Biopolymers from marine prokaryotes.

Biopolymers from marine prokaryotes, both Bacteria and Archaea, offer a number of novel material properties and commercial opportunities. The characteristics of marine exopolysaccharides and melanins that enhance the survival ability of the organisms producing them can be exploited for a number of products ranging from emulsifiers to adhesives. In the prokaryotes, the polyhydroxyalkanoates form carbon-storage molecules, but their technological application is entirely different, serving as a potential base material for biodegradable plastics. Marine biopolymers are a significant and undeveloped biological resource.

Characterization of melA: a gene encoding melanin biosynthesis from the marine bacterium Shewanella colwelliana.

A recombinant plasmid with the ability to impart melanin synthesis to an Escherichia coli host was isolated from a Shewanella colwelliana genomic library. The genetic determinant of the Mel+ phenotype is carried on a 1.3-kb DNA fragment and sequence analysis of this revealed a single intact open reading frame that was sufficient for melanin synthesis (mel). This gene is expressed as a monocistronic transcript and a putative transcription start point is located 115 nucleotides upstream from the translational start codon. The mel gene encoded a protein of 39.5 kDa [346 amino acids (aa)] that showed no aa sequence homology with other proteins known to mediate melanin synthesis (e.g., tyrosinases).

Capsular polysaccharide surrounds smooth and rugose types of Salmonella enterica serovar Typhimurium DT104.

The biofilms and rugose colony morphology of Salmonella enterica serovar Typhimurium strains are usually associated with at least two different exopolymeric substances (EPS), curli and cellulose. In this study, another EPS, a capsular polysaccharide (CP) synthesized constitutively in S. enterica serovar Typhimurium strain DT104 at 25 and 37 degrees C, has been recognized as a biofilm matrix component as well. Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis indicated that the CP is comprised principally of glucose and mannose, with galactose as a minor constituent. The composition differs from that of known colanic acid-containing CP that is isolated from cells of Escherichia coli and other enteric bacteria grown at 37 degrees C. The reactivity of carbohydrate-specific lectins conjugated to fluorescein isothiocyanate or gold particles with cellular carbohydrates demonstrated the cell surface localization of CP. Further, lectin binding also correlated with the FACE analysis of CP. Immunoelectron microscopy, using specific antibodies against CP, confirmed that CP surrounds the cells. Confocal microscopy of antibody-labeled cells showed greater biofilm formation at 25 degrees C than at 37 degrees C. Since the CP was shown to be produced at both 37 degrees C and 25 degrees C, it does not appear to be significantly involved in attachment during the early formation of the biofilm matrix. Although the attachment of S. enterica serovar Typhimurium DT104 does not appear to be mediated by its CP, the capsule does contribute to the biofilm matrix and may have a role in other features of this organism, such as virulence, as has been shown previously for the capsules of other gram-negative and gram-positive bacteria.

Phylogenetic characterization of marine bacterium strain 2-40, a degrader of complex polysaccharides.

The marine bacterium strain 2-40 was isolated from the salt marsh cord grass, Spartina alterniflora, in the Chesapeake Bay watershed, VA, USA. It is Gram-negative, requires sea salts and is a strict aerobe. It degrades numerous complex polysaccharides and synthesizes eumelanin. By 16S rDNA analysis, the isolate was shown to be a member of the gamma-subclass of the Proteobacteria, related to Microbulbifer hydrolyticus and to a cellulolytic nitrogen-fixing bacterium.

Hyphomonas spp., Shewanella spp., and Other Marine Bacteria Lack Heterogeneous (Ladderlike) Lipopolysaccharides.

The lipopolysaccharides (LPS) of 19 marine bacteria were examined for size heterogeneities by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in conjunction with an LPS-specific silver staining method. Fifteen marine bacteria had an R-type LPS instead of the ladderlike LPS array characteristic of most bacteria. In addition, three marine bacteria also had a single large LPS molecule. Without constraints (e.g., surface masking), R-type LPS, a more hydrophobic molecule, predominates in Shewanella and Hyphomonas species and in other marine bacteria.

Evidence for the Adhesive Function of the Exopolysaccharide of Hyphomonas Strain MHS-3 in Its Attachment to Surfaces.

Hyphomonas strain MHS-3 (MHS-3) is a marine procaryote with a biphasic life cycle and which has prosthecate stages that adhere to submerged substrata. We found that adherent forms produced an exopolysaccharide (EPS) capsule that bound Glycine max lectin, Arachis hypogaea lectin, and Bauhinia purpurea lectin (BPA), each having affinity for N-acetyl-d-galactosamine. It also bound the dye Calcofluor. BPA and Calcofluor were tested for the ability to hinder MHS-3 adhesion to glass surfaces; they reduced attachment by >50 and >85%, respectively. Periodate treatment also reduced attachment (by >80%), but pronase treatment did not. Furthermore, an EPS(sup-) variant, Hyphomonas strain MHS-3 rad, did not attach well to surfaces. These results suggest that the MHS-3 EPS capsule is an adhesin.

Interactions between Shewanella colwelliana, Oyster Larvae, and Hydrophobic Organophosphate Pesticides.

Shewanella colwelliana (strain D) is a periphytic estuarine bacterium that forms biofilms beneficial to oyster set. Our study examined whether these and other films concentrated two hydrophobic, organophosphate pesticides, Abate and malathion, that are detected in Chesapeake Bay oyster waters. Both biofilms and purified exopolysaccharide of S. colwelliana did not adsorb more of the Abate or malathion than could be accounted for by adsorption to control surfaces. Similar results were obtained by using Deleya marina, Hyphomonas MHS3, and autochthonous biofilms. Conversely, decapsulated S. colwelliana D cells, prepared in the laboratory, bioconcentrated Abate. Significantly, the S. colwelliana D biofilms exposed to Abate did not inhibit the settlement and metamorphosis of Crassostrea gigas larvae.

Modulation of adenylate energy charge during the swarmer cycle of Hyphomicrobium neptunium.

Adenylate energy charge was measured in the budding bacterium Hyphomicrobium neptunium through the course of the swarmer cycle. The energy charge was modulated, being low in swarm cells (0.64) and in cells initiating bud formation (0.57), an event which coincides with a round of DNA replication.

Isolation and characterization of S-layer proteins from a vent prosthecate bacterium.

MWapp 116,000 and 29,000 proteins (p116 and p29), major outer membrane proteins of Hyphomonas jannaschiana reproductive cells, were extracted from cell envelopes by dialysis against EDTA, 2 M urea or distilled water. These proteins were precipitated by divalent cations and resolubilized by EDTA-Na, reflecting alternate monomer, multimer states. From two-dimensional gel electrophoresis it was determined that p116 and p29 had a pl of 4.5. Both were glycoproteins. Results suggest that p116 and p29 are surface layer (S-layer) proteins, with p116 a tetramer of the p29. The S-layer could protect the adherent H. jannaschiana reproductive cell from exoenzyme activity, antibiotics and other bacteriocidal molecules produced in the bacterial films formed on many marine surfaces.

The melA gene is essential for melanin biosynthesis in the marine bacterium Shewanella colwelliana.

The surface-adhering, Gram-negative marine bacterium Shewanella colwelliana synthesizes a red-brow melanin in the late stage of exponential growth in laboratory culture. Previous studies identified a single gene, melA, from S. colwelliana that could impart the ability to produce melanin to an E. coli host. However, these studies did not demonstrate a requirement for melA during melanization in S. colwelliana. In this paper, genetic analyses, using a broad host range conjugation system to generate specific lesions, reveal that melA null mutants fail to synthesize pigment. The wild-type melA gene provided in trans on a low copy number plasmid complemented these null mutations, as well as a spontaneous pigment variant, to wild-type melanin synthesis. Polyclonal antibodies, raised against a MelA-LacZ fusion protein, were used to confirm the presence of the melA gene product in wild-type S. colwelliana and verify its absence in the non-pigmented mutants. In addition, detection of the MelA protein over the course of growth in batch culture revealed a constant steady-state level of MelA protein, suggesting that the timing of melanization and the quantity of melanin synthesized is not controlled at the level of melA expression.

Production and characterization of monoclonal antibodies specific for Shewanella colwelliana exopolysaccharide.

Six monoclonal antibodies were produced to whole cells of Shewanella colwelliana (Aco1 to Aco6) and two (Aco22 to Aco23) to purified exopolysaccharide (EPS). Aco1, -4 to -6, -22, and -23 bound to both the cell surface and the purified EPS, while Aco2 and -3 bound to cells only. The EPS of S. colwelliana was antigenically unique from those of nine other species of marine bacteria that were tested. Mapping studies revealed that all of the EPS-specific monoclonal antibodies bound to the same epitope. This EPS epitope was sensitive to cleavage of ester bonds, but neither pyruvate, acetate, nor terminal nonreducing sugars were required for antigenicity. When S. colwelliana was grown on rich media, most of its EPS was loosely associated with the cell surface.

Inhibition of deoxyribonucleic acid synthesis and bud formation by nalidixic acid in Hyphomicrobium neptunium.

The relationship between chromosome replication and morphogenesis in the budding bacterium Hyphomicrobium neptunium has been investigated. Nalidixic acid was found to completely inhibit deoxyribonucleic acid synthesis, but not ribonucleic acid synthesis. The antibiotic was bacteriostatic to the organism for the initial 5 h of exposure; thereafter it was bacteriocidal. Observation of inhibited cultures revealed cells that had produced abnormally long stalks, but no buds. These results indicate that bud formation is coupled to chromosome replication in H. neptunium. They do not exclude the possibilities that cross wall formation and bud separation may also be coupled to chromosome replication.

Major outer membrane proteins unique to reproductive cells of Hyphomonas jannaschiana.

Separation on the basis of molecular weight resolved three proteins specific to the swarmer cell of Hyphomonas jannaschiana. In the reproductive cell, 4 major proteins were identified as cytoplasmic and 10 were identified as envelope. Of these envelope proteins, one was common to both the inner and outer membranes, four were common to the inner membrane, and five were common to the outer membrane. Four of these outer membrane proteins were specific to the reproductive cell, and two of these proteins, with apparent molecular weights of 116,000 and 29,000, constituted 19% of the total cell protein and 54% of the outer membrane protein.

Semipermissive Replication of a Nuclear Polyhedrosis Virus of Autographa californica in a Gypsy Moth Cell Line.

Several gypsy moth cell lines have been previously described as nonpermissive for the multiple-embedded nuclear polyhedrosis virus of Autographa californica (AcMNPV). In this report, we demonstrate the semipermissive infection of a gypsy moth cell line, IPLB-LD-652Y, with AcMNPV. IPLB-LD-652Y cells infected with AcMNPV produced classic cytopathic effects but failed to yield infectious progeny virus. Results of experiments employing DNA-DNA dot hybridization suggested that AcMNPV DNA synthesis was initiated from 8 to 12 h postinfection (p.i.), continued at a maximum rate from 12 to 20 h p.i., and declined from 20 to 36 h p.i. The rate of AcMNPV DNA synthesis approximated that observed in the permissive TN-368 cell line. AcMNPV-infected IPLB-LD-652Y cells, pulse-labeled with [(35)S]methionine at various time intervals p.i. and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed four virus-induced proteins, one novel to the semipermissive system and three early alpha proteins, synthesized from 1 to 20 h p.i. Thereafter, both host and viral protein synthesis was completely suppressed. These results suggest that AcMNPV adsorbed, penetrated, and initiated limited macromolecular synthesis in the semipermissive gypsy moth cell line. However, the infection cycle was restricted during the early phase of AcMNPV replication.

Microbial-invertebrate interactions and potential for biotechnology.

CONCLUSION: As the interactions between marine invertebrates and their bacterial commensals and symbionts are better understood, the application of biotechnology will enhance both environmental and economic benefit. In the immediate future, marine bacteria, either selected or genetically engineered, will play a significant role in enhancing the development of selected invertebrates in aquaculture and in the field. Luck may also favor discovery of mechanisms to suppress the development of biofouling species, perhaps by making it possible to coat submerged surfaces with bacterial films designed to repell larvae and/or interfere with larval morphogenesis. In any case, the future is appealing.

The polar polysaccharide capsule of Hyphomonas adhaerens MHS-3 has a strong affinity for gold.

Select groups of bacteria, including prothescate species, have an unusual capacity to sequester gold and bioconcentrate it to very high levels. Hyphomonas adhaerens MHS-3 (MHS-3) is one such species, as demonstrated by Energy Dispersive Spectroscopy. Transmission electron microscopy revealed that the binding site was specific on the polar polysaccharide capsule. A capsuleless mutant and periodate-treated wild type did not sequester gold. The gold may interact with the same sites in the capsule that naturally adhere MHS-3 to surfaces in the marine environment.

Characterization of the agarase system of a multiple carbohydrate degrading marine bacterium.

A marine bacterium strain 2-40 (2-40) degraded numerous complex carbohydrates, such as agar, chitin and alginate. It may play an important role in altering carbon fluxes in marine environments. End-product analyses revealed that 2-40 synthesized an agarase system that consisted of at least three enzymes, beta-agarase I, beta-agarase II and alpha-agarase, which acted in concert to degrade polymeric agar to D-galactose and 3,6-anhydro-L-galactose. The agarase system was shown to be both cell envelope-associated and extracellular, with the relative concentrations depending on the growth phase. The principal depolymerase, a beta-agarase I, hydrolysed agar to both neoagarotetrose and neoagarobiose, as identified by thin layer chromatography. This agarase had a mass of 98 kD and a Pi of 4.3. The agarase system was repressed by D-glucose and D-galactose and induced by agar, agarose, neoagarobiose, neoagarotetrose and neoagarohexose.

Upstream sequence of the parahydroxyphenyl-pyruvate dioxygenase (melA) coding region leading to enhanced expression in Shewanella colwelliana.

Hyperexpression of the enzyme, parahydroxyphenylpyruvate dioxygenase (pHPPH; melA), in the tyrosine degradative pathway yields excess homogentisic acid which oxidatively polymerizes to pyomelanin. Depression of melA in Shewanella colwelliana strain D was found to result from a single base pair transition upstream of the promoter. This was the sole lesion detected in pHPPH hyperexpressing strains. It is suggested that a T to C transition alters the mRNA structure, exposing the ribosome binding site, thereby enhancing translational efficiency.

Development of Defined, Minimal, and Complete Media for the Growth of Hyphomicrobium neptunium.

A complete synthetic medium containing 15 amino acids, a minimal synthetic medium (GAMS) containing 4 amino acids, and a supplemented minimal medium (GAMS + calcium pantothenate) have been developed for the cultivation of Hyphomicrobium neptunium ATCC 15444. Depending on the complexity of the synthetic media, generation times were approximately 2 to 3 times longer, and maximum cell densities were 0.3 to 0.9 log(10) lower than in ZoBell marine broth 2216. The fates of C-labeled amino acids in GAMS were monitored. Results suggested that H. neptunium was auxotrophic for methionine, utilized glutamic acid as a primary energy source, and readily anabolized and catabolized serine and aspartic acid. Individual amino acid concentrations above 125 mM induced prolonged lag periods, whereas only methionine was not growth limiting at a concentration as low as 2 mM.

Characterization of a Marine Bacterium Associated with Crassostrea virginica (the Eastern Oyster).

A gram-negative bacterium found to be closely associated with oysters has been isolated and characterized. The organism, designated LST, has a generation time of 106 min in Marine broth under optimal growth conditions at 25 degrees C. During the decline phase of growth, it exhibits a morphological transition from a motile rod (ca. 1 mum in length) to an elongated, 3- to 40-mum, nonmotile, tightly coiled helix. LST synthesizes and releases a pigment in the stationary and decline phases of growth. Identified as melanin on the basis of chemical properties and UV absorbance maxima, the pigment comprises polymers of heterogeneous molecular weights, ranging from 12,000 to 120,000. The guanosine-plus-cytosine content of the LST DNA is 46%, and results of phenetic analysis and DNA-DNA hybridization indicate that this bacterium represents a new species. LST adheres to a variety of surfaces, including glass, plastics, and oyster shell, and has been shown to promote the settlement of oyster larvae.