved in borate buffer prior to compositional analysis. An equivalent amount of the alkylated sample was dialyzed to exchange the sample buffer and incubated with trypsin overnight, followed by reverse phase high performance liquid chromatography to isolate peptides (Hewlett Packard model HP1090) and Edman degradation analysis. Preparative Isoelectric Focusing—A sample (39 mg of total protein) at a pH of 6.5 was added to 16 ml of deionized 5 m urea and 1.8-ml ampholytes (Bio-Rad) and loaded onto a Mini Rotofor Cell (Bio-Rad) for initial fractionation in the range pH 3-5. Constant power (10 W) was applied for 5 h, with the system cooled to 4 °C, and was terminated when the voltage had stabilized (1982 V) for about 30 min. Fractions were collected for protein determinations and SDS-PAGE. After evaluation, fractions of interest were pooled, diluted in additional 5 m urea, and separated further in the Mini Rotofor Cell. LC-MS/MS—Mass spectrometric analyses were as described (19Hunsucker S.W. Klage K. Slaughter S.M. Potts M. Helm R.F. Biochem. Biophys. Res. Commun. 2004; 317: 1121-1127Crossref PubMed Scopus (30) Google Scholar). Full details are presented in supplemental materials and methods. Nucleic Acid Manipulations and Analyses—Genomic DNAs from N. commune DRH1 and N. commune UTEX 584 were isolated in the presence of cetyltrimethylammonium bromide, and purified through cesium chloride buoyant-density ultracentrifugation as described (11Shirkey B. Kovarcik D.P. Wright D.J. Wilmoth G. Prickett T.F. Helm R.F. Gregory E.M. Potts M. J. Bacteriol. 2000; 182: 189-197Crossref PubMed Scopus (129) Google Scholar, 20Shirkey B. McMaster N.J. Smith S.C. Wright D.J. Rodriguez H. Jaruga P. Birincioglu M. Helm R.F. Potts M. Nucleic Acids Res. 2003; 31: 2995-3005Crossref PubMed Scopus (89) Google Scholar). Detailed protocols for polymerase chain reaction assay, construction of gene libraries, synthesis of hybridization probes, screening of gene libraries, Northern analysis, and primer extension assays are provided in supplemental materials and methods. Accession Number—The nucleotide and protein sequence data described in this study were deposited in NCBI with assigned accession numbers DQ155425 and DQ155426. Primary Sequence Analysis of Native WspA—To isolate wspA we first obtained data on the primary amino acid sequence of the native WspA from N. commune CHEN/1986 through limited endoproteolysis and Edman degradation assays. In multiple independent sequence assays, with seven of eight peptide preparations, the yield (pmol cycle-1) at cycles 10, 11, and 12 dropped sharply (to 90% sequence identity to putative proteins in N. punctiforme ATCC 29133. The translation stop codon of orfT34 and initiation codon of orfT35 overlap and presumably function through translation coupling; in addition the putative product of orfT34 contains 10% phenylalanine. Structural Organization of wspA and Its Flanking Regions—The full-length wspA ORF has three potential ATG translation initiation codons within the first 40 codons (Fig. 2B). The third ATG codon precedes immediately the sequence identified in multiple trials at the N terminus of WspA through protein sequencing (Fig. 1A, ALYGY ...), and is located 10-15 bases downstream of the putative Shine-Dalgarno sequence 5′-AAGGAG-3′. Inspection of the N-terminal region of WspA failed to identify potential sequences that would permit the protein to enter the secretory pathway (21Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4928) Google Scholar). However, a 14-residue putative signal sequence, fulfilling criteria of SignalP for Gram-positive bacteria, was identified at the C terminus with a putative cleavage site between residues 309 and 310 in one of three regions predicted to be disordered (Fig. 2C, arrowhead). Following isolation of wspA from N. commune DRH1, PCR was used to amplify the gene from different sources of N. commune that were characterized through phylogenetic analysis based upon their Group I intron sequences (3Wright D. Prickett T. Helm R.F. Potts M. Int. J. Syst. Evol. Microbiol. 2001; 51: 1839-1852Crossref PubMed Scopus (45) Google Scholar); our analysis confirmed microheterogeneity in this collection of genomic wspA clones (Fig. 2D). The wspA Promoter Region—We focused our attention on the region proximal to and upstream of wspA, as well as physiological profiling to determine under which conditions wspA was transcribed, and questioned whether wspA occurred in a mono- or polycistronic operon. Primer extension assay of wspA mRNA (Fig. 3, A and B) identified a primary reverse transcriptase-PCR product of ∼475 bp and additional products ∼345, ∼250, and ∼200 bp in low yield. Inspection of sequences upstream of the sites of extension termination, and comparison of the intergenic region with -35 and -10 promoter sequences identified in a detailed study of the genome of cyanobacterium Prochlorococcus MED4 (22Vogel J. Axmann I.M. Herzel H. Hess W.R. Nucleic Acids Res. 2003; 31: 2890-2899Crossref PubMed Scopus (36) Google Scholar), located two regions of interest (Fig. 3, A and C). Region one, predicted as a putative wspA promoter contained 5′-TATAGT-3′ (-10), spaced 16 nucleotides from a 14-bp region that corresponded with a consensus -35 element found in Prochlorococcus genes including petH2 and ftsZ (Fig. 3C). A second 5′-TATAGT-3′ sequence in region two was 16 nucleotides from a putative -35 sequence that corresponded in sequence, and again in spacing, with the -35 region of other Prochlorococcus genes including ccmK and kaiB. The latter gene also had 5′-TATAGT-3′ as its -10 sequence (22Vogel J. Axmann I.M. Herzel H. Hess W.R. Nucleic Acids Res. 2003; 31: 2890-2899Crossref PubMed Scopus (36) Google Scholar). Interestingly, the AT-rich region from nucleotides 9003 to 9326 (upstream of wspA; Fig. 2B) contains 18 sequences that conform to the consensus -10 sequence 5′-TANNNT-3′, and 13 of these are very frequent -10 elements in the Prochlorococcus genome. wspA Is Found Solely in N. commune—WspA has no counterpart in the proteomes of those cyanobacteria for which full genome sequence data are available, and shares no obvious identity with sequences in the public databases. The top BLASTP scores (∼20% identity for residues ∼40 through 280) included: a putative surface-exposed protein in Burkholderia pseudomallei (accession YP_110805); the Hep_Hag family protein/hemagglutinin motif and/YadA-like domain protein in Burkholderia mallei ATCC 23344 (accession YP_105401); type I flagellin FliC in Burkholderia cepacia (accession AAC38200); muconate cycloisomerase in Solfolobus solfataricus P2 (accession AAK43295); and nucleoporin, autotransporter, and adhesion sequences. The sequence DRD, which was recalcitrant to sequence assay (Fig. 1A), occurs in the most hydrophilic region of WspA; in a region predicted to be disordered (supplemental materials Fig. S1) (23Linding R. Russell R.B. Neduva V. Gibson T.J. Nucleic Acids Res. 2003; 31: 3701-3708Crossref PubMed Scopus (825) Google Scholar). There was little commonality in the codon usage of wspA, sodF, icfA, or orfT71 (see Fig. 2A). Other Proteins in the Extracellular Matrix—A fraction of WspA and SodF, in relatively pure form, is secreted by desiccated field materials of N. commune CHEN/1986, simply upon rehydration, but upon cell lysis the majority of the protein is distributed in a dark brown to black, highly viscous extract, which requires considerable purification before resolution in SDS-PAGE gels (Fig. 4A) (9Scherer S. Potts M. J. Biol. Chem. 1989; 264: 12546-12553Abstract Full Text PDF PubMed Google Scholar, 10Hill D.R. Hladun S.L. Scherer S. Potts M. J. Biol.