The pangenome of (Antarctic) Pseudoalteromonas bacteria: evolutionary and functional insights.

BACKGROUND: Pseudoalteromonas is a genus of ubiquitous marine bacteria used as model organisms to study the biological mechanisms involved in the adaptation to cold conditions. A remarkable feature shared by these bacteria is their ability to produce secondary metabolites with a strong antimicrobial and antitumor activity. Despite their biotechnological relevance, representatives of this genus are still lacking (with few exceptions) an extensive genomic characterization, including features involved in the evolution of secondary metabolites production. Indeed, biotechnological applications would greatly benefit from such analysis. RESULTS: Here, we analyzed the genomes of 38 strains belonging to different Pseudoalteromonas species and isolated from diverse ecological niches, including extreme ones (i.e. Antarctica). These sequences were used to reconstruct the largest Pseudoalteromonas pangenome computed so far, including also the two main groups of Pseudoalteromonas strains (pigmented and not pigmented strains). The downstream analyses were conducted to describe the genomic diversity, both at genus and group levels. This allowed highlighting a remarkable genomic heterogeneity, even for closely related strains. We drafted all the main evolutionary steps that led to the current structure and gene content of Pseudoalteromonas representatives. These, most likely, included an extensive genome reduction and a strong contribution of Horizontal Gene Transfer (HGT), which affected biotechnologically relevant gene sets and occurred in a strain-specific fashion. Furthermore, this study also identified the genomic determinants related to some of the most interesting features of the Pseudoalteromonas representatives, such as the production of secondary metabolites, the adaptation to cold temperatures and the resistance to abiotic compounds. CONCLUSIONS: This study poses the bases for a comprehensive understanding of the evolutionary trajectories followed in time by this peculiar bacterial genus and for a focused exploitation of their biotechnological potential.

Antagonistic interactions between endophytic cultivable bacterial communities isolated from the medicinal plant Echinacea purpurea.

In this work we have studied the antagonistic interactions existing among cultivable bacteria isolated from three ecological niches (rhizospheric soil, roots and stem/leaves) of the traditional natural medicinal plant Echinacea purpurea. The three compartments harboured different taxonomic assemblages of strains, which were previously reported to display different antibiotic resistance patterns, suggesting the presence of differential selective pressure due to antagonistic molecules in the three compartments. Antagonistic interactions were assayed by the cross-streak method and interpreted using a network-based analysis. In particular 'within-niche inhibition' and 'cross-niche inhibition' were evaluated among isolates associated with each compartment as well as between isolates retrieved from the three different compartments respectively. Data obtained indicated that bacteria isolated from the stem/leaves compartment were much more sensitive to the antagonistic activity than bacteria from roots and rhizospheric soil. Moreover, both the taxonomical position and the ecological niche might influence the antagonistic ability/sensitivity of different strains. Antagonism could play a significant role in contributing to the differentiation and structuring of plant-associated bacterial communities.

Antimicrobial Activity of Monoramnholipids Produced by Bacterial Strains Isolated from the Ross Sea (Antarctica).

Microorganisms living in extreme environments represent a huge reservoir of novel antimicrobial compounds and possibly of novel chemical families. Antarctica is one of the most extraordinary places on Earth and exhibits many distinctive features. Antarctic microorganisms are well known producers of valuable secondary metabolites. Specifically, several Antarctic strains have been reported to inhibit opportunistic human pathogens strains belonging to Burkholderia cepacia complex (Bcc). Herein, we applied a biodiscovery pipeline for the identification of anti-Bcc compounds. Antarctic sub-sea sediments were collected from the Ross Sea, and used to isolate 25 microorganisms, which were phylogenetically affiliated to three bacterial genera (Psychrobacter, Arthrobacter, and Pseudomonas) via sequencing and analysis of 16S rRNA genes. They were then subjected to a primary cell-based screening to determine their bioactivity against Bcc strains. Positive isolates were used to produce crude extracts from microbial spent culture media, to perform the secondary screening. Strain Pseudomonas BNT1 was then selected for bioassay-guided purification employing SPE and HPLC. Finally, LC-MS and NMR structurally resolved the purified bioactive compounds. With this strategy, we achieved the isolation of three rhamnolipids, two of which were new, endowed with high (MIC < 1 µg/mL) and unreported antimicrobial activity against Bcc strains.

Genome-scale metabolic reconstruction and constraint-based modelling of the Antarctic bacterium Pseudoalteromonas haloplanktis†TAC125.

The Antarctic strain Pseudoalteromonas haloplanktis TAC125 is one of the model organisms of cold-adapted bacteria and is currently exploited as a new alternative expression host for numerous biotechnological applications. Here, we investigated several metabolic features of this strain through in silico modelling and functional integration of -omics data. A genome-scale metabolic model of P. haloplanktis TAC125 was reconstructed, encompassing information on 721 genes, 1133 metabolites and 1322 reactions. The predictive potential of this model was validated against a set of experimentally determined growth rates and a large dataset of growth phenotypic data. Furthermore, evidence synthesis from proteomics, phenomics, physiology and metabolic modelling data revealed possible drawbacks of cold-dependent changes in gene expression on the overall metabolic network of P. haloplanktis TAC125. These included, for example, variations in its central metabolism, amino acid degradation and fatty acid biosynthesis. The genome-scale metabolic model described here is the first one reconstructed so far for an Antarctic microbial strain. It allowed a system-level investigation of variations in cellular metabolic fluxes following a temperature downshift. It represents a valuable platform for further investigations on P. haloplanktis TAC125 cellular functional states and for the design of more focused strategies for its possible biotechnological exploitation.

Origin, duplication and reshuffling of plasmid genes: Insights from Burkholderia vietnamiensis G4 genome.

Using a computational pipeline based on similarity networks reconstruction we analysed the 1133 genes of the Burkholderia vietnamiensis (Bv) G4 five plasmids, showing that gene and operon duplication played an important role in shaping the plasmid architecture. Several single/multiple duplications occurring at intra- and/or interplasmids level involving 253 paralogous genes (stand-alone, clustered or operons) were detected. An extensive gene/operon exchange between plasmids and chromosomes was also disclosed. The larger the plasmid, the higher the number and size of paralogous fragments. Many paralogs encoded mobile genetic elements and duplicated very recently, suggesting that the rearrangement of the Bv plastic genome is ongoing. Concerning the "molecular habitat" and the "taxonomical status" (the Preferential Organismal Sharing) of Bv plasmid genes, most of them have been exchanged with other plasmids of bacteria belonging (or phylogenetically very close) to Burkholderia, suggesting that taxonomical proximity of bacterial strains is a crucial issue in plasmid-mediated gene exchange.

Phenotypic and genomic characterization of the Antarctic bacterium Gillisia sp. CAL575, a producer of antimicrobial compounds.

Microorganisms from Antarctica have evolved particular strategies to cope with cold. Moreover, they have been recently reported as producers of antimicrobial compounds, which inhibit the growth of other bacteria. In this work we characterized from different viewpoints the Gillisia sp. CAL575 strain, a psychrotrophic bacterium that produces microbial volatile organic compounds involved in the growth inhibition of Burkholderia cepacia complex members. Sequencing and analysis of the whole genome of Gillisia sp. CAL575 revealed that it includes genes that are involved in secondary metabolite production, adaptation to cold conditions, and different metabolic pathways for the production of energy. All these features make Gillisia sp. CAL575 a possible tool for biotechnology.

Manila clams from Hg polluted sediments of Marano and Grado lagoons (Italy) harbor detoxifying Hg resistant bacteria in soft tissues.

A mechanism of mercury detoxification has been suggested by a previous study on Hg bioaccumulation in Manila clams (Ruditapes philippinarum) in the polluted Marano and Grado lagoons and in this study we demonstrate that this event could be partly related to the detoxifying activities of Hg-resistant bacteria (MRB) harbored in clam soft tissues. Therefore, natural clams were collected in six stations during two different periods (winter and spring) from Marano and Grado Lagoons. Siphons, gills and hepatopancreas from acclimatized clams were sterile dissected to isolate MRB. These anatomical parts were glass homogenized or used for whole, and they were lying on a solid medium containing 5mgl(-1) HgCl2 and incubated at 30°C. A total of fourteen bacterial strains were isolated and were identified by 16S rDNA sequencing and analysis, revealing that strains were representative of eight bacterial genera, four of which were Gram-positive (Enterococcus, Bacillus, Jeotgalicoccus and Staphylococcus) and other four were Gram-negative (Stenotrophomonas, Vibrio, Raoultella and Enterobacter). Plasmids and merA genes were found and their sequences determined. Fluorescence in situ hybridization (FISH) technique shows the presence of Firmicutes, Actinobacteria and Gammaproteobacteria by using different molecular probes in siphon and gills. Bacterial clumps inside clam flesh were observed and even a Gram-negative endosymbiont was disclosed by transmission electronic microscope inside clam cells. Bacteria harbored in cavities of soft tissue have mercury detoxifying activity. This feature was confirmed by the determination of mercuric reductase in glass-homogenized siphons and gills.

Draft genome sequence of the hydrocarbon-degrading and emulsan-producing strain Acinetobacter venetianus RAG-1T.

We report the draft genome sequence of Acinetobacter venetianus strain RAG-1(T), which is able to degrade hydrocarbons and to synthesize a powerful biosurfactant (emulsan) that can be employed for oil removal and as an adjuvant for vaccine delivery. The genome sequence of A. venetianus RAG-1(T) might be useful for bioremediation and/or clinical purposes.

Draft genome sequence of the volatile organic compound-producing Antarctic bacterium Arthrobacter sp. strain TB23, able to inhibit cystic fibrosis pathogens belonging to the Burkholderia cepacia complex.

Arthrobacter sp. strain TB23 was isolated from the Antarctic sponge Lissodendoryx nobilis. This bacterium is able to produce antimicrobial compounds and volatile organic compounds (VOCs) that inhibit the growth of other Antarctic bacteria and of cystic fibrosis opportunistic pathogens, respectively. Here we report the draft genome sequence of Arthrobacter sp. TB23.

Exploring the HME and HAE1 efflux systems in the genus Burkholderia.

BACKGROUND: The genus Burkholderia includes a variety of species with opportunistic human pathogenic strains, whose increasing global resistance to antibiotics has become a public health problem. In this context a major role could be played by multidrug efflux pumps belonging to Resistance Nodulation Cell-Division (RND) family, which allow bacterial cells to extrude a wide range of different substrates, including antibiotics. This study aims to i) identify rnd genes in the 21 available completely sequenced Burkholderia genomes, ii) analyze their phylogenetic distribution, iii) define the putative function(s) that RND proteins perform within the Burkholderia genus and iv) try tracing the evolutionary history of some of these genes in Burkholderia. RESULTS: BLAST analysis of the 21 Burkholderia sequenced genomes, using experimentally characterized ceoB sequence (one of the RND family counterpart in the genus Burkholderia) as probe, allowed the assembly of a dataset comprising 254 putative RND proteins. An extensive phylogenetic analysis revealed the occurrence of several independent events of gene loss and duplication across the different lineages of the genus Burkholderia, leading to notable differences in the number of paralogs between different genomes. A putative substrate [antibiotics (HAE1 proteins)/heavy-metal (HME proteins)] was also assigned to the majority of these proteins. No correlation was found between the ecological niche and the lifestyle of Burkholderia strains and the number/type of efflux pumps they possessed, while a relation can be found with genome size and taxonomy. Remarkably, we observed that only HAE1 proteins are mainly responsible for the different number of proteins observed in strains of the same species. Data concerning both the distribution and the phylogenetic analysis of the HAE1 and HME in the Burkholderia genus allowed depicting a likely evolutionary model accounting for the evolution and spreading of HME and HAE1 systems in the Burkholderia genus. CONCLUSION: A complete knowledge of the presence and distribution of RND proteins in Burkholderia species was obtained and an evolutionary model was depicted. Data presented in this work may serve as a basis for future experimental tests, focused especially on HAE1 proteins, aimed at the identification of novel targets in antimicrobial therapy against Burkholderia species.

Antimicrobial activity of six essential oils against Burkholderia cepacia complex: insights into mechanism(s) of action.

AIM: To investigate the activity and mechanisms of action of six essential oils (EOs) against Burkholderia cepacia complex, opportunistic human pathogens highly resistant to antibiotics. MATERIALS & METHODS: Minimal inhibitory concentration of EOs alone, plus antibiotics or efflux pump inhibitors was determined. RESULTS: Origanum vulgare, Thymus vulgaris and Eugenia caryophyllata EOs resulted to be more active than the other EOs. EOs did not enhance antibiotic activity against the model strain B. cenocepacia J2315. EOs resulted more active in the presence of an efflux pump inhibitor acting on Resistance-Nodulation Cell Division efflux pumps and against B. cenocepacia J2315 Resistance-Nodulation Cell Division knocked-out mutants. CONCLUSION: EOs showed intracellular mechanisms of action and, thus, the efflux pumps inhibitor addition could boost their activity.

Antagonism and antibiotic resistance drive a species-specific plant microbiota differentiation in Echinacea spp.

A key factor in the study of plant-microbes interactions is the composition of plant microbiota, but little is known about the factors determining its functional and taxonomic organization. Here we investigated the possible forces driving the assemblage of bacterial endophytic and rhizospheric communities, isolated from two congeneric medicinal plants, Echinacea purpurea (L.) Moench and Echinacea angustifolia (DC) Heller, grown in the same soil, by analysing bacterial strains (isolated from three different compartments, i.e. rhizospheric soil, roots and stem/leaves) for phenotypic features such as antibiotic resistance, extracellular enzymatic activity, siderophore and indole 3-acetic acid production, as well as cross-antagonistic activities. Data obtained highlighted that bacteria from different plant compartments were characterized by specific antibiotic resistance phenotypes and antibiotic production, suggesting that the bacterial communities themselves could be responsible for structuring their own communities by the production of antimicrobial molecules selecting bacterial-adaptive phenotypes for plant tissue colonization.

Preliminary data on antibacterial activity of Echinacea purpurea-associated bacterial communities against Burkholderia cepacia complex strains, opportunistic pathogens of Cystic Fibrosis patients.

Burkholderia cepacia complex bacteria (Bcc) represent a serious threat for immune-compromised patient affected by Cystic Fibrosis (CF) since they are resistant to many substances and to most antibiotics. For this reason, the research of new natural compounds able to inhibit the growth of Bcc strains has raised new interest during the last years. A source of such natural compounds is represented by medicinal plants and, in particular, by bacterial communities associated with these plants able to produce molecules with antimicrobial activity. In this work, a panel of 151 (endophytic) bacteria isolated from three different compartments (rhizospheric soil, roots, and stem/leaves) of the medicinal plant Echinacea purpurea were tested (using the cross-streak method) for their ability to inhibit the growth of 10 Bcc strains. Data obtained revealed that bacteria isolated from the roots of E. purpurea are the most active in the inhibition of Bcc strains, followed by bacteria isolated from the rhizospheric soil, and endophytes from stem/leaf compartment. At the same time, Bcc strains of environmental origin showed a higher resistance toward inhibition than the Bcc strains with clinical (i.e. CF patients) origin. Differences in the inhibition activity of E. purpurea-associated bacteria are mainly linked to the environment -the plant compartment- rather than to their taxonomical position.

Pseudoalteromonas haloplanktis produces methylamine, a volatile compound active against Burkholderia cepacia complex strains.

The Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 has been reported to produce several Volatile Organic Compounds (VOCs), which are able to inhibit the growth of Burkholderia cepacia complex (Bcc) strains, opportunistic pathogens responsible for the infection of immune-compromised patients. However, no specific antibacterial VOCs have been identified to date. The purpose of the present study was to identify specific VOCs that contribute to Bcc inhibition by the Antarctic strain. When grown on defined medium containing D-gluconate and L-glutamate as carbon, nitrogen and energy sources, P. haloplanktis TAC125 is unable to inhibit the growth of Bcc strains. However, single addition of several amino acids to the defined medium restores the P. haloplanktis TAC125 inhibition ability. With the aim of identifying specific volatile compound/s responsible for Bcc inhibition, we set up an apparatus for VOC capture, accumulation, and storage. P. haloplanktis TAC125 was grown in an automatic fermenter which was connected to a cooling system to condense VOCs present in the exhaust air outlet. Upon addition of methionine to the growth medium, the VOC methylamine was produced by P. haloplanktis TAC125. Methylamine was found to inhibit the growth of several Bcc strains in a dose-dependent way. Although it was reported that P. haloplanktis TAC125 produces VOCs endowed with antimicrobial activity, this is the first demonstration that methylamine probably contributes to the anti-Bcc activity of P. haloplanktis TAC125 VOCs.

Is the plant-associated microbiota of Thymus spp. adapted to plant essential oil?

We examined whether the microbiota of two related aromatic thyme species, Thymus vulgaris and Thymus citriodorus, differs in relation to the composition of the respective essential oil (EO). A total of 576 bacterial isolates were obtained from three districts (leaves, roots and rhizospheric soil). They were taxonomically characterized and inspected for tolerance to the EO from the two thyme species. A district-related taxonomic pattern was found. In particular, high taxonomic diversity among the isolates from leaves was detected. Moreover, data obtained revealed a differential pattern of resistance of the isolates to EOs extracted from T. vulgaris and T. citriodorus, which was interpreted in terms of differing chemical composition of the EO of their respective host plants. In conclusion, we suggest that bacterial colonization of leaves in Thymus spp. is influenced by the EO present in leaf glandular tissue as one of the selective forces shaping endophytic community composition.

Phenotypic and genomic characterization of the antimicrobial producer Rheinheimera sp. EpRS3 isolated from the medicinal plant Echinacea purpurea: insights into its biotechnological relevance.

In recent years, there has been increasing interest in plant microbiota; however, despite medicinal plant relevance, very little is known about their highly complex endophytic communities. In this work, we report on the genomic and phenotypic characterization of the antimicrobial compound producer Rheinheimera sp. EpRS3, a bacterial strain isolated from the rhizospheric soil of the medicinal plant Echinacea purpurea. In particular, EpRS3 is able to inhibit growth of different bacterial pathogens (Bcc, Acinetobacter baumannii, and Klebsiella pneumoniae) which might be related to the presence of gene clusters involved in the biosynthesis of different types of secondary metabolites. The outcomes presented in this work highlight the fact that the strain possesses huge biotechnological potential; indeed, it also shows antimicrobial effects upon well-described multidrug-resistant (MDR) human pathogens, and it affects plant root elongation and morphology, mimicking indole acetic acid (IAA) action.

Draft Genome Sequences of the Antimicrobial Producers Pseudomonas sp. TAA207 and Pseudomonas sp. TAD18 Isolated from Antarctic Sediments.

We report here the draft genome sequence of the Pseudomonas sp. TAA207 and Pseudomonas sp. TAD18 strains, isolated from Antarctic sediments during a summer campaign near coastal areas of Terra Nova Bay (Antarctica). Genome sequence knowledge allowed the identification of genes associated with the production of bioactive compounds and antibiotic resistance. Furthermore, it will be instrumental for comparative genomics and the fulfillment of both basic and application-oriented investigations.

Genomic and phenotypic characterization of the species Acinetobacter venetianus.

Crude oil is a complex mixture of hydrocarbons and other organic compounds that can produce serious environmental problems and whose removal is highly demanding in terms of human and technological resources. The potential use of microbes as bioremediation agents is one of the most promising fields in this area. Members of the species Acinetobacter venetianus have been previously characterized for their capability to degrade n-alkanes and thus may represent interesting model systems to implement this process. Although a preliminary experimental characterization of the overall hydrocarbon degradation capability has been performed for five of them, to date, the genetic/genomic features underlying such molecular processes have not been identified. Here we have integrated genomic and phenotypic information for six A. venetianus strains, i.e. VE-C3, RAG-1(T), LUH 13518, LUH 7437, LUH 5627 and LUH 8758. Besides providing a thorough description of the A. venetianus species, these data were exploited to infer the genetic features (presence/absence patterns of genes) and the short-term evolutionary events possibly responsible for the variability in n-alkane degradation efficiency of these strains, including the mechanisms of interaction with the fuel droplet and the subsequent catabolism of this pollutant.

Draft Genome Sequence of Pseudomonas sp. EpS/L25, Isolated from the Medicinal Plant Echinacea purpurea and Able To Synthesize Antimicrobial Compounds.

We announce here the draft genome sequence of Pseudomonas sp. strain EpS/L25, isolated from the stem/leaves of the medicinal plant Echinacea purpurea This genome will allow for comparative genomics in order to identify genes associated with the production of bioactive compounds and antibiotic resistance.

Essential Oil from Origanum vulgare Completely Inhibits the Growth of Multidrug-Resistant Cystic Fibrosis Pathogens.

Essential oils (EOs) are known to inhibit the growth of a wide range of microorganisms. Particularly interesting is the possible use of EOs to treat multidrug-resistant cystic fibrosis (CF) pathogens. We tested the essential oil (EO) from Origanum vulgare for in vitro antimicrobial activity, against three of the major human opportunistic pathogens responsible for respiratory infections in CF patients; these are methicillin-resistant Staphylococcus aureus, Stenotrophomonas maltophilia and Achromobacter xylosoxidans. Antibiotic susceptibility of each strain was previously tested by the standard disk diffusion method. Most strains were resistant to multiple antibiotics and could be defined as multi-drug-resistant (MDR). The antibacterial activity of O. vulgare EO (OEO) against a panel of 59 bacterial strains was evaluated, with MIC and MBC determined at 24, 48 and 72 hours by a microdilution method. The OEO was effective against all tested strains, although to a different extent. The MBC and MIC of OEO for S. aureus strains were either lower or equal to 0.50%, v/v, for A. xylosoxidans strains were lower or equal to 1% and 0.50%, v/v, respectively; and for S. maltophilia strains were lower or equal to 0.25%, v/v. The results from this study suggest that OEO might exert a role as an antimicrobial in the treatment of CF infections.