Cobetia Marina Research Articles

Evidence for grazing-mediated production of dissolved surface-active material by marine protists

Surface-active organic compounds or surfactants play important roles in a variety of upper ocean processes, including surface microlayer physics and gas exchange and the aggregation of colloidal material. Although surfactants are presumed to be produced primarily by phytoplankton , production by protozoan grazers has not been investigated. In general, the processes controlling surfactant abundance in the field are poorly understood. In this study, a two-phase laboratory system containing protists and prey was used to examine the possibility of surfactant production during protozoan grazing. Three protist species were examined, a scuticociliate, Uronema sp. (10–15 μm), and two flagellates, Cafeteria sp. (2–4 μm), and Paraphysomonas imperforata (4–8 μm). For all experimental cultures , protozoan inocula were added to rinsed bacterial suspensions ( Halomonas halodurans ) in sterile seawater . Surfactants, dissolved organic carbon (DOC) and population dynamics were monitored until protists had reached stationary growth. Surfactant activities increased during protozoan exponential growth. Surfactant production in the ciliate cultures was significantly higher than in either of the flagellate cultures. Bacterial controls maintained low DOC concentrations and surfactant activities. Estimates of protozoan surfactant production rates range from 10 −8 to 10 −9 mg protist −1 h −1 (Triton X-100 equivalents). Under non-bloom conditions (10 3 protozoan cells ml −1 ), we estimated a surfactant production rate of 10 −5 –10 −6 mg h −1 (within 1 ml of seawater), which is comparable to estimates of phytoplankton production of surface-active material during blooms. Thus, protozoan grazers constitute a potentially significant source of surface-active material in areas where protists are abundant, such as the sediment–water interface and microbial loop-dominated oligotrophic regimes.

Marine protozoa produce organic matter with a high affinity for PCBs during grazing.

Processes influencing organic carbon distribution and composition can control the speciation of organic contaminants such as polychlorinated biphenyls (PCBs) and ultimately determine their residence time in aquatic environments. Protozoan grazers are active in the remineralization and recycling of organic material both in the water column and at the sediment-water interface. Thus, they influence the quality and quantity of potential PCB binding substrates in the suspended and dissolved phases of aqueous systems. In this study, common headspace systems were used to compare the chlorobiphenyl-binding affinity of dissolved organic carbon (DOC) in protozoan and bacterial culture filtrates (<0.2 microm) relative to DOC in a seawater control. Culture filtrates from three marine protozoan species were compared-Uronema sp., Cafeteria sp., and Paraphysomonas imperforata. Each protozoan species was fed the same bacterial prey, Halomonas halodurans, which was also used as a bacterial control. Affinities of culture DOC for [14C]3,3',4,4'-tetrachlorobiphenyl (IUPAC 77) were normalized to DOC and surfactant concentrations. Values of DOC equilibrium partition coefficients (K(DOC)) ranged from 10(4.6) in seawater (Vineyard Sound, MA) to 10(5.4) and 10(5.5) in protist cultures, indicating that grazer-modified DOC was a better sorbent for PCBs than DOC in bacterial or seawater controls.

Settlement inhibition of fouling invertebrate larvae by metabolites of the marine bacterium halomonas marina within a polyurethane coating

The marine bacterium, Halomonas marina (ATCC 27129), was shown to inhibit settlement and development of the sessile invertebrates Balanus amphitrite and Bugula neritina. Different bacterial treatments were employed to investigate this interaction. Filmed bacteria and liquid suspensions of whole cells, lysed cells and culture filtrate all reduced settlement of B. amphitrite. Polyurethane coatings containing whole cells were partially inhibitory while lysed cells caused complete inhibition of B. amphitrite larval settlement. In contrast, culture filtrate in a polyurethane matrix stimulated settlement of B. amphitrite larvae. Whole cells, culture filtrate, and lysed cells embedded in a polyurethane coating also controlled B. neritina settlement and maturation.

Analysis and expression of algL, which encodes alginate lyase in Pseudomonas syringae Pv. syringae.

Pseudomonas syringae produces alginate, an exopolysaccharide that contributes to the virulence and epiphytic fitness of this phytopathogenic bacterium. P. syringae also produces the algL-encoded alginate lyase, which cleaves the alginate biopolymer via a beta-elimination reaction. The algL gene from P. syringae maps to a 1134 bp region within the alginate biosynthetic operon, and is similar to algL from Halomonas marina, P. aeruginosa, Azotobacter chroococcum, and A. vinelandii. algL from P. syringae was over expressed in Escherichia coli; two periplasmic forms of AlgL were overproduced (40 and 37 kDa). Both forms were enzymatically active and recognized by antibodies raised against AlgL from P. aeruginosa. Analysis of the regions flanking algL revealed significant homology to algX and algI, genes previously identified in the biosynthetic operon of other alginate-producing bacteria.

Substratum/bacterial interactions and larval attachment: Films and exopolysaccharides of Halomonas marina (ATCC 25374) and their effect on barnacle cyprid larvae, Balanus amphitrite Darwin

Laboratory experiments were conducted to study the interaction between adhesion of the bacterium Halomonas marina to substrata of different wettabilities, the combination of which has been demonstrated to influence the attachment response of cyprid larvae of the barnacle Balanus amphitrite. Cyprid attachment in the presence of bacterial films was shown to be inhibited when films were on polystyrene but not on tissue‐culture polystyrene or glass. Using an enzyme‐linked lectin assay, bacteria on polystyrene showed an increase in binding of the lectin concanavalin A compared to bacteria on tissue‐culture treated polystyrene, indicating a difference in surface polymers associated with H. marina when attached to different substrata. Although bacterial growth supernatants when adsorbed to polystyrene were inhibitory to barnacle attachment, exopolysaccharides, to which the lectins may be binding, were not inhibitory. The data indicate that adhesion of films of bacteria to polystyrene alters the exopolymer production by H. marina and it is suggested that this change may be involved in the inhibition of cyprid attachment. However, the inhibition of cyprid larvae does not appear to be associated with the exopolysaccharides of the bacterium.

Importance of passive diffusion in the uptake of polychlorinated biphenyls by phagotrophic protozoa.

Unicellular protozoan grazers represent a size class of organisms where a transition in the mechanism of chlorobiphenyl (CB) introduction, from diffusion through surface membranes to ingestion of contaminated prey, could occur. This study compares the relative importance of these two processes in the overall uptake of polychlorinated biphenyls by protists. Uptake rates and steady-state concentrations were compared in laboratory cultures of grazing and nongrazing protozoa. These experiments were conducted with a 10-microm marine scuticociliate (Uronema sp.), bacterial prey (Halomonas halodurans), and a suite of 21 CB congeners spanning a range of aqueous solubilities. The dominant pathway of CB uptake by both grazing and nongrazing protozoa was diffusion. Organic-carbon-normalized CB concentrations (in the protozoan cell) were equivalent in grazing and nongrazing protozoa for all congeners studied. Rate constants for uptake into and loss from the protozoan cell were independently determined by using [3,3',4, 4'-(14)C]tetrachlorobiphenyl (IUPAC no. 77), 0.38 +/- 0.03 min(-1) and (1.1 +/- 0.1) x 10(-5) (g of organic carbon)(-1) min(-1), respectively. Magnitudes of the uptake and loss processes were calculated and compared by using a numerical model. The model result was consistent with data from the bioaccumulation experiment and supported the hypothesis that diffusive uptake is faster than ingestive uptake in phagotrophic unicellular protozoa.

Surface-grafted, environmentally sensitive polymers for biofilm release.

Controlling bacterial biofouling is desirable for almost every human enterprise in which solid surfaces are introduced into nonsterile aqueous environments. One approach that is used to decrease contamination of manufactured devices by microorganisms is using materials that easily slough off accumulated material (i.e., fouling release surfaces). The compounds currently used for this purpose rely on low surface energy to inhibit strong attachment of organisms. In this study, we examined the possible use of environmentally responsive (or "smart") polymers as a new class of fouling release agents; a surface-grafted thermally responsive polymer, poly(N-isopropylacrylamide) (PNIPAAM), was used as a model compound. PNIPAAM is known to have a lower critical solubility temperature of approximately 32 degrees C (i.e., it is insoluble in water at temperatures above 32 degrees C and is soluble at temperatures below 32 degrees C). Under experimental conditions, >90% of cultured microorganisms (Staphylococcus epidermidis, Halomonas marina) and naturally occurring marine microorganisms that attached to grafted PNIPAAM surfaces during 2-, 18-, 36-, and 72-h incubations were removed when the hydration state of the polymer was changed from a wettability that was favorable for attachment to a wettability that was less favorable. Of particular significance is the observation that an organism known to attach in the greatest numbers to hydrophobic substrata (i.e., H. marina) was removed when transition of PNIPAAM to a more hydrated state occurred, whereas an organism that attaches in the greatest numbers to hydrophilic substrata (i.e., S. epidermidis) was removed when the opposite transition occurred. Neither solvated nor desolvated PNIPAAM exhibited intrinsic fouling release properties, indicating that the phase transition was the important factor in removal of organisms. Based on our observations of the behavior of this model system, we suggest that environmentally responsive polymers represent a new approach for controlling biofouling release.

Lower critical solubility temperature materials as biofouling release agents

Poly(N-isopropylacrylamide) (PNIPAAM) exhibits a lower critical solubility temperature (LCST) of 32°C. Using thin films of this compound as a model system, the potential of ‘smart polymers’ as biofouling-release agents was examined. PNIPAAM-coated glass slides were incubated in artificial sea water containing the marine bacterium Halomonas marina or in natural bay water at a temperature above the LCST. Upon rinsing of the biofouled samples with artificial sea water below the LCST, the dissolution of the coating released over 90% of the attached fouling material, a significant increase over the release obtained for glass controls. These experiments demonstrate the potential of PNIPAAM and similar polymers as possible fouling-release agents, and suggest that tethered PNIPAAM (or similar polymers) may be useful as regenerable fouling-release surfaces.

Unification of the Genera Deleya (Baumann et al. 1983), Halomonas (Vreeland et al. 1980), and Halovibrio (Fendrich 1988) and the Species Paracoccus halodenitrificans (Robinson and Gibbons 1952) into a Single Genus, Halomonas, and Placement of the Genus Zymobacter in the Family Halomonadaceae

We determined the 16S rRNA sequences of the type strains of species belonging to the genera Deleya and Halomonas for which no sequence data were available previously. We also determined the 16S rRNA sequence of ACAM 21, a representative strain of a biovar of Halomonas subglaciescola. The members of the genera Deleya, Halomonas, and Halovibrio and the misnamed organism Paracoccus halodenitrificans formed a monophyletic group within the gamma subclass of the Proteobacteria. The 16S rRNA sequences of the members of this group contained all of the signature features previously identified as characteristic of the group. The frequency of occurrence of these signature features among other members of the gamma subclass has remained stable during the expansion of the database of rRNA sequences. The levels of 16S rRNA sequence similarity between members of the species belonging to the genera Deleya, Halomonas, and the misnamed organism P. halodenitrificans ranged from 91.5 to 100%; however, the level of sequence similarity for members of well-resolved monophyletic subgroups which might represent separate genera was 98%. At a sequence similarity level of 98% 10 subgroups were resolved, but these groups could not be differentiated on the basis of chemotaxonomic or phenotypic characteristics. In this paper we propose that members of the genera Deleya, Halomonas, and Halovibrio should be placed in a single genus, the genus Halomonas, and we emend the description of this genus. The resulting new combinations are Halomonas aquamarina (Deleya aquamarina Akagawa and Yamasato 1989), Halomonas variabilis (Halovibrio variabilis Fendrich 1988), Halomonas venusta (Deleya venusta Baumann et al. 1983), Halomonas cupida (Deleya cupida Baumann et al. 1983), Halomonas pacifica (Deleya pacifica Baumann et al. 1983), Halomonas marina (Deleya marina Baumann et al. 1983), Halomonas halophila (Deleya halophila Quesada et al. 1984), and Halomonas salina (Deleya salina Valderrama et al. 1991). We transfer the misnamed organism P. halodenitrificans to the genus Halomonas as Halomonas halodenitrificans comb. nov. (P. halodenitrificans Robinson and Gibbons 1952). The genus Zymobacter is closely related to the genus Halomonas. While the genus Zymobacter can be clearly distinguished from the genus Halomonas, these two taxa share important genotypic, chemotaxonomic, and phenotypic characteristics. We propose that the genus Zymobacter should be transferred to the family Halomonadaceae and emend the description of the family Halomonadaceae. The 16S rRNA sequence of Halomonas subglaciescola ACAM 21 was significantly different from the 16S rRNA sequence of the type strain of Halomonas subglaciescola (strain ACAM 12) but was nearly identical to the 16S rRNA sequence of Halomonas halodurans.

Description of two new species of Halomonas: Halomonas israelensis sp.nov. and Halomonas canadensis sp.nov.

Six well-known strains of halotolerant bacteria, including two strains previously identified only as NRCC 41227 and Ba1, have been compared using 125 phenotypic characters and DNA-DNA hybridization. Although these strains represent some of the most heavily studied salt-tolerant bacteria, they have never been taxonomically compared. The data presented show that these bacteria form a relatively homogeneous group related at the genus level. The taxonomic comparison showed that these six organisms represented four distinct species all related above the 65% Jaccard coefficient level. In addition to two previously identified bacterial species, Halomonas elongata (ATCC 33173T) and Halomonas halodurans (ATCC 29686T), the strains included in this study represent two previously unnamed Halomonas species. These two new taxa have been assigned the names Halomonas israelensis (ATCC 43985T) and Halomonas canadensis (NRCC 41227T = ATCC 43984). DNA-DNA hybridization show that these two species are related to the type species H. elongata at 54.9 and 48.9%, respectively.

Development of a gene reporter system in moderately halophilic bacteria by employing the ice nucleation gene of Pseudomonas syringae

The expression of the ice nucleation gene inaZ of Pseudomonas syringae in several moderate halophiles was investigated to establish its utility as a reporter for promoter activity and gene expression studies in these biotechnologically and environmentally important bacteria. A promoterless version of inaZ was introduced in two different restriction sites and at both orientations in a recombinant plasmid able to replicate in moderate halophiles and, in particular, within the sequence of its pHE1 part, a native plasmid of Halomonas elongata. One orientation of both recombinant constructs expressed high levels of ice nucleation activity in H. elongata and Volcaniella eurihalina cells, indicating that inaZ was probably introduced in the correct orientation downstream of putative native promoters. A recombinant construct carrying a tandem duplication of inaZ at the same orientation gave significantly higher ice nucleation activity, showing that inaZ is appropriate for gene dosage studies. The ice nucleation gene was also expressed in H. elongata and V. eurihalina under the control of Pbla (the promoter of the beta-lactamase gene of Escherichia coli) and Ppdc (the promoter of the pyruvate decarboxylase gene of Zymomonas mobilis). One of the inaZ reporter plasmids expressing high levels of ice nucleation activity under the control of a native putative promoter was also transferred in Halomonas subglaciescola, Halomonas meridiana, Halomonas halodurans, and Deleya halophila. In all cases, Ice+ transconjugants were successfully isolated, demonstrating that inaZ is expressed in a wide spectrum of moderately halophilic species.

Isotopic fractionation of oxygen by respiring marine organisms

We measured the respiratory isotope effect ϵresp for seven representative unicellular marine organisms. The bacterium Pseudomonas halodurans, the diatom Phaeodactylum tricornutum, the phytoflagellates Cryptomonas baltica and Dunaliella tertiolecta, the heterotrophic flagellates Paraphysomonas imperforata and Bodo sp., and the ciliate Uronema sp. exhibit ϵresp values in the range 14‐26‰. We also measured ϵresp for three metazoans. The ϵresp for the copepod Acartia tonsa ranged from 17 to 25‰, while two larger organisms, the mollusk Mercenaria mercenaria and the salmon Salmo salmar, respire with a smaller ϵresp of 5‐10‰. The average respiratory isotope effect of the dominant marine respirers (the bacteria, microalgae and zooplankton) is about 20 ± 3‰. An ϵresp of this magnitude supports the hypothesis that the photosynthesis‐respiration cycle is responsible for the 23.5‰ enrichment in the δ18O ratio of atmospheric O2 relative to seawater (the Dole effect). The large value and high variability in the average ϵresp limits the usefulness of a proposed method using the δ18O of naturally fractionated dissolved O2 in seawater as a tracer of primary production in the oligotrophic ocean.

Phenotypic Comparison of Halotolerant Bacteria: Halomonas halodurans sp. nov., nom. rev., comb. nov.

Several strains of halotolerant bacteria from North America were compared for 125 characteristics. Although these organisms had several characteristics in common, they are a relatively heterogeneous group. The 12 strains could be divided into at least three and perhaps as many as five taxa. Halomonas elongata ATCC 33173T (T = type strain) and “Pseudomonas halodurans” ATCC 29686T had 79% phenotypic similarity, indicating that these organisms are probably separate species of the same genus. We conclude that this relationship is sufficient to warrant assignment of “Pseudomonas halodurans” to the genus Halomonas as Halomons halodurans sp. nov., nom rev., comb nov.

Novel function of eubacterial flagella: role in aggregation of a marine bacterium

Pseudomonas marina (ATCC 27 129) rapidly aggregates when suspended in buffered artificial seawater (ASW). Light microscopic observations of stained preparations, showed that flagella-flagella contact was responsible for this phenomenon. Aggregation did not occur if flagella were sheared off, or if motility was inhibited with NaN3. Aggregates were not observed when Mg2+ was omitted from ASW, even though the bacteria remained motile. Other divalent cations, including Ca2+, Mn2+, and Ba2+ could replace Mg2+. However, there is no absolute requirement for divalent cations, since aggregation occurred in ASW containing Cs+ or Li+ instead of Mg2+. P. marina aggregates developed from pH 5.8–8.4, but not below pH 5.8 even though motility continued unimpaired to pH 4.5.

Uptake and Conversion of Selenium by a Marine Bacterium

Bio-conversion of Se was examined by incubating Pseudomonas marina in seawater containing either selenite (Na2SeIVO3) or selenate (Na2SeVIO4). At the concentrations of selenite and selenate used (10−4–10−7 mol/L), the growth of P. marina was not inhibited. Under these conditions, selenite was taken up by P. marina, but selenate was not found to enter the cells. Pseudomonas marina incorporated selenite from filtered seawater into sub-cellular fractions, primarily protein (30–50%) and amino acids (44–70%). When incubated in marine broth, P. marina incorporated 75selenite primarily into protein (up to 75%), with a lesser amount into the amino acid fraction (approximately 25%). Insignificant amounts were associated with the bacterial lipid fraction. SeIV was found in the protein and amino acid fractions within 10 min of incubation in medium containing selenite. In uptake studies the level of SeIV in the incubating medium decreased markedly, corresponding presumably to SeIV entering the bacterial cells from the medium. However, simultaneous measurements of total Se in the medium (gas–liquid chromatography following photooxidation) revealed an increasing amount of non-SeIV species of Se in the medium throughout the same period. These results were interpreted as due to the bioconversion of selenite by P. marina into water-soluble non-SeIV metabolite(s) and their subsequent release back into the medium. Up to 35% of the total Se found in the medium after 24 h can be accounted for by this conversion of SeIV into another oxidation state. Pseudomonas marina is also capable of reducing SeIV to elemental Se; this pathway becomes increasingly evident at higher concentrations of Na2SeO3.

Description of Deleya gen. nov. Created to Accommodate the Marine Species Alcaligenes aestus, A. pacificus, A. cupidus, A. venustus, and Pseudomonas marina

Recent investigations of the evolutionary relationships of marine species assigned to the genera Alcaligenes (Alcaligenes aestus, Alcaligenes pacificus, Alcaligenes cupidus, and Alcaligenes venustus) and Pseudomonas (Pseudomonas marina) are reviewed. Data obtained by a number of different methods indicate that these five species are part of a single evolutionary lineage distinct from their close relatives, the fluorescent pseudomonads, and unrelated to the type species of Alcaligenes (Alcaligenes faecalis). Consequently, all five species are transferred to a new genus, Deleya, with Deleya aesta designated as the type species. The phenotype properties of D. aesta, Deleya pacifica, Deleya cupida, Deleya venusta, and Deleya marina are presented, together with the distinguishing traits of these organisms.

Pseudomonas halodurans sp. nov., a halotolerant bacterium

Twenty-nine (29) isolates (strains) were obtained from the Great Bay Estuary, New Hampshire, using a synthetic seawater medium with NaCl concentrations between 0.30 and 2.65 M. All the strains were gram-negative; did not accumulate or hydrolyze poly-β-hydroxybutyrate; demonstrated ortho ring cleavage on aromatic compounds and showed the following homogeneous characteristics: utilization of substrates, antibiotic response, acid production from sugars, colony morphology, catalase and oxidase production, lack of pigmentation and flourescence; and had a mean guanine-plus-cytosine content of deoxyribonucleic acid composition of 63.2 ± 1.1 mol %. The strains demonstrated flagellated dimorphism; being motile by a singular polar flagellum at NaCl concentrations up to 0.8 M, and nonmotile and aflagellated at higher NaCl concentrations. A total of 150 biochemical, cultural, morphological, nutritional, and physiological traits were tested for the 29 strains and the type strain of six pseudomonad reference species. The 29 strains and reference strains were then compared with other reference and nonreference strains of the following genera: Aeromonas, Alcaligenes, Alteromonas, Arthrobacter, Flavobacterium, Pseudomonas, and Vibrio using 125 unit characters and employing numerical taxonomy by the Jaccard (SJ) coefficient and single linkage clustering method. The 29 strains were similar at the 95% level and clustered with several known Pseudomonas strains between the 70–75% similarity level (S) but clustered separately at S greater than 75% level. A type strain has been deposited with the American Type Culture Collection (ATCC 29686) and has been named Pseudomonas halodurans sp. nov.

Assimilatory sulfur metabolism in marine microorganisms: Sulfur metabolism, growth, and protein synthesis of Pseudomonas halodurans and Alteromonas luteo-violaceus during sulfate limitation

Sulfate concentration in the growth medium exerted a strong influence on the sulfur content of protein in two marine bacteria, Pseudomonas halodurans and Alteromonasluteo-violaceus, but the distribution of sulfur in major biochemical fractions was not affected. 90% of the total cellular sulfur was contained in low molecular weight organic compounds and protein; inorganic sulfate was not an important component. The sulfur content of isolated protein and total cellular sulfur increased in proportion to the external sulfate concentration for both bacteria, reaching a maximum at about 100–250 μM. The growth rate of P. halodurans only was dependent on the sulfate concentration.

Assimilatory sulfur metabolism in marine microorganisms: Sulfur metabolism, protein synthesis, and growth of Pseudomonas halodurans and Alteromonas luteo-violaceus during unperturbed batch growth

Analysis of the distribution of 35S-sulfate and 14C-glutamate in major biochemical components of the two marine bacteria, Pseudomonas halodurans and Alteromonas luteo-violaceus, was compared with cell density and total cellular protein during exponential growth in batch culture. For both organisms, the sulfur distribution was restricted principally to the low molecular weight organic and protein fractions, which together accounted for over 90% of the total sulfur. Carbon was more widely distributed, with these two fractions containing only 70% of the total label.

Assimilatory sulfur metabolism in marine microorganisms: characteristics and regulation of sulfate transport in Pseudomonas halodurans and Alteromonas luteo-violaceus.

Sulfate transport capacity was not regulated by cysteine, methionine, or glutathione in Pseudomonas halodurans, but growth on sulfate or thiosulfate suppressed transport. Subsequent sulfur starvation of cultures grown on all sulfur sources except glutathione stimulated uptake. Only methionine failed to regulate sulfate transport in Alteromonas luteo-violaceus, and sulfur starvation of all cultures enhanced transport capacity. During sulfur starvation of sulfate-grown cultures of both bacteria, the increase in transport capacity was mirrored by a decrease in the low-molecular-weight organic sulfur pool. Little metabolism of endogenous inorganic sulfate occurred. Cysteine was probably the major regulatory compound in A. luteo-violaceus, but an intermediate in sulfate reduction, between sulfate and cysteine, controlled sulfate transport in P. halodurans. Kinetic characteristics of sulfate transport in the marine bacteria were similar to those of previously reported nonmarine systems in spite of significant regulatory differences. Sulfate and thiosulfate uptake in P. halodurans responded identically to inhibitors, were coordinately regulated by growth on various sulfur compounds and sulfur starvation, and were mutually competitive inhibitors of transport, suggesting that they were transported by the same mechanism. The affinity of P. halodurans for thiosulfate was much greater than for sulfate.