Enhanced copper-resistance gene repertoire in Alteromonas macleodii strains isolated from copper-treated marine coatings.

Copper is prevalent in coastal ecosystems due to its use as an algaecide and as an anti-fouling agent on ship hulls. Alteromonas spp. have previously been shown to be some of the early colonizers of copper-based anti-fouling paint but little is known about the mechanisms they use to overcome this initial copper challenge. The main models of copper resistance include the Escherichia coli chromosome-based Cue and Cus systems; the plasmid-based E. coli Pco system; and the plasmid-based Pseudomonas syringae Cop system. These were all elucidated from strains isolated from copper-rich environments of agricultural and/or enteric origin. In this work, copper resistance assays demonstrated the ability of Alteromonas macleodii strains CUKW and KCC02 to grow at levels lethal to other marine bacterial species. A custom database of Hidden Markov Models was designed based on proteins from the Cue, Cus, and Cop/Pco systems and used to identify potential copper resistance genes in CUKW and KCC02. Comparative genomic analyses with marine bacterial species and bacterial species isolated from copper-rich environments demonstrated that CUKW and KCC02 possess genetic elements of all systems, oftentimes with multiple copies, distributed throughout the chromosome and mega-plasmids. In particular, two copies of copA (the key player in cytoplasmic detoxification), each with its own apparent MerR-like transcriptional regulator, occur on a mega-plasmid, along with multiple copies of Pco homologs. Genes from both systems were induced upon exposure to elevated copper levels (100 µM- 3 mM). Genomic analysis identified one of the merR-copA clusters occurs on a genomic island (GI) within the plasmid, and comparative genomic analysis found that either of the merR-copA clusters, which also includes genes coding for a cupredoxin domain-containing protein and an isoprenylcysteine methyltransferase, occurs on a GI across diverse bacterial species. These genomic findings combined with the ability of CUKW and KCC02 to grow in copper-challenged conditions are couched within the context of the genome flexibility of the Alteromonas genus.

A multi-step approach for testing non-toxic amphiphilic antifouling coatings against marine microfouling at different levels of biological complexity.

Marine biofouling on artificial surfaces such as ship hulls or fish farming nets causes enormous economic damage. The time for the developmental process of antifouling coatings can be shortened by reliable laboratory assays. For designing such test systems, it is important that toxic effects can be excluded, that multiple parameters can be addressed simultaneously and that mechanistic aspects can be included. In this study, a multi-step approach for testing antifouling coatings was established employing photoautotrophic biofilm formation of marine microorganisms in micro- and mesoscoms. Degree and pattern of biofilm formation was determined by quantification of chlorophyll fluorescence. For the microcosms, co-cultures of diatoms and a heterotrophic bacterium were exposed to fouling-release coatings. For the mesocosms, a novel device was developed that permits parallel quantification of a multitude of coatings under defined conditions with varying degrees of shear stress. Additionally, the antifouling coatings were tested for leaching of potential compounds and finally tested in sea trials. This multistep-approach revealed that the individual steps led to consistent results regarding antifouling activity of the coatings. Furthermore, the novel mesocosm system can be employed for advanced antifouling analysis including metagenomic approaches for determination of microbial diversity attaching to different coatings under changing shear forces.

Benefit from decline: the primary transcriptome of Alteromonas macleodii str. Te101 during Trichodesmium demise.

Interactions between co-existing microorganisms deeply affect the physiology of the involved organisms and, ultimately, the function of the ecosystem as a whole. Copiotrophic Alteromonas are marine gammaproteobacteria that thrive during the late stages of phytoplankton blooms in the marine environment and in laboratory co-cultures with cyanobacteria such as Trichodesmium. The response of this heterotroph to the sometimes rapid and transient changes in nutrient supply when the phototroph crashes is not well understood. Here, we isolated and sequenced the strain Alteromonas macleodii str. Te101 from a laboratory culture of Trichodesmium erythraeum IMS101, yielding a chromosome of 4.63 Mb and a single plasmid of 237 kb. Increasing salinities to ≥43 ppt inhibited the growth of Trichodesmium but stimulated growth of the associated Alteromonas. We characterized the transcriptomic responses of both microorganisms and identified the complement of active transcriptional start sites in Alteromonas at single-nucleotide resolution. In replicate cultures, a similar set of genes became activated in Alteromonas when growth rates of Trichodesmium declined and mortality was high. The parallel activation of fliA, rpoS and of flagellar assembly and growth-related genes indicated that Alteromonas might have increased cell motility, growth, and multiple biosynthetic activities. Genes with the highest expression in the data set were three small RNAs (Aln1a-c) that were identified as analogs of the small RNAs CsrB-C in E. coli or RsmX-Z in pathogenic bacteria. Together with the carbon storage protein A (CsrA) homolog Te101_05290, these RNAs likely control the expression of numerous genes in responding to changes in the environment.

Transcriptional response of Prochlorococcus to co-culture with a marine Alteromonas: differences between strains and the involvement of putative infochemicals.

Interactions between marine microorganisms may determine the dynamics of microbial communities. Here, we show that two strains of the globally abundant marine cyanobacterium Prochlorococcus, MED4 and MIT9313, which belong to two different ecotypes, differ markedly in their response to co-culture with a marine heterotrophic bacterium, Alteromonas macleodii strain HOT1A3. HOT1A3 enhanced the growth of MIT9313 at low cell densities, yet inhibited it at a higher concentration, whereas it had no effect on MED4 growth. The early transcriptomic responses of Prochlorococcus cells after 20 h in co-culture showed no evidence of nutrient starvation, whereas the expression of genes involved in photosynthesis, protein synthesis and stress responses typically decreased in MED4 and increased in MIT313. Differential expression of genes involved in outer membrane modification, efflux transporters and, in MIT9313, lanthipeptides (prochlorosins) suggests that Prochlorococcus mount a specific response to the presence of the heterotroph in the cultures. Intriguingly, many of the differentially-expressed genes encoded short proteins, including two new families of co-culture responsive genes: CCRG-1, which is found across the Prochlorococcus lineage and CCRG-2, which contains a sequence motif involved in the export of prochlorosins and other bacteriocin-like peptides, and are indeed released from the cells into the media.

Production of enzymes by Alteromonas sp. A321 to degrade polysaccharides from Enteromorpha prolifera.

Polysaccharides from Enteromorpha prolifera (PE) are becoming increasingly popular due to its bioactivity and abundant source. Screening novel microorganisms which could secrete enzymes to degrade PE efficiently for oligosaccharides production is a promising solution to improve its application. In this study, a marine bacterium that can produce enzymes to degrade PE specifically was selected. It was identified as Alteromonas sp. A321, based on the biochemical properties and 16S rDNA gene sequencing. In order to maximize the activity of degradase for polysaccharides from E. prolifera (DPE), the effects of medium composition and culture conditions were investigated. The highest DPE production was obtained in the medium consisting of K2HPO4 0.15%, PE 0.9%, NaNO3 0.4%, NaCl 1.0% and MgSO4 0.05%. The degradase activity was enhanced from original 0.391 U/ml to 0.744 U/ml. DPE show high efficiency and substrate specificity to PE with 63.53% of reducing sugar production in the 7 h hydrolysis.

Induction of antifouling diterpene production by Streptomyces cinnabarinus PK209 in co-culture with marine-derived Alteromonas sp. KNS-16.

An active antifouling diterpene was isolated from marine actinomycete strain PK209 and productivity was induced in a co-culture experiment. The active constituent was identified as the diterpene lobocompactol by interpretion of nuclear magnetic resonance and mass spectroscopy data. A PK209 co-culture was designed and a lobocompactol-resistant bacterium, KNS-16, was selected as co-culture competitor to induce lobocompactol production. Adding a small volume of 16-h-old KNS-16 culture to the 96-h-old PK209 culture caused rapid induction of lobocompactol production. The final yield was 2.7 mg/L, 10.4-fold higher than that collected from a single PK209 culture. The two bacteria, strains PK209 and KNS-16, were identified as Streptomyces cinnabarinus and Alteromonas sp. based on 16S rDNA sequencing. Lobocompactol showed significant antifouling activity, of 0.18 and 0.43 µg/mL, for EC50 against the macroalga Ulva pertusa and the diatom Navicula annexa respectively. It showed activity with MIC of 61-112 µg/mL against fouling bacteria.

Efficacy of UV treatment in the management of bacterial adhesion on hard surfaces.

The efficacy of UV treatment to control bacterial adhesion onto hard surfaces was investigated in laboratory conditions. The major characteristics necessary for biofilm formation like extracellular polymeric substance (EPS) production, carbohydrate and protein concentration in EPS, and adhesion ability onto hard surface were studied using two bacterial strains isolated from marine biofilms. The results showed that there was a considerable difference between the control and UV treated bacterial cultures in their viability, production of EPS, and adhesion ability. The protein and carbohydrate concentration of the EPS and the adhesion of bacterial cells to surface were also considerably reduced due to UV treatment. This study indicates that treatment of water with UV light may be used to control biofilm development on hard surfaces.

Genetic analysis of the Alteromonas macleodii [NiFe]-hydrogenase.

Alteromonas macleodii Deep ecotype is a marine, heterotrophic, gammaproteobacterium isolated in the Mediterranean Sea between depths of 1000 and 3500 m. The sequenced strain was previously reported to contain a [NiFe] hydrogenase. We verified the presence of this hydrogenase in other strains of A. macleodii Deep ecotype that were previously isolated from several bathypelagic microenvironments. We developed a system for the genetic manipulation of A. macleodii Deep ecotype using conjugation and used this system to create mutant strains that lack the [NiFe] hydrogenase structural genes (hynSL). The mutants did not possess hydrogenase activity, and complementation of the mutant strain with the hynSL genes successfully restored hydrogenase activity. Both the mutant and the wild-type strains grew at the same rate in a variety of media and under different environmental conditions, indicating little effect of the hydrogenase mutation under the conditions tested.

Determination of lipid degradation by marine lipase-producing bacteria: critical evaluation of lipase activity assays.

With the aim of obtaining a better understanding of lipids-lipases interactions in bacterioplankton communities in oceans, we used different methods for measuring lipase activities in pure cultures of the marine strain Alteromonas macleodii. The decay of tripalmitate added to cultures was followed chemically over time. In an enzymatic approach, lipase activities were measured using the fluorogenic lipid analogs MUF-palmitate and ELF-palmitate. When hydrolyzed by lipase, the non-fluorescent substrates release MUF and ELF Alcohol (ELFA) which are fluorescent. As shown by spectrofluorometry, ELF-palmitate was an efficient competitor for MUF-palmitate. However, the activities reached using these two fluorogenic substrates were different, but still much higher than the tripalmitate hydrolysis rate, measured chemically. MUF- and ELF-palmitate would not be hydrolyzed by lipase sensu stricto (defined as triacylglycerol acylesterase E.C. 3.1.1.3) but rather reflects lipolytic activities in a broad sense. ELFA is also water-insoluble and theoretically precipitates in the external membrane of bacteria causing its hydrolysis, which would allow microscopic identification of active cells. By epifluorescence microscopy, the accumulation of ELFA fluorescence over time was detected (as large, diffuse halos), but no precipitates were clearly associated with bacteria on slide preparations, neither for pure cultures of Alteromonas macleodii nor for natural samples from the Bay of Marseille, France. Among possible biases, those related to the hydrophobic/hydrophilic conditions required for precipitation are discussed.

Monitoring of algicidal bacterium, Alteromonas sp. strain A14 in its application to natural Cochlodinium polykrikoides blooming seawater using fluorescence in situ hybridization.

The red tide of dinoflagellate, Cochlodinium polykrikoides has frequently occurred in coastal waters, causing severe damage to fisheries. In the present study, the algicidal bacterium Alteromonas sp. A14 isolated from the southern coast of Korea was applied to a red tide of C. polykrikoides in a laboratory experiment. In the experiment, the abundance of the strain A14 was monitored using fluorescence in situ hybridization. Inoculation of the A14 at a final cell density of 9.0 x 10(5) cells/ml caused a significant decrease in C. polykrikoides abundance from 1,830 to 700 cells/ml during 2 days, while abundances of harmless diatoms rapidly increased from 3 days. Abundances of both A14 and other bacteria increased to 1 day. After 1 day, with flagellate abundance increased, bacterial abundance decreased. Finally, algicidal bacterial abundance decreased to 3.5 x 10(4) cells/ml. In the biological control of harmful algal blooms, in addition to decrease in target algal abundance and not occurrence of other harmful blooms, decrease in abundance of utilized organism is also important. This study emphasizes the importance of monitoring the inoculated bacterium when applying bacterium to natural seawater.

Facilitation of robust growth of Prochlorococcus colonies and dilute liquid cultures by "helper" heterotrophic bacteria.

Axenic (pure) cultures of marine unicellular cyanobacteria of the Prochlorococcus genus grow efficiently only if the inoculation concentration is large; colonies form on semisolid medium at low efficiencies. In this work, we describe a novel method for growing Prochlorococcus colonies on semisolid agar that improves the level of recovery to approximately 100%. Prochlorococcus grows robustly at low cell concentrations, in liquid or on solid medium, when cocultured with marine heterotrophic bacteria. Once the Prochlorococcus cell concentration surpasses a critical threshold, the "helper" heterotrophs can be eliminated with antibiotics to produce axenic cultures. Our preliminary evidence suggests that one mechanism by which the heterotrophs help Prochlorococcus is the reduction of oxidative stress.

Maltose adaptive mutant of heterotrophic marine bacterium, Alteromonas espejiana Bal-31: changes in the growth and the induction of extracellular alpha-amylase.

Growth of the heterotrophic marine bacterium, Alteromonas espejiana Bal-31 was inhibited in the presence of sucrose, maltose and even glucose, but not with starch. Extracellular alpha-amylase was induced with a lag phase of 2 h in the presence of starch. In contrast, cell growth of the S2a mutant was not affected by the addition of maltose, and starch was ineffective in the induction of extracellular alpha-amylase in this mutant. Activity of extracellular alpha-amylase was induced from the S2a mutant with a 4-h lag phase in the presence of maltose, and the high level of enzyme activity was maintained for at least 24 h. Activity of alpha-amylase induced by both wild type starch and S2a mutant maltose cultures were mainly observed in extracellular locations. This activity could be stopped by tetracycline treatment, indicating that enzyme induction was dependant on gene expression and not on enzyme protein secretory mechanisms. Our results showed that the mutation in S2a changed the growth and the modulation of the specific alpha-amylase in response to carbon nutrients.

Roles of four chitinases (chia, chib, chic, and chid) in the chitin degradation system of marine bacterium Alteromonas sp. strain O-7.

Alteromonas sp. strain O-7 secretes four chitinases (ChiA, ChiB, ChiC, and ChiD) in the presence of chitin. To elucidate why the strain produces multiple chitinases, we studied the expression levels of the four genes and proteins, their enzymatic properties, and their synergistic effects on chitin degradation. Among the four chitinases, ChiA was produced in the largest quantities, followed by ChiD, and the production of ChiB and ChiC changed at lower levels than those of ChiA and ChiD. The expression of the chiA, chiB, chiC, and chiD genes was investigated at the transcriptional level. The RNA transcript of chiA was most strongly induced in the presence of chitin, the expression of chiD followed, and the RNA transcripts of chiB and chiC changed at low levels. The hydrolyzing activities of the four chitinases against various substrates were examined. ChiA was the most active enzyme against powdered chitin, whereas ChiC was the most active against soluble chitin among the four chitinases. ChiD had activities closer to those of ChiA than to those of ChiB and ChiC. ChiB showed no distinctive feature against the chitinous substrates tested. When powdered chitin was treated with the proper combination of four chitinases, an approximately 2.0-fold increase in the hydrolytic activity was observed. These results, together with the results described above, indicate that ChiA plays a central role in chitin degradation for this strain.

Biosynthesis of the marine antibiotic pentabromopseudilin. 2. The pyrrole ring.

The biosynthesis of the potent marine antibiotic, pentabromopseudilin (1), was investigated. Feeding studies with Alteromonas luteoviolaceus were performed on a defined medium. D,L-[5-(13)C]proline was incorporated symmetrically, demonstrating that the pyrrole ring of pentabromopseudilin is derived from proline.

Culturable and nonculturable bacterial symbionts in the toxic benthic dinoflagellate Ostreopsis lenticularis.

The toxic benthic dinoflagellate Ostreopsis lenticularis hosts a variety of symbiont bacterial flora. Laboratory cultured Ostreopsis clones require the presence of symbiotic Pseudomonas/Alteromonas bacterial strains for growth and toxicity development. Three culturable bacterial strains associated with Ostreopsis were identified as Pseudomonas/Alteromonas strain 1, Pseudomonas/Alteromonas strain 2 and Acinetobacter. Denaturing gradient gel electrophoresis (DGGE) analyses of extracted Ostreopsis associated bacterial DNAs indicated that there were three culturable and four non-culturable associated bacterial strains. The results presented here are the first report of the presence of unculturable bacterial symbionts in a toxic benthic dinoflagellate. Ostreopsis lost toxicity when exposed to elevated temperatures in the field and laboratory culture and subsequently recovered toxicity at reduced temperatures. Ostreopsis associated culturable Pseudomonas/Alteromonas bacterial strains were significantly reduced in dinoflagellate cultures exposed to elevated temperatures. The decreased toxicity of O. lenticularis exposed to elevated temperatures and their subsequent recovery of toxicity in periods of reduced thermal stress may have resulted from the effects of elevated temperature on the spectrum of culturable and unculturable bacterial species interacting with their Ostreopsis host.

Tolerance to various toxicants by marine bacteria highly resistant to mercury.

Bacteria highly resistant to mercury isolated from seawater and sediment samples were tested for growth in the presence of different heavy metals, pesticides, phenol, formaldehyde, formic acid, and trichloroethane to investigate their potential for growth in the presence of a variety of toxic xenobiotics. We hypothesized that bacteria resistant to high concentrations of mercury would have potential capacities to tolerate or possibly degrade a variety of toxic materials and thus would be important in environmental pollution bioremediation. The mercury-resistant bacteria were found to belong to Pseudomonas, Proteus, Xanthomonas, Alteromonas, Aeromonas, and Enterobacteriaceae. All these environmental bacterial strains tolerant to mercury used in this study were capable of growth at a far higher concentration (50 ppm) of mercury than previously reported. Likewise, their ability to grow in the presence of toxic xenobiotics, either singly or in combination, was superior to that of bacteria incapable of growth in media containing 5 ppm mercury. Plasmid-curing assays done in this study ascertained that resistance to mercury antibiotics, and toxic xenobiotics is mediated by chromosomally borne genes and/or transposable elements rather than by plasmids.

A novel polymer produced by a bacterium isolated from a deep-sea hydrothermal vent polychaete annelid.

AIMS: The objective of the present work was to describe an aerobic, mesophilic and heterotrophic marine bacterium, designated HYD657, able to produce an exopolysaccharide (EPS). It was isolated from a East Pacific Rise deep-sea hydrothermal vent polychaete annelid. METHODS AND RESULTS: This micro-organism, on the basis of the phenotypical features and genotypic investigations, can be clearly assigned to the Alteromonas macleodii species and the name A. macleodii subsp. fijiensis biovar deepsane is proposed. Optimal growth occurs between 30 and 35 degrees C, at pH between 6.5 and 7.5 and at ionic strengths between 20 and 40 g x l(-1) NaCl. The G + C content of DNA was 46.5%. This bacterium excreted, under laboratory conditions, an EPS consisting of glucose, galactose, rhamnose, fucose and mannose as neutral sugars along with glucuronic and galacturonic acids and a diacidic hexose identified as a 3-0-(1 carboxyethyl)-D-glucuronic acid. Its average molecular mass was 1.6 x 10(6) Da. CONCLUSIONS: The bacterium HYD657, for which the name A. macleodii subsp. fijiensis biovar deepsane is proposed, produces an unusual EPS in specific medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to its interesting biological activities, applications have been found in cosmetics. Its probable contribution to the filamentous microbial mat in the Alvinella pompejana microenvironment can be also mentioned.

Stimulation of Alexandrium fundyense growth by bacterial assemblages from the Bay of Fundy.

AIMS: To evaluate the influence of marine bacterial assemblages from the Bay of Fundy on Alexandrium fundyense str. CB301 growth. METHODS AND RESULTS: Bacterial assemblages were collected from the Bay of Fundy during an Alexandrium spp. bloom, serially diluted to extinction and inoculated into axenic CB301 cultures. Bacterial assemblages dramatically enhanced CB301 growth. Retrieval and analysis of 16S rDNA fragments revealed an Alteromonas sp. strain to be the only detectable bacterium in all assemblages that promoted A. fundyense and the sole bacterium found in the most dilute inoculum that promoted A. fundyense. While this bacterium has not yet been isolated, other isolates obtained from the assemblages did not stimulate A. fundyense, indicating that the observed stimulation was not a general effect of marine bacteria. CONCLUSIONS: Bay of Fundy marine bacterial assemblages dominated by a member of the family Alteromonadaceae were found to dramatically stimulate growth of A. fundyense. SIGNIFICANCE AND IMPACT OF THE STUDY: These results show that native bacteria have the potential to dramatically promote the growth of A. fundyense and may play an important role in influencing A. fundyense dynamics in the Bay of Fundy.

Succession of pelagic marine bacteria during enrichment: a close look at cultivation-induced shifts.

Enrichment experiments with North Sea bacterioplankton were performed to test if rapid incubation-induced changes in community structure explain the frequent isolation of members of a few particular bacterial lineages or if readily culturable bacteria are common in the plankton but in a state of dormancy. A metabolic inhibitor of cell division (nalidixic acid [NA]) was added to substrate-amended (S+) and unamended (S-) grazer-free seawater samples, and shifts in community composition and per cell DNA and protein content were compared with untreated controls. In addition, starvation survival experiments were performed on selected isolates. Incubations resulted in rapid community shifts towards typical culturable genera rather than in the activation of either dormant cells or the original DNA-rich bacterial fraction. Vibrio spp. and members of the Alteromonas/Colwellia cluster (A/C) were selectively enriched in S+ and S-, respectively, and this trend was even magnified by the addition of NA. These increases corresponded with the rise of cell populations with distinctively different but generally higher protein and DNA content in the various treatments. Uncultured dominant gamma-proteobacteria affiliating with the SAR86 cluster and members of the culturable genus Oceanospirillum were not enriched or activated, but there was no indication of substrate-induced cell death, either. Strains of Vibrio and A/C maintained high ribosome levels in pure cultures during extended periods of starvation, whereas Oceanospirillum spp. did not. The life strategy of rapidly enriched culturable gamma-proteobacteria could thus be described as a "feast and famine" existence involving different activation levels of substrate concentration.

A serine protease-encoding gene (aprII) of Alteromonas sp. Strain O-7 is regulated by the iron uptake regulator (Fur) protein.

The ferric uptake regulator (Fur) box-like sequence was located upstream of the serine protease-encoding gene (aprII) from a marine bacterium, Alteromonas sp. strain O-7. To clarify whether the production of AprII (the gene product of aprII) is regulated by the environmental iron concentrations, this strain was cultured under iron-depleted or iron-rich conditions and the level of AprII in the culture supernatant was analyzed by Western blotting. The production of AprII was significantly repressed under iron-rich conditions. Northern hybridization analysis demonstrated that AprII biosynthesis was regulated by iron through the control of transcription. These results indicate that aprII is a new member of the iron regulon and plays an important role in the iron acquisition system of the strain. Furthermore, the gene encoding Fur was cloned and sequenced. The deduced amino acid sequence of the cloned Fur showed high sequence similarity with that from gram-negative bacteria.