Nancy I. López

Antimicrobial activity and surface bacterial film in marine sponges

The antimicrobial activity of three sponge species was tested against marine benthic bacteria and the presence of epibiotic bacteria on their surfaces was investigated. The aim of the present study was to determine whether there is a correlation between antimicrobial activities and the presence of a bacterial film. Seven benthic bacterial strains were isolated from the vicinity of the sponges and used as assay organisms. Gram-positive and Gram-negative bacteria were equally affected by all the sponge extracts. The encrusting sponge Crambe crambe featured the strongest antimicrobial activity in the assays and no bacteria were found on its surface. The other two sponges, Ircinia fasciculata and Spongia officinalis, featured lower antimicrobial activity than C. crambe and the number of bacteria found on their surfaces was of the same order of magnitude as that found on immersed glass slides used as controls. It was concluded that antimicrobial activities detected in laboratory assays were effective as mechanisms to combat microfouling in only some cases, and other possible interpretations are considered.

Polyhydroxyalkanoate degradation is associated with nucleotide accumulation and enhances stress resistance and survival of Pseudomonas oleovorans in natural water microcosms

ABSTRACT Pseudomonas oleovorans GPo1 and its polyhydroxyalkanoic acid (PHA) depolymerization-minus mutant, GPo500 phaZ, residing in natural water microcosms, were utilized to asses the effect of PHA availability on survival and resistance to stress agents. The wild-type strain showed increased survival compared to the PHA depolymerase-minus strain. The appearance of a round cellular shape, characteristic of bacteria growing under starvation conditions, was delayed in the wild type in comparison to the mutant strain. Percent survival at the end of ethanol and heat challenges was always higher in GPo1 than in GPo500. Based on these results and on early experiments (H. Hippe, Arch. Mikrobiol. 56:248–277, 1967) that suggested an association of PHA utilization with respiration and oxidative phosphorylation, we investigated the association between PHA degradation and nucleotide accumulation. ATP and guanosine tetraphosphate (ppGpp) production was analyzed under culture conditions leading to PHA depolymerization. A rise in the ATP and ppGpp levels appeared concomitant with PHA degradation, while this phenomenon was not observed in the mutant strain unable to degrade the polymer. Complementation of the phaZ mutation restored the wild-type phenotype.

Polyhydroxyalkanoates are essential for maintenance of redox state in the Antarctic bacterium Pseudomonas sp. 14-3 during low temperature adaptation

Polyhydroxyalkanoates (PHAs) are highly reduced bacterial storage compounds that increase fitness in changing environments. We have previously shown that phaRBAC genes from the Antarctic bacterium Pseudomonas sp. 14-3 are located in a genomic island containing other genes probably related with its adaptability to cold environments. In this paper, Pseudomonas sp. 14-3 and its PHA synthase-minus mutant (phaC) were used to asses the effect of PHA accumulation on the adaptability to cold conditions. The phaC mutant was unable to grow at 10°C and was more susceptible to freezing than its parent strain. PHA was necessary for the development of the oxidative stress response induced by cold treatment. Addition of reduced compounds cystine and gluthathione suppressed the cold sensitive phenotype of the phaC mutant. Cold shock produced very rapid degradation of PHA in the wild type strain. The NADH/NAD ratio and NADPH content, estimated by diamide sensitivity, decreased strongly in the mutant after cold shock while only minor changes were observed in the wild type. Accordingly, the level of lipid peroxidation in the mutant strain was 25-fold higher after temperature downshift. We propose that PHA metabolism modulates the availability of reducing equivalents, contributing to alleviate the oxidative stress produced by low temperature.

A Polyhydroxybutyrate-Producing Pseudomonas sp. Isolated from Antarctic Environments with High Stress Resistance

Pseudomonas sp. 14-3, a strain that accumulates large quantities of polyhydroxybutyrate (PHB) when grown on octanoate, was isolated from Antarctic environments. This isolate was characterized on the basis of phenotypic features and partial sequencing of its 16S ribosomal RNA gene. Pseudomonas sp. 14-3 showed increased tolerance to both thermal and oxidative stress compared with three other Pseudomonas species. Stress tolerance of Pseudomonas sp. 14-3 was analyzed in polyhydroxyalkanoate accumulating and non-accumulating conditions, and increased levels of stress resistance were observed when PHB was produced. Pseudomonas sp. 14-3 was isolated from Antarctic regions, a habitat normally exposed to extreme conditions. An association between high PHB accumulation and high stress resistance in bacteria adapted to extreme environments is suggested.

The effect of nutrient additions on bacterial activity in seagrass (Posidonia oceanica) sediments

The influence of nutrient additions on benthic bacterial activity under seagrass meadows was tested by enriching five seagrass (Posidonia oceanica) meadows with nutrients over one year. We found a highly significant response of benthic bacterial activity to nutrient additions, which was reflected in greater (about two-fold) ammonification rates and, to a smaller extent, a significant tendency for a greater exoenzymatic activity. Nutrient additions significantly raised bacterial activity, without altering the seasonal changes in bacterial activity. As a result of the increased bacterial activity, the organic content of the sediments declined significantly, by about 33%, after one year of nutrient addition. Hence, nutrient additions to the seagrass meadows enhance seagrass production but also accelerate bacterial decomposition of seagrass carbon, thereby reducing the capacity of the sediments to store organic carbon. These results demonstrate that sediment nutrient availability limits bacterial activity in these Posidonia oceanica meadows, and identify bacteria as important nutrient consumers in these systems.

Pseudomonas extremaustralis sp. nov., a Poly(3-hydroxybutyrate) Producer Isolated from an Antarctic Environment

A Gram-negative, mobile, rod-shaped, non-spore-forming bacterium (strain 14-3T) was isolated from a temporary pond in Antarctica. On the basis of 16S rRNA gene sequence similarity, strain 14-3T was shown to belong to the genus Pseudomonas sensu stricto. Physiological and biochemical tests supported the phylogenetic affiliation. Strain 14-3T is closely related to Pseudomonas veronii DSM 11331T, sharing 99.7% sequence similarity. DNA–DNA hybridization experiments between the two strains showed only moderate reassociation similarity (35.1%). Tests for arginine dihydrolase and nitrate reduction were positive, while those for denitrification, indol production, glucose acidification, urease, ß-galactosidase, esculin, caseine and gelatin hydrolysis were negative. Growth of this bacterium occurred in a range from 4 to 37°C but not at 42°C. It accumulated poly(3-hydroxybutyrate) when grown on sodium octanoate medium. Strain 14-3T therefore represents the type strain of a new species, for which the name Pseudomonas extremaustralis sp. nov. is proposed. The type strain 14-3T has been deposited as DSM 17835T and as CIP 109839T.

Bacterial activity in NW Mediterranean seagrass (Posidonia oceanica) sediments

Abstract We examine here the hypothesis that benthic bacterial activity in seagrass [ Posidonia oceanica (L.) Delile] meadows is dependent on seagrass growth and availability of inorganic nutrients in the sediments. This was achieved by measuring bacterial activity (ammonification rates, and exoproteolytic and exoglucosidase activities) during an annual cycle in five P. oceanica meadows in the NW Mediterranean. Benthic bacterial activity was high, and tended to increase with increasing seagrass production. This trend is likely to involve a direct effect derived from the greater supply of organic carbon in productive meadows, and an indirect effect derived from the fact that productive meadows develop over nutrient-rich sediments and yield nutrient-rich detritus. Phosphorus availability to bacteria was low, for plant detritus was deficient in phosphorus relative to bacterial requirements, and bacterial activity was reduced after seagrasses depleted inorganic phosphorus from the sediments at the onset of exponential plant growth. These results indicate that, on local and annual time scales, benthic bacterial activity is directly related to seagrass production in the NW Mediterranean, because of enhanced inputs of organic matter by the seagrasses, while on seasonal scales, bacteria and seagrass metabolism are inversely related, apparently because of competition for inorganic nutrients.

rpoS Gene Expression in Carbon-Starved Cultures of the Polyhydroxyalkanoate-Accumulating Species Pseudomonas oleovorans

The expression of the rpoS gene during PHA depolymerization was monitored in Pseudomonas oleovorans GPo1 and its mutant defective in PHA degradation by analyzing the tolerance to oxidative and thermal stresses and the RpoS intracellular content. An increase in the tolerance to H2O2 and heat shock was observed coincidentally with PHA degradation. Western blotting experiments performed in carbon-starved cultures showed that the RpoS levels were higher in the wild type than in the mutant strain. Complementation of the phaZ mutation restores the wild-type RpoS levels. These results suggest a probable association between PHA depolymerization and the stress tolerance phenotype controlled by RpoS.

Poly(3-hydroxybutyrate) influences biofilm formation and motility in the novel Antarctic species Pseudomonas extremaustralis under cold conditions

Polyhydroxyalkanoates (PHAs) are highly reduced bacterial storage compounds that increase fitness in changing environments. It has previously shown that polyhydroxybutyrate (PHB) accumulation is essential during the growth under cold conditions. In this work, the relationship between PHB accumulation and biofilm development at low temperature was investigated. P. extremaustralis, an Antarctic strain able to accumulate PHB, and its phaC mutant, impaired in the synthesis of this polymer, were used to analyze microaerobic growth, biofilm development, EPS content and motility. PHB accumulation increased motility and survival of planktonic cells in the biofilms developed by P. extremaustralis under cold conditions. Microaerobic conditions rescued the cold growth defect of the mutant strain. The PHB accumulation capability could constitute an adaptative advantage for the colonization of new ecological niches in stressful environments.

Biofilm lifestyle enhances diesel bioremediation and biosurfactant production in the Antarctic polyhydroxyalkanoate producer Pseudomonas extremaustralis

Diesel is a widely distributed pollutant. Bioremediation of this kind of compounds requires the use of microorganisms able to survive and adapt to contaminated environments. Pseudomonas extremaustralis is an Antarctic bacterium with a remarkable survival capability associated to polyhydroxyalkanoates (PHAs) production. This strain was used to investigate the effect of cell growth conditions—in biofilm versus shaken flask cultures—as well as the inocula characteristics associated with PHAs accumulation, on diesel degradation. Biofilms showed increased cell growth, biosurfactant production and diesel degradation compared with that obtained in shaken flask cultures. PHA accumulation decreased biofilm cell attachment and enhanced biosurfactant production. Degradation of long-chain and branched alkanes was observed in biofilms, while in shaken flasks only medium-chain length alkanes were degraded. This work shows that the PHA accumulating bacterium P. extremaustralis can be a good candidate to be used as hydrocarbon bioremediation agent, especially in extreme environments.