Benefits and Drawbacks of Harboring Plasmid pP32BP2, Identified in Arctic Psychrophilic Bacterium Psychrobacter sp. DAB_AL32B.

Psychrobacter sp. DAB_AL32B, originating from Spitsbergen island (Arctic), carries the large plasmid pP32BP2 (54,438 bp). Analysis of the pP32BP2 nucleotide sequence revealed the presence of three predicted phenotypic modules that comprise nearly 30% of the plasmid genome. These modules appear to be involved in fimbriae synthesis via the chaperone-usher pathway (FIM module) and the aerobic and anaerobic metabolism of carnitine (CAR and CAI modules, respectively). The FIM module was found to be functional in diverse hosts since it facilitated the attachment of bacterial cells to abiotic surfaces, enhancing biofilm formation. The CAI module did not show measurable activity in any of the tested strains. Interestingly, the CAR module enabled the enzymatic breakdown of carnitine, but this led to the formation of the toxic by-product trimethylamine, which inhibited bacterial growth. Thus, on the one hand, pP32BP2 can enhance biofilm formation, a highly advantageous feature in cold environments, while on the other, it may prevent bacterial growth under certain environmental conditions. The detrimental effect of harboring pP32BP2 (and its CAR module) seems to be conditional, since this replicon may also confer the ability to use carnitine as an alternative carbon source, although a pathway to utilize trimethylamine is most probably necessary to make this beneficial. Therefore, the phenotype determined by this CAR-containing plasmid depends on the metabolic background of the host strain.

Exploring the genome of Arctic Psychrobacter sp. DAB_AL32B and construction of novel Psychrobacter-specific cloning vectors of an increased carrying capacity.

Cold-active bacteria are currently of great interest in biotechnology, and their genomic and physiological features have been extensively studied. One of the model psychrotolerant bacteria are Psychrobacter spp. Analysis of Arctic psychrophilic Psychrobacter sp. DAB_AL32B genome content provided an insight into its overall stress response, and genes conferring protection against various life-limiting factors (i.e., low temperature, increased ultraviolet radiation, oxidative stress and osmotic pressure) were recognized and described. Moreover, it was revealed that the strain carries a large plasmid pP32BP2. Its replication system was used for the construction of two novel shuttle vectors (pPS-NR-Psychrobacter-Escherichia coli-specific plasmid and pPS-BR-Psychrobacter-various Proteobacteria-specific plasmid) of an increased carrying capacity, which may be used for genetic engineering of Psychrobacter spp.

Genome-Guided Characterization of Ochrobactrum sp. POC9 Enhancing Sewage Sludge Utilization-Biotechnological Potential and Biosafety Considerations.

Sewage sludge is an abundant source of microorganisms that are metabolically active against numerous contaminants, and thus possibly useful in environmental biotechnologies. However, amongst the sewage sludge isolates, pathogenic bacteria can potentially be found, and such isolates should therefore be carefully tested before their application. A novel bacterial strain, Ochrobactrum sp. POC9, was isolated from a sewage sludge sample collected from a wastewater treatment plant. The strain exhibited lipolytic, proteolytic, cellulolytic, and amylolytic activities, which supports its application in biodegradation of complex organic compounds. We demonstrated that bioaugmentation with this strain substantially improved the overall biogas production and methane content during anaerobic digestion of sewage sludge. The POC9 genome content analysis provided a deeper insight into the biotechnological potential of this bacterium and revealed that it is a metalotolerant and a biofilm-producing strain capable of utilizing various toxic compounds. The strain is resistant to rifampicin, chloramphenicol and ß-lactams. The corresponding antibiotic resistance genes (including blaOCH and cmlA/floR) were identified in the POC9 genome. Nevertheless, as only few genes in the POC9 genome might be linked to pathogenicity, and none of those genes is a critical virulence factor found in severe pathogens, the strain appears safe for application in environmental biotechnologies.

Plasmids of Psychrotolerant Polaromonas spp. Isolated From Arctic and Antarctic Glaciers - Diversity and Role in Adaptation to Polar Environments.

Cold-active bacteria of the genus Polaromonas (class Betaproteobacteria) are important components of glacial microbiomes. In this study, extrachromosomal replicons of 26 psychrotolerant Polaromonas strains, isolated from Arctic and Antarctic glaciers, were identified, sequenced, and characterized. The plasmidome of these strains consists of 13 replicons, ranging in size from 3,378 to 101,077 bp. In silico sequence analyses identified the conserved backbones of these plasmids, composed of genes required for plasmid replication, stable maintenance, and conjugal transfer. Host range analysis revealed that all of the identified plasmids are narrow-host-range replicons, only able to replicate in bacteria of closely related genera (Polaromonas and Variovorax) of the Comamonadaceae family. Special attention was paid to the identification of plasmid auxiliary genetic information, which may contribute to the adaptation of bacteria to environmental conditions occurring in glaciers. Detailed analysis revealed the presence of genes encoding proteins potentially involved in (i) protection against reactive oxygen species, ultraviolet radiation, and low temperatures; (ii) transport and metabolism of organic compounds; (iii) transport of metal ions; and (iv) resistance to heavy metals. Some of the plasmids also carry genes required for the molecular assembly of iron-sulfur [Fe-S] clusters. Functional analysis of the predicted heavy metal resistance determinants demonstrated that their activity varies, depending on the host strain. This study provides the first molecular insight into the mobile DNA of Polaromonas spp. inhabiting polar glaciers. It has generated valuable data on the structure and properties of a pool of plasmids and highlighted their role in the biology of psychrotolerant Polaromonas strains and their adaptation to the environmental conditions of Arctic and Antarctic glaciers.

Genome content, metabolic pathways and biotechnological potential of the psychrophilic Arctic bacterium Psychrobacter sp. DAB_AL43B, a source and a host of novel Psychrobacter-specific vectors.

Psychrobacter sp. DAB_AL43B, isolated from ornithogenic soil collected on the Arctic island of Spitsbergen, is a newly sequenced psychrophilic strain susceptible to conjugation and electrotransformation. Its genome consists of a circular chromosome (3.3 Mb) and four plasmids (4.4-6.4kb). In silico genome mining and microarray-based phenotypic analysis were performed to describe the metabolic potential of this strain and identify possible biotechnological applications. Metabolic reconstruction indicated that DAB_AL43B prefers low-molecular-weight carboxylates and amino acids as carbon and energy sources. Genetic determinants of heavy-metal resistance, anthracene degradation and possible aerobic denitrification were also identified. Comparative analyses revealed a relatively close relationship between DAB_AL43B and other sequenced Psychrobacter species. In addition, the plasmids of this strain were used as the basis for the construction of Escherichia coli-Psychrobacter spp. shuttle vectors. Taken together, the results of this work suggest that DAB_AL43B is a promising candidate as a new model strain for studies on Psychrobacter spp.

Identification and Characterization of Putative Integron-Like Elements of the Heavy-Metal-Hypertolerant Strains of Pseudomonas spp.

Pseudomonas strains isolated from the heavily contaminated Lubin copper mine and Zelazny Most post-flotation waste reservoir in Poland were screened for the presence of integrons. This analysis revealed that two strains carried homologous DNA regions composed of a gene encoding a DNA_BRE_C domain-containing tyrosine recombinase (with no significant sequence similarity to other integrases of integrons) plus a three-component array of putative integron gene cassettes. The predicted gene cassettes encode three putative polypeptides with homology to (i) transmembrane proteins, (ii) GCN5 family acetyltransferases, and (iii) hypothetical proteins of unknown function (homologous proteins are encoded by the gene cassettes of several class 1 integrons). Comparative sequence analyses identified three structural variants of these novel integron-like elements within the sequenced bacterial genomes. Analysis of their distribution revealed that they are found exclusively in strains of the genus Pseudomonas.

Identification of miniature plasmids in psychrophilic Arctic bacteria of the genus Variovorax.

The Svalbard archipelago (Spitsbergen Island) is the northernmost landmass in the European Arctic and has a variety of small- and medium-sized glaciers. The plasmidome of eleven psychrophilic strains of Variovorax spp. isolated from the ice surface of Hans and Werenskiold Glaciers of Spitsbergen Island, was defined. This analysis revealed the presence of six plasmids whose nucleotide sequences have been determined. Four of them, exhibiting high reciprocal sequence similarity, possess unique structures, since their genomes lack any recognized genes. These miniature replicons, not exceeding 1 kb in size, include pHW69V1 (746 bp), which is the smallest autonomous replicon so far identified in free-living bacteria. The miniature plasmids share no similarity with known sequences present in the databases. In silico and experimental analyses identified conserved DNA regions essential for the initiation of replication of these replicons.

Sequence determination and analysis of three plasmids of Pseudomonas sp. GLE121, a psychrophile isolated from surface ice of Ecology Glacier (Antarctica).

Pseudomonas sp. GLE121 (a psychrophilic Antarctic strain) carries three plasmids: pGLE121P1 (6899 bp), pGLE121P2 (8330 bp) and pGLE121P3 (39,583 bp). Plasmids pGLE121P1 and pGLE121P2 show significant sequence similarity to members of the IncP-9 and IncP-7 incompatibility groups, respectively, while the largest replicon, pGLE121P3, is highly related to plasmid pNCPPB880-40 of Pseudomonas syringae pathovar tomato NCPPB880. All three plasmids have a narrow host range, limited to members of the genus Pseudomonas. Plasmid pGLE121P3 encodes a conjugal transfer system, while pGLE121P1 carries only a putative MOB module, conserved in many mobilizable plasmids. Plasmid pGLE121P3 contains an additional load of genetic information, including a pair of genes with homology to the rulAB operon, responsible for ultraviolet radiation (UVR) tolerance. Given the increasing UV exposure in Antarctic regions, the expression of these genes is likely to be an important adaptive response.