Stanislava Králová

Red-pink pigmented Hymenobacter coccineus sp. nov., Hymenobacter lapidarius sp. nov. and Hymenobacter glacialis sp. nov., isolated from rocks in Antarctica

Four rod-shaped and Gram-stain-negative bacterial strains, CCM 8647, CCM 8649T, CCM 8643T and CCM 8648T, were isolated from rock samples collected on James Ross Island, Antarctica. Extensive biotyping, fatty acid profiling, chemotaxonomy, 16S rRNA gene sequencing and whole-genome sequencing was applied to isolates to clarify their taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that all four isolates belonged to the genus Hymenobacter. Strains CCM 8649T and CCM 8647 were most closely related to Hymenobacter arizonensis OR362-8T (94.4 % 16S rRNA gene sequence similarity), strain CCM 8643T to Hymenobacter terrae DG7AT (96.3 %) and strain CCM 8648T to Hymenobacter glaciei VUG-A130T (96.3 %). The predominant fatty acids of CCM 8649T and CCM 8647 were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C16 : 1ω5c and iso-C15 : 0, whereas those of CCM 8643T and CCM 8648T were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and C16 : 1ω5c. The quinone systems contained exclusively menaquinone MK-7. The major polyamine was sym-homospermidine. All four strains contained the major polar lipid phosphatidylethanolamine. The G+C content of genomic DNA ranged from 60-63 mol%. Whole-genome sequencing data supported the finding that isolates represented distinct species of the genus Hymenobacter. On the basis of the results obtained, three novel species are proposed for which the names Hymenobacter coccineus sp. nov., Hymenobacter lapidarius sp. nov. and Hymenobacter glacialis sp. nov. are suggested, with the type strains CCM 8649T (=LMG 29441T=P5239T), CCM 8643T (=LMG 29435T=P3150T) and CCM 8648T (=LMG 29440T=P5086T), respectively.

Classification of strain CCM 4446T as Rhodococcus degradans sp. nov.

Strain CCM 4446T, with notable biodegradation capabilities, was investigated in this study in order to elucidate its taxonomic position. Chemotaxonomic analyses of quinones, polar lipids, mycolic acids, polyamines and the diamino acid of the cell-wall peptidoglycan corresponded with characteristics of the genus Rhodococcus. Phylogenetic analysis, based on the 16S rRNA gene sequence, assigned strain CCM 4446T to the genus Rhodococcus and placed it in the Rhodococcus erythropolis 16S rRNA gene clade. Further analysis of catA and gyrB gene sequences, automated ribotyping with EcoRI restriction endonuclease, whole-cell protein profiling, DNA-DNA hybridization and extensive biotyping enabled differentiation of strain CCM 4446T from all phylogenetically closely related species, i.e., Rhodococcus baikonurensis, Rhodococcus qingshengii, Rhodococcus erythropolis and Rhodococcus globerulus. The results obtained show that the strain investigated represents a novel species within the genus Rhodococcus, for which the name Rhodococcus degradans sp. nov., is proposed. The type strain is CCM 4446T ( = LMG 28633T).

Mucilaginibacter terrae sp. nov., isolated from Antarctic soil

A bacterial strain designated CCM 8645T was isolated from a soil sample collected nearby a mummified seal carcass in the northern part of James Ross Island, Antarctica. The cells were short rods, Gram-stain-negative, non-motile, catalase and oxidase positive, and produced a red-pink pigment on R2A agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, extensive biotyping using conventional tests and commercial identification kits and chemotaxonomic analyses were applied to clarify its taxonomic position. Phylogenetic analysis based on the 16S rRNA gene placed strain CCM 8645T in the genus Mucilaginibacter with the closest relative being Mucilaginibacter daejeonensis Jip 10T, exhibiting 96.5 % 16S rRNA pairwise similarity which was clearly below the 97 % threshold value recommended for species demarcation. The major components in fatty acid profiles were Summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C15 : 0 iso and C17 : 0 iso 3OH. The cellular quinone content was exclusively menaquinone MK-7. The major polyamine was sym-homospermidine and predominant polar lipids were phosphatidylethanolamine and phosphatidylserine. Based on presented results, we propose a novel species for which the name Mucilaginibacter terrae sp. nov. is suggested, with the type strain CCM 8645T (=LMG 29437T).

Pedobacter jamesrossensis sp. nov., Pedobacter lithocola sp. nov., Pedobacter mendelii sp. nov. and Pedobacter petrophilus sp. nov., isolated from the Antarctic environment

A taxonomic study performed on 17 Gram-stain-negative rod-shaped bacterial strains originating from the Antarctic environment is described. Initial phylogenetic analysis using 16S rRNA gene sequencing differentiated the strains into four groups belonging to the genus Pedobacter but they were separated from all hitherto described Pedobacter species. Group I (n=8) was closest to Pedobacter aquatilis (97.8 % 16S rRNA gene sequence similarity). Group II (n=2) and group III (n=4) were closely related (98.8 % 16S rRNA gene sequence similarity) and had Pedobacter jejuensis as their common nearest neighbour. Group IV (n=3) was distantly delineated from the remaining Pedobacter species. Differentiation of the analysed strains into four clusters was further confirmed by repetitive sequence-based PCR fingerprinting, ribotyping, DNA-DNA hybridization and phenotypic traits. Common to representative strains for the four groups were the presence of major menaquinone MK-7, sym-homospermidine as the major polyamine, phosphatidylethanolamine, two unidentified lipids (L2, L5) and an unidentified aminolipid (AL2) as the major polar lipids, presence of an alkali-stable lipid, and C16:1ω7c/C16:1ω6c (summed feature 3), iso-C15:0 and iso-C 17:0 3-OH as the major fatty acids, which corresponded to characteristics of the genus Pedobacter. The obtained results showed that the strains analysed represent four novel species of the genus Pedobacter, for which the names Pedobacter jamesrossensis sp. nov. (type strain CCM 8689T=LMG 29684T), Pedobacter lithocola sp. nov. (CCM 8691T=LMG 29685T), Pedobacter mendelii sp. nov. (CCM 8685T=LMG 29688T) and Pedobacter petrophilus sp. nov. (CCM 8687T=LMG 29686T) are proposed.

Role of fatty acids in cold adaptation of Antarctic psychrophilic Flavobacterium spp.

Cold-loving microorganisms developed numerous adaptation mechanisms allowing them to survive in extremely cold habitats, such as adaptation of the cell membrane. The focus of this study was on the membrane fatty acids of Antarctic Flavobacterium spp., and their adaptation response to cold-stress. Fatty acids and cold-response of Antarctic flavobacteria was also compared to mesophilic and thermophilic members of the genus Flavobacterium. The results showed that the psychrophiles produced more types of major fatty acids than meso- and thermophilic members of this genus, namely C15:1 iso G, C15:0 iso, C15:0 anteiso, C15:1ω6c, C15:0 iso 3OH, C17:1ω6c, C16:0 iso 3OH and C17:0 iso 3OH, summed features 3 (C16:1ω7cand/or C16:1ω6c) and 9 (C16:0 10-methyl and/or C17:1 iso ω9c). It was shown that the cell membrane of psychrophiles was composed mainly of branched and unsaturated fatty acids. The results also implied that Antarctic flavobacteria mainly used two mechanisms of membrane fluidity alteration in their cold-adaptive response. The first mechanism was based on unsaturation of fatty acids, and the second mechanism on de novo synthesis of branched fatty acids. The alteration of the cell membrane was shown to be similar for all thermotypes of members of the genus Flavobacterium.

Pedobacter psychrophilus sp. nov., isolated from fragmentary rock

Strain P4487AT was isolated during investigation of cultivable bacterial populations of environmental materials sampled at James Ross Island, Antarctica. It revealed Gram-stain-negative short rod-shaped cells producing a pink pigment. Phylogenetic analysis based on 16S rRNA gene sequences allocated strain P4487AT to the genus Pedobacter but showed that the strain represents a distinct intrageneric phylogenetic lineage clearly separated from remaining Pedobacter species. Phylogenetically, strain P4487AT formed a common branch with the Pedobacter arcticus and Pedobacter lignilitoris cluster while the highest value of 94.4 % 16S rRNA gene sequence similarity suggested that Pedobacter lentus is the most closely related species. Biochemical and physiological test results enabled the differentiation of strain P4487AT from all phylogenetically closely related species. Chemotaxonomic analyses of strain P4487AT showed MK-7 as the respiratory menaquinone, sym-homospermidine as the major polyamine, phosphatidylethanolamine and two unidentified lipids as the major polar lipids, presence of sphingolipids, and C16 : 1ω7c/C16 : 1ω6c (summed feature 3), iso-C15 : 0 and iso-C17 : 0 3-OH as the major fatty acids, all of which corresponded with characteristics of the genus Pedobacter. The results showed that strain P4487AT represents a novel species within the genus Pedobacter, for which the name Pedobacter psychrophilus sp. nov. is proposed. The type strain is P4487AT (=CCM 8644T=LMG 29436T).

Staphylococcus edaphicus sp. nov., Isolated in Antarctica, Harbors the mecC Gene and Genomic Islands with a Suspected Role in Adaptation to Extreme Environments

ABSTRACT Two Gram-stain-positive, coagulase-negative staphylococcal strains were isolated from abiotic sources comprising stone fragments and sandy soil in James Ross Island, Antarctica. Here, we describe properties of a novel species of the genus Staphylococcus that has a 16S rRNA gene sequence nearly identical to that of Staphylococcus saprophyticus. However, compared to S. saprophyticus and the next closest relatives, the new species demonstrates considerable phylogenetic distance at the whole-genome level, with an average nucleotide identity of <85% and inferred DNA-DNA hybridization of <30%. It forms a separate branch in the S. saprophyticus phylogenetic clade as confirmed by multilocus sequence analysis of six housekeeping genes, rpoB, hsp60, tuf, dnaJ, gap, and sod. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and key biochemical characteristics allowed these bacteria to be distinguished from their nearest phylogenetic neighbors. In contrast to S. saprophyticus subsp. saprophyticus, the novel strains are pyrrolidonyl arylamidase and β-glucuronidase positive and β-galactosidase negative, nitrate is reduced, and acid produced aerobically from d-mannose. Whole-genome sequencing of the 2.69-Mb large chromosome revealed the presence of a number of mobile genetic elements, including the 27-kb pseudo-staphylococcus cassette chromosome mec of strain P5085T (ψSCCmecP5085), harboring the mecC gene, two composite phage-inducible chromosomal islands probably essential to adaptation to extreme environments, and one complete and one defective prophage. Both strains are resistant to penicillin G, ampicillin, ceftazidime, methicillin, cefoxitin, and fosfomycin. We hypothesize that antibiotic resistance might represent an evolutionary advantage against beta-lactam producers, which are common in a polar environment. Based on these results, a novel species of the genus Staphylococcus is described and named Staphylococcus edaphicus sp. nov. The type strain is P5085T (= CCM 8730T = DSM 104441T). IMPORTANCE The description of Staphylococcus edaphicus sp. nov. enables the comparison of multidrug-resistant staphylococci from human and veterinary sources evolved in the globalized world to their geographically distant relative from the extreme Antarctic environment. Although this new species was not exposed to the pressure of antibiotic treatment in human or veterinary practice, mobile genetic elements carrying antimicrobial resistance genes were found in the genome. The genomic characteristics presented here elucidate the evolutionary relationships in the Staphylococcus genus with a special focus on antimicrobial resistance, pathogenicity, and survival traits. Genes encoded on mobile genetic elements were arranged in unique combinations but retained conserved locations for the integration of mobile genetic elements. These findings point to enormous plasticity of the staphylococcal pangenome, shaped by horizontal gene transfer. Thus, S. edaphicus can act not only as a reservoir of antibiotic resistance in a natural environment but also as a mediator for the spread and evolution of resistance genes.

Rufibacter ruber sp. nov., isolated from fragmentary rock

A red-pigmented, Gram-stain-negative, rod-shaped, aerobic bacterium, designated strain CCM 8646T, was isolated from stone fragments in James Ross Island, Antarctica. Strain CCM 8646T was able to grow from 10 to 40 °C, in the presence of up to 1 % (w/v) NaCl and at pH 7.0-11.0. Analysis of the 16S rRNA gene sequence placed strain CCM 8646T in the genus Rufibacter with the closest relative being Rufibacter roseus H359T (97.07 % 16S rRNA gene sequence similarity). The digital DNA-DNA hybridization values between strain CCM 8646T and R. roseus H359T were low (21.30±2.34 %). The major quinone was menaquinone MK-7. The polar lipids comprised phosphatidylethanolamine, an unknown aminoglycolipid and six unknown polar lipids. The G+C content of strain CCM 8646T was 51.54 mol%. On the basis of phenotypic, chemotaxonomic and genotyping results, strain CCM 8646T is considered to represent a novel species within the genus Rufibacter, for which the name Rufibacter ruber sp. nov. is proposed. The type strain is CCM 8646T (=LMG 29438T).

Flavobacterium chryseum sp. nov. and Flavobacterium psychroterrae sp. nov., novel environmental bacteria isolated from Antarctica

A group of rod-shaped, aerobic, Gram-stain-negative, gliding bacteria producing flexirubin-type pigment was isolated from environmental samples collected in Antarctica in 2009-2014. Phylogenetic analysis of the almost complete 16S rRNA gene sequences revealed two separated branches belonging to the genus Flavobacterium. Group I (n=8), represented by strain CCM 8826T, shared the highest sequence similarity to Flavobacterium collinsii 983-08T (98.8 %) and Flavobacterium saccharophilum DSM 1811T (98.4 %), and group II (n=4) represented by strain CCM 8827T shared the highest similarity to Flavobacterium aquidurense WB 1.1-56T (99.6 %). High genetic homogeneity of both groups, separation from each other and from phylogenetically close Flavobacterium species was verified by the rep-PCR fingerprinting method. DNA-DNA hybridization confirmed low genomic relatedness between strain CCM 8826T and F. collinsii 983-08T and F. saccharophilum DSM 1811T (18 and 28 %, respectively) and between strain CCM 8827T and F. aquidurense WB 1.1-56T (27 %). Chemotaxonomic analyses of strains CCM 8826T and CCM 8827T revealed the respiratory quinone to be MK-6, the major identified polar lipid was phosphatidylethanolamine and the predominant polyamine was sym-homospermidine. The common major fatty acids were C15 : 0 iso, C17 : 0 iso 3OH, C15 : 1 iso G, Summed Feature 3 (C16 : 1ω7c/C16 : 1ω6c), C15 : 0 iso 3OH and additionally, C15 : 0 anteiso among group II members. All analyses confirmed that strains of group I and II represent two novel species of the genus Flavobacterium, for which the names Flavobacterium chryseum sp. nov. (type strain CCM 8826T=P3160T=LMG 30615T) and Flavobacterium psychroterrae sp. nov. (type strain CCM 8827T=P3922T=LMG 30616T) are proposed.