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.

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.

Salmonella enterica serovar Typhimurium DT104 displays a rugose phenotype.

Rugose phenotypes, such as those observed in Vibrio cholerae, have increased resistance to chlorine, oxidative stress, and complement-mediated killing. In this study we identified and defined a rugose phenotype in Salmonella enterica serovar Typhimurium DT104 and showed induction only on certain media at 25 degrees C after 3 days of incubation. Incubation at 37 degrees C resulted in the appearance of the smooth phenotype. Observation of the ultrastructure of the rugose form and a stable smooth variant (Stv), which was isolated following a series of passages of the rugose cells, revealed extracellular substances only in cells from the rugose colony. Observation of the extracellular substance by scanning electron microscopy (SEM) was correlated with the appearance of corrugation during development of rugose colony morphology over a 4-day incubation period at 25 degrees C. In addition, the cells also formed a pellicle in liquid broth, which was associated with the appearance of interlacing slime and fibrillar structures, as observed by SEM. The pellicle-forming cells were completely surrounded by capsular material, which bound cationic ferritin, thus indicating the presence of an extracellular anionic component. The rugose cells, in contrast to Stv, showed resistance to low pH and hydrogen peroxide and an ability to form biofilms. Based on these results and analogy to the rugose phenotype in V. cholerae, we propose a possible role for the rugose phenotype in the survival of S. enterica serovar Typhimurium DT104.

Hyphomonas adhaerens sp. nov., Hyphomonas johnsonii sp. nov. and Hyphomonas rosenbergii sp. nov., marine budding and prosthecate bacteria.

Three strains of prosthecate, budding bacteria, MHS-2T, MHS-3T and VP6T, were isolated from marine habitats including the open ocean (the pelagic zone), the offshore region (the neritic zone) and the hydrothermal vent region. A polyphasic approach including 16S rDNA sequencing, phenotypic analyses, serology, fatty acid analyses, membrane protein profiles and DNA-DNA hybridizations was used to place these strains in the genus Hyphomonas, a taxon of the alpha-Proteobacteria. The results of these analyses also showed that strains MHS-3T, MHS-2T and VP6T each represent a new species of Hyphomonas. The names Hyphomonas adhaerens (type strain MHS-3T, ATCC 43965T), Hyphomonas johnsonii (type strain MHS-2T, ATCC 43964T) and Hyphomonas rosenbergii (type strain VP6T, ATCC 43869T) are proposed for the new species. With these additions, Hyphomonas now contains eight species.

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.

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.

Spatial and Temporal Deposition of Adhesive Extracellular Polysaccharide Capsule and Fimbriae by Hyphomonas Strain MHS-3.

Hyphomonas strain MHS-3, a member of a genus of primary colonizers of surfaces immersed in marine water, synthesizes two structures that mediate adhesion to solid substrata, namely, capsular exopolysaccharide and fimbriae. Specific stains, gold-labelled lectins, and monoclonal antibodies, along with transmission electron microscopy of synchronized populations, revealed that both structures are polarly and temporally expressed. The timed synthesis and placement of the fimbriae and capsule correlated with the timing and locus of MHS-3 adhesion.

Magnification of tributyl tin toxicity to oyster larvae by bioconcentration in biofilms of Shewanella colwelliana.

The toxic effects of dissolved versus bioconcentrated tributyl tin (TBT) on oyster larvae were compared. Water column TBT levels, which had no effect in solution, inhibited natural attachment and metamorphosis of oyster larvae on bottom surfaces due to bioconcentration by biofilms. This mechanism should be considered when evaluating heavy metal toxicity in the environment.

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.

Fine-structure evidence for cell membrane partitioning of the nucleoid and cytoplasm during bud formation in Hyphomonas species.

Hyphomonas spp. reproduce by budding from the tip of the prosthecum, distal to the main body of the reproductive cell; thus, the chromosome must travel through the prosthecum to enter the progeny, the swarm cell. When viewed by electron microscopy, negatively stained whole cells, ultrathin-sectioned cells, and freeze-etched and frozen hydrated cells all had marked swellings of the cytoplasmic membrane (CM) in the prosthecum which are termed pseudovesicles (PV). PV were separated by constrictions in the contiguous CM. In replicating cells, PV housed ribosomes and DNA, which was identified by its fibrillar appearance and by lactoferrin-gold labeling. The micrographs also revealed that the CM bifurcates at the origin of the prosthecum so that one branch partitions the main body of the reproductive cell from the prosthecum and swarm cell. The results of this fine-structure analysis suggest models explaining DNA segregation and the marked asymmetric polarity of the budding reproductive cell.

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.

Influence of protein conditioning films on binding of a bacterial polysaccharide adhesin from Hyphomonas MHS-3.

A putative polysaccharide adhesin which mediates non-specific attachment of Hyphomonas MHS-3 (MHS-3) to hydrophilic substrata has been isolated and partially characterized. A polysaccharide-enriched portion of the extracellular polymeric substance (EPS(P)) from MHS-3 was separated into four fractions using high performance size exclusion chromatography (HPSEC). Comparison of chromatograms of EPS(P) from MHS-3 and a reduced adhesion strain (MHS-3 rad) suggested that one EPS(P) fraction, which consisted of carbohydrate, served as an adhesin. Adsorption of this fraction to germanium (Ge) was investigated using attenuated total reflection Fourier transform infrared (ATR/FT-IR) spectrometry. Binding curves indicated that the isolated fraction had a relatively high affinity for Ge when ranked against an adhesive protein from Mytilis edulis, mussel adhesive protein (MAP) and an acidic polysaccharide (alginate from Macrocystis pyrifera). Spectral features were used to identify the fraction as a polysaccharide previously reported to adsorb preferentially out of the EPS(P) mixture. Conditioning the Ge substratum with either bovine serum albumin (BSA) or MAP decreased the adsorption of the adhesive polysaccharide significantly. Conditioning Ge with these proteins also decreased adhesion of whole cells.

Adhesion of biofilms to inert surfaces: A molecular level approach directed at the marine environment.

Protein/ligand interactions involved in mediating adhesion between microorganisms and biological surfaces have been well-characterized in some cases (e.g. pathogen/host interactions). The strategies microorganisms employ for attachment to inert surfaces have not been so clearly elucidated. An experimental approach is presented which addresses the issues from the point of view of molecular interactions occurring at the interface.

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.

Homogentisic acid is the primary precursor of melanin synthesis in Vibrio cholerae, a Hyphomonas strain, and Shewanella colwelliana.

The enzyme p-hydroxyphenylpyruvate hydroxylase (HPPH) is involved in pigmentation (pyomelanin) via homogentisic acid (HGA). Pyomelanin formation is correlated with HGA production and expression of HPPH in three disparate marine species: Vibrio cholerae, a Hyphomonas strain, and Shewanella colwelliana. Induction of pigmentation in V. cholerae 569B by nutrient limitation also correlated with production of HGA.

Homogentisic acid is the product of MelA, which mediates melanogenesis in the marine bacterium Shewanella colwelliana D.

Shewanella colwelliana D is a marine procaryote which produces a diffusible brown pigment that correlates with melA gene expression. Previously, melA had been cloned, sequenced, and expressed in Escherichia coli; however, the reaction product of MelA had not been identified. This report identifies that product as homogentisic acid, provides evidence that the pigment is homogentisic acid-melanin (pyomelanin), and suggests that MelA is p-hydroxyphenylpyruvate hydroxylase. This is the first report of pyomelanin in an obligate marine bacterium.

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.

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.

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).