Identification and classification of marine bacteria associated with poly(ethylene terephthalate) degradation

Poly(ethylene terephthalate) (PET) is a manmade synthetic polymer that has been widely used in a variety of industries. The high production and usage of PET, together with the inappropriate handling of PET wastes are becoming a global environmental issue, especially in the marine environment. Detailed taxonomic investigations of marine bacteria that have the potential to degrade PET were the focus of this thesis. Taxonomic studies based on comparative phylogenetic and genomic analyses, traditional physiological and biochemical studies, together with newly developed modern taxonomic tools, namely multi-locus sequence analysis and MALDI-TOF MS revealed three novel bacterial species, Alteromonas australica H17T, Marinobacter similis A3d10T and Marinobacter salarius R9SW1T, in which M. similis A3d10T has shown to have the ability to degrade the PET trimer in this study

Antibacterial properties of multi-walled carbon nanotube-silver nanoparticles composite thin films

Composite thin films containing multi-walled carbon nanotube (MWCNT) and electro-deposited silver nanoparticles (Ag NPs) with varied size and density were prepared. The antimicrobial properties of the thin films were evaluated against two Staphylococcus aureus strains. Bacterial suspensions were put in contact with the MWCNT/Ag NP films and NBT assay was used to determine the antibacterial effectiveness of the surfaces following incubation. A significant reduction in the number of viable cells of both microorganisms was observed compared to bare glass and MWCNT surface. The results also showed that the density of Ag NPs had an effect on the antibacterial activity of MWCNT/Ag NP films

Alteromonas australica sp. nov., a poly(ethylene terephthalate) (PET) degrading bacterium, isolated from the Tasman Sea, Pacific Ocean, Victoria, Australia

The first volume of Bergey's Manual of Systematic Bacteriology (1984) described only one genus and one species of Gram-negative aerobic heterotrophic marine bacteria with one polar flagellum, Alteromonas Baumann, Baumann, Mandel, and Allen 1972. During its dynamic and turbulent taxonomic history, the genus underwent detailed investigations and currently comprises 9 recognised species: Alteromonas macleodii (Baumann et al., 1972), A. marina (Yoon et al., 2003), A. stellipolaris (Van Trappen et al., 2004), A. litorea (Yoon et al., 2004), A. hispanica (Martinez-Checa et al., 2005), A. addita (Ivanova et al., 2005), A. simiduii, A. tagae (Chiu et al., 2007) and A. genovensis (Vandecandelaere et al., 2008). In this study, a new species belonging to the genus Alteromonas is presented. This bacterium was isolated from a seawater sample collected from Port Philip Bay (the Tasman Sea, Pacific Ocean) in the course of a taxonomic survey of free-living microbial populations capable of metabolising poly (ethylene terephthalate) (PET) reported by Webb et al. (2009). The new bacterium, designated as strain H17T, exhibited characteristics typical for the genus, including phenotypic, chemotaxonomic (phospholipids and cellular fatty acids; the latter had the high proportion of 16:

Development of multilocus sequence analysis (MLSA) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for Alteromonas species classification

Identification and classification of novel bacterial species is traditionally based on the polyphasic approach relying on phenotypic (e.g., growth and morphology), chemotaxonomic (e.g. mol% G + C composition, lipid and fatty acid composition), genotypic (e.g., chromosomal DNA - DNA hybridization (DDH)), and phylogenetic (e.g., 16S rRNA gene sequence analysis) characteristics. With fast growing numbers of newly described bacteria and correspondent sequencing data it has become apparent that the 16S rRNA gene sequence analysis may not be sufficient for species discrimination; the commonly used threshold value of 97% 16S rRNA gene sequence similarity was found to be not satisfactory as some distinct species share up to 99.9% of their 16S rRNA gene sequence similarity. Other molecular approaches which may be adopted in bacterial systematics are currently under intensive exploration in an attempt to improve efficiency and accuracy of the identification of novel species. Recently, multilocus sequence analysis (MLSA) has been introduced into bacterial systematics, in which multiple housekeeping genes (usually 5 to 7) are assessed as a group for discrimination of phylogenetically close bacteria. MLSA has been shown to have better taxonomic resolution for classification of closely related bacteria on the species level. Another technique, matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS), has been shown to be reliable, easy to perform, cost effective, can be used across different conditions, and may even be used for subspecies discrimination. This study aimed to take the advantages of MLSA and MALDI-TOF MS for the identification of Alteromonas-related bacteria. A collection of marine bacteria belonging to the genus Alteromonas, as confirmed by the 16S rRNA gene sequence analysis, was isolated from sea water samples collected in Port Philip Bay (the Tasman Sea, Pacific Ocean). Bacterial identification based on the polyphasic approach indicated that some of these bacteria may be novel species. In order to evaluate the applicability of MLSA and MALDI-TOF MS for Alteromonas-related bacteria identification, a comparative study of newly isolated bacteria and other validly described species from phylogenetically close genera was performed. In the MLSA study, a group of housekeeping genes (gyrB, rpoB, dnaK, sucC, glyA, pmg, and metG) were amplified using previously and newly designed oligonucleotide primers. For mass spectral analysis, individual colonies were extracted and transferred directly onto the MALDI target plate. Samples were then introduced into the mass spectrometer and results were generated by the MALDI Biotyper software. The results obtained in this study indicated that both molecular techniques generated reliable data useful for Alteromonas species identification as alternative to DDH

Updating the taxonomic toolbox: classification of Alteromonas spp. using multilocus phylogenetic analysis and MALDI-TOF mass spectrometry

Bacteria of the genus Alteromonas are Gram-negative, strictly aerobic, motile, heterotrophic marine bacteria known for their versatile metabolic activities. Identification and classification of novel species belonging to the genus Alteromonas generally involves DNA-DNA hybridization (DDH) as distinct species often fail to be resolved at the 97% threshold value of the 16S rRNA gene sequence similarity. In this study, the applicability of Multilocus Phylogenetic Analysis (MLPA) and Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for the differentiation of Alteromonas species has been evaluated. Phylogenetic analysis incorporating five house-keeping genes (dnaK, sucC, rpoB, gyrB, and rpoD) revealed a threshold value of 98.9% that could be considered as the species cut-off value for the delineation of Alteromonas spp. MALDI-TOF MS data analysis reconfirmed the Alteromonas species clustering. MLPA and MALDI-TOF MS both generated data that were comparable to that of the 16S rRNA gene sequence analysis and may be considered as useful complementary techniques for the description of new Alteromonas species

Nova religio : NR ; the journal of alternative and emergent religions

Two non-pigmented, motile, Gram-negative marine bacteria designated R9SW1T and A3d10T were isolated from sea water samples collected from Chazhma Bay, Gulf of Peter the Great, Sea of Japan, Pacific Ocean, Russia and St. Kilda Beach, Port Phillip Bay, the Tasman Sea, Pacific Ocean, respectively. Both organisms were found to grow between 4 °C and 40 °C, between pH 6 to 9, and are moderately halophilic, tolerating up to 20% (w/v) NaCl. Both strains were found to be able to degrade Tween 40 and 80, but only strain R9SW1T was found to be able to degrade starch. The major fatty acids were characteristic for the genus Marinobacter including C16:0, C16:1ω7c, C18:1ω9c and C18:1ω7c. The G+C content of the DNA for strains R9SW1T and A3d10T were determined to be 57.1 mol% and 57.6 mol%, respectively. The two new strains share 97.6% of their 16S rRNA gene sequences, with 82.3% similarity in the average nucleotide identity (ANI), 19.8% similarity in the in silico genome-to-genome distance (GGD), 68.1% similarity in the average amino acid identity (AAI) of all conserved protein-coding genes, and 31 of the Karlin's genomic signature dissimilarity. A phylogenetic analysis showed that R9SW1T clusters with M. algicola DG893T sharing 99.40%, and A3d10T clusters with M. sediminum R65T sharing 99.53% of 16S rRNA gene sequence similarities. The results of the genomic and polyphasic taxonomic study, including genomic, genetic, phenotypic, chemotaxonomic and phylogenetic analyses based on the 16S rRNA, gyrB and rpoD gene sequence similarities, the analysis of the protein profiles generated using MALDI-TOF mass spectrometry, and DNA-DNA relatedness data, indicated that strains R9SW1T and A3d10(T) represent two novel species of the genus Marinobacter. The names Marinobacter salarius sp. nov., with the type strain R9SW1(T) ( =  LMG 27497(T)  =  JCM 19399(T)  =  CIP 110588(T)  =  KMM 7502(T)) and Marinobacter similis sp. nov., with the type strain A3d10(T) ( =  JCM 19398(T)  =  CIP 110589(T)  =  KMM 7501T), are proposed

The genomic signatures between strains R9SW1^T, A3d10^T, <i>M. adhaerens</i> HP15^T and <i>M. hydrocarbonoclasticus</i> ATCC 49840^T.

<p>Data in the lower triangular corresponds to ANI/AAI (%) and data in the upper triangular corresponds to GGD (%)/Karlin signature.</p><p>The genomic signatures between strains R9SW1^T, A3d10^T, <i>M. adhaerens</i> HP15^T and <i>M. hydrocarbonoclasticus</i> ATCC 49840^T.</p

Differential characteristics between strains R9SW1^T, A3d10^T, their close phylogenetic neighbors and type species of the genus <i>Marinobacter</i>.

<p>Strains: 1, strain R9SW1^T; 2, <i>M. algicola</i> LMG 23835^T; 3, strain A3d10^T; 4, <i>M. sediminum</i> LMG 23833^T; 5, <i>M. salsuginis</i> CIP 109893^T; 6, <i>M. adhaerens</i> CIP 110141^T; 7, <i>M. flavimaris</i> CIP 108615^T; 8, <i>M. lipolyticus</i> SM19^T; 9, <i>M. gudaonensis</i> SL014B61A^T; 10, <i>M. goseongensis</i> En6^T; 11, <i>M. xestospongiae</i> UST090418-1611^T; 12, <i>M. guineae</i> M3B^T; 13, <i>M. hydrocarbonoclasticus</i> SP.17^T.</p><p>Data for nitrate and nitrite reduction, starch hydrolysis, fermentation, indole and acid production, organic substrates utilisation, and enzyme activities for strains R9SW1^T, <i>M. algicola</i> LMG 23835^T, A3d10^T, <i>M. sediminum</i> LMG 23833^T, <i>M. salsuginis</i> CIP 109893^T, <i>M. adhaerens</i> CIP 110141^T, <i>M. flavimaris</i> CIP 108615^T and <i>M. hydrocarbonoclasticus</i> SP. 17^T are from this study. The data in brackets are from previously published work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106514#pone.0106514-Gauthier1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106514#pone.0106514-Green1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106514#pone.0106514-Romanenko1" target="_blank">[62]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106514#pone.0106514-Montes1" target="_blank">[70]</a>.</p><p>+, Positive; -, Negative; w, Weak reaction; ND, No Data available.</p><p>Differential characteristics between strains R9SW1^T, A3d10^T, their close phylogenetic neighbors and type species of the genus <i>Marinobacter</i>.</p