Structure and biosynthesis of carotenoids produced by a novel Planococcus sp. isolated from South Africa.

BACKGROUND: The genus Planococcus is comprised of halophilic bacteria generally reported for the production of carotenoid pigments and biosurfactants. In previous work, we showed that the culturing of the orange-pigmented Planococcus sp. CP5-4 isolate increased the evaporation rate of industrial wastewater brine effluent, which we attributed to the orange pigment. This demonstrated the potential application of this bacterium for industrial brine effluent management in evaporation ponds for inland desalination plants. Here we identified a C30-carotenoid biosynthetic gene cluster responsible for pigment biosynthesis in Planococcus sp. CP5-4 through isolation of mutants and genome sequencing. We further compare the core genes of the carotenoid biosynthetic gene clusters identified from different Planococcus species' genomes which grouped into gene cluster families containing BGCs linked to different carotenoid product chemotypes. Lastly, LC-MS analysis of saponified and unsaponified pigment extracts obtained from cultures of Planococcus sp. CP5-4, revealed the structure of the main (predominant) glucosylated C30-carotenoid fatty acid ester produced by Planococcus sp. CP5-4. RESULTS: Genome sequence comparisons of isolated mutant strains of Planococcus sp. CP5-4 showed deletions of 146 Kb and 3 Kb for the non-pigmented and "yellow" mutants respectively. Eight candidate genes, likely responsible for C30-carotenoid biosynthesis, were identified on the wild-type genome region corresponding to the deleted segment in the non-pigmented mutant. Six of the eight candidate genes formed a biosynthetic gene cluster. A truncation of crtP was responsible for the "yellow" mutant phenotype. Genome annotation revealed that the genes encoded 4,4'-diapolycopene oxygenase (CrtNb), 4,4'- diapolycopen-4-al dehydrogenase (CrtNc), 4,4'-diapophytoene desaturase (CrtN), 4,4'- diaponeurosporene oxygenase (CrtP), glycerol acyltransferase (Agpat), family 2 glucosyl transferase 2 (Gtf2), phytoene/squalene synthase (CrtM), and cytochrome P450 hydroxylase enzymes. Carotenoid analysis showed that a glucosylated C30-carotenoid fatty acid ester, methyl 5-(6-C17:3)-glucosyl-5, 6'-dihydro-apo-4, 4'-lycopenoate was the main carotenoid compound produced by Planococcus sp. CP5-4. CONCLUSION: We identified and characterized the carotenoid biosynthetic gene cluster and the C30-carotenoid compound produced by Planococcus sp. CP5-4. Mass-spectrometry guided analysis of the saponified and unsaponified pigment extracts showed that methyl 5-glucosyl-5, 6-dihydro-apo-4, 4'-lycopenoate esterified to heptadecatrienoic acid (C17:3). Furthermore, through phylogenetic analysis of the core carotenoid BGCs of Planococcus species we show that various C30-carotenoid product chemotypes, apart from methyl 5-glucosyl-5, 6-dihydro-apo-4, 4'-lycopenoate and 5-glucosyl-4, 4-diaponeurosporen-4'-ol-4-oic acid, may be produced that could offer opportunities for a variety of applications.

Whole genome sequencing and comparative genomic analyses of Planococcus alpniumensis MSAK28401T, a new species isolated from Antarctic krill.

BACKGROUND: Extremophiles have attracted much attention in the last few decades, as they possess different properties by producing certain useful metabolites. However, the secondary metabolism of the extremophiles of Antarctic krill has received little attention. RESULTS: In this study, a new bacterial strain MSAK28401T from Antarctic krill was isolated and identified. The results of analysis on phenotypic, chemotaxonomic, and genomic characteristics showed that the strain MSAK28401T belongs to the genus Planococcus. Cells of this strain were coccoid (0.89-1.05 µm) and aerobic. The majority of the fatty acid content was C15:0 anteiso (37.67 ± 0.90%) followed by C16:1 ω7c alcohol (10.37 ± 1.22%) and C16:0 iso (9.36 ± 0.71%). The calculated average nucleotide identity and DNA-DNA hybridization values between the strain MSAK28401T and type strains P. citreus DSM 20549T and P. rifietoensis M8T were lower than 91 and 70%, respectively. The strain MSAK28401T (=KCTC 43283T and MCCC 1k05448T) represented a new member of the genus Planococcus and was named P. alpniumensis sp. nov. Moreover, genes involved in the degradation of aromatic compounds (e.g., salicylate, gentisate, and quinate) were found in the genome, implying that strain MSAK28401T has an aromatic compound as its potential metabolite. This work will help us understand the genomic characteristics and potential metabolic pathway of Planococcus from Antarctic krill. CONCLUSIONS: This study reported the genomic information and phenotypic characteristics of the new strain P. alpniumensis MSAK28401T isolated from Antarctic krill, and provided the genome information of Planococcus strains for further studying the function roles in aromatic compound metabolism.

Genome-Based Insights into the Production of Carotenoids by Antarctic Bacteria, Planococcus sp. ANT_H30 and Rhodococcus sp. ANT_H53B.

Antarctic regions are characterized by low temperatures and strong UV radiation. This harsh environment is inhabited by psychrophilic and psychrotolerant organisms, which have developed several adaptive features. In this study, we analyzed two Antarctic bacterial strains, Planococcus sp. ANT_H30 and Rhodococcus sp. ANT_H53B. The physiological analysis of these strains revealed their potential to produce various biotechnologically valuable secondary metabolites, including surfactants, siderophores, and orange pigments. The genomic characterization of ANT_H30 and ANT_H53B allowed the identification of genes responsible for the production of carotenoids and the in silico reconstruction of the pigment biosynthesis pathways. The complex manual annotation of the bacterial genomes revealed the metabolic potential to degrade a wide variety of compounds, including xenobiotics and waste materials. Carotenoids produced by these bacteria were analyzed chromatographically, and we proved their activity as scavengers of free radicals. The quantity of crude carotenoid extracts produced at two temperatures using various media was also determined. This was a step toward the optimization of carotenoid production by Antarctic bacteria on a larger scale.

Genomic insights into the salt tolerance and cold adaptation of Planococcus halotolerans SCU63T.

Planococcus halotolerans, recently described as a novel species with SCU63T as the type strain, is capable of thriving in up to 15% NaCl and temperatures as low as 0 °C. To better understand its adaptation strategies at the genomic level, strain SCU63T was subjected to whole-genome sequencing and data mining. The high-quality assembly yielded 17 scaffolds with a genome size of 3,622,698 bp. Its genome harbors 3683 protein-coding sequences and 127 RNA genes, as well as three biosynthetic gene clusters and 25 genomic islands. The phylogenomic tree provided compelling insights into the evolutionary relationships of Planococcus. Comparative genomic analysis revealed key similarities and differences in the functional gene categories among Planococcus species. Strain SCU63T was shown to have diverse stress response systems for high salt and cold habitats. Further comparison with three related species showed the presence of numerous unique gene clusters in the SCU63T genome. The strain might serve as a good model for using extremozymes in various biotechnological processes.

Planococcus lenghuensis sp. nov., an oil-degrading bacterium isolated from petroleum-contaminated soil.

A Gram-staining-positive and aerobic coccus with the ability to degrade petroleum bacterium, designated Y42T, was isolated from the Lenghu oil field located in the northern margin of the Qaidam Basin. Phylogenetic and signature nucleotides analyses revealed that strain Y42T belongs to the genus Planococcus. The multiple sequence alignments of 16S rRNA and housekeeping genes showed that strain Y42T formed a distinct lineage with the other Planococcus clade. The average nucleotide identity (ANI) and DNA-DNA hybridization values (DDH) between strain Y42T and the reference strains were 69.5-70.1 and 19.4-21.7%, respectively, which values were below the threshold for species delineation. The major fatty acids of strain Y42T were anteiso-C15:0. The respiratory quinone was MK-7 (71.8%) as the predominant menaquinone followed the MK-6 (28.2%) and the cell-wall hydrolysates contained LL-diaminopimelic acid. The polar lipid was composed of diphosphatidyl glycerol, phosphatidyl glycerol, phosphoglycolipid, aminophospholipid and four unidentified lipids. The peptidoglycan type was A4α (L-Lys-D-Glu). The strain Y42T possessed larger genome (approximately 4 MB) and revealed obvious differences for the abundance of the COG categories compared with the other Planococcus bacteria. Also, the strain Y42T also possessed more unique orthologous proteins. The structural characteristics of the strain Y42T genome provided a competitive advantage for better survival in petroleum-polluted environments. Combined with the 16S rRNA gene and genome sequence, phenotypic as well as chemotaxonomic characterisations, strain Y42T is considered to represent a novel species of the genus Planococcus, for which the name Planococcus lenghuensis sp. nov. be proposed. The type strain is Y42T (= CGMCC 1.15921T = JCM 32719T).

Characteristics of Planococcus antioxidans sp. nov., an antioxidant-producing strain isolated from the desert soil in the Qinghai-Tibetan Plateau.

Strain Y74T was an isolate from the sandy soil in the town of Huatugou, Qinghai-Tibet Plateau, China. An analysis of this strain's phenotypic, chemotaxonomic, and genomic characteristics established the relationship of the isolate with the genus Planococcus. Strain Y74T was able to grow between 4 and 42°C (with an optimum temperature of 28°C) at pH values of 6-8.5 and in 0%-7% (w/v) NaCl. The dominant quinones were MK-8 and MK-7. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, and an unknown phospholipid. The majority of the fatty acid content was anteiso-C15:0 (28.8%) followed by C16:1 ω7c alcohol (20.9%) and iso-C14:0 (13.4%). The 16S rRNA gene sequence similarity analysis demonstrated a stable branch formed by strain Y74T and Planococcus halotolerans SCU63T (99.66%). The digital DNA-DNA hybridization between these two strains was 57.2%. The G + C content in the DNA of Y74T was 44.5 mol%. In addition, the morphological, physiological, and chemotaxonomic pattern clearly differentiated the isolates from their known relatives. In conclusion, the strain Y74T (=JCM 32826T  = CICC24461T ) represents a novel member of the genus Planococcus, for which the name Planococcus antioxidans sp. nov. is proposed. Strain Y74T was found to have potent antioxidant activity via its hydrogen peroxide tolerance and its 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity. The DPPH radical-scavenging activity was determined to be 40.2 ± 0.7%. The genomic analysis indicated that six peroxidases genes, one superoxide dismutase gene, and one dprA (DNA-protecting protein) are present in the genome of Y74T .

Planococcus halotolerans sp. nov., isolated from a saline soil sample in China.

A novel Gram-stain-positive, coccoid or short rod-shaped, moderate-orange-pigmented, halotolerant and psychrotolerant bacterium, designated strain SCU63T, was isolated from a saline soil sample in China, and characterized by a polyphasic taxonomic approach. 16S rRNA gene sequence similarity of strain SCU63T to species in the genera Planococcus and Planomicrobium ranged from 96.5 to 98.6 %. Phylogenetic trees as well as diagnostic signature nucleotides in the 16S rRNA gene sequence supported the view that this strain should be assigned to the genus Planococcus. Further, average nucleotide identity and digital DNA-DNA hybridization analyses confirmed the separate species status of strain SCU63T relative to the closely related taxa. The isolate grew at 0-40 °C (optimum, 30-35 °C), at pH 6.5-9.0 (pH 7.0-7.5) and in the presence of 0-15 % (w/v) NaCl (3 %). The principal fatty acids were anteiso-C15 : 0, C16 : 1ω7c alcohol, iso-C16 : 0 and iso-C14 : 0, and the dominant isoprenoid quinones were MK-8 and MK-7. The peptidoglycan type was determined to be A4α (l-Lys-d-Glu), and the polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminophospholipid and one unidentified lipid. The DNA G+C content was 44.6 mol%. Based on the genotypic, phenotypic and chemotaxonomic data, strain SCU63T can be classified as a novel species in the genus Planococcus, for which the name Planococcushalotolerans sp. nov. is proposed. The type strain is SCU63T (=CGMCC 1.13628T=KCTC 43001T).

Analysis of Novel Antioxidant Sesquarterpenes (C35 Terpenes) Produced in Recombinant Corynebacterium glutamicum.

Novel synthetic isoprenoids have been synthesized in engineered microbial hosts by evolving terpene synthase or expressing heterologous terpene synthases. Recently, the native operon, crtNaNcM derived from Planococcus sp. PAMC 21323, has isolated for potential industrial applications of C35 carotenoids. For the first time, novel C35 carotenoids (sesquarterpene) were synthesized in Corynebacterium glutamicum expressing the crtNaNcM genes. The recombinant strains accumulate various sesquarterpene including 4-apolycopene (red color), 4-aponeurosporene (yellow color), and no pigmentation, depending on the expression of the genetic elements of the crtNaNcM genes. Subsequently, the carotenoid extract from the cells harboring pCES-H36-CrtNaNcM was analyzed, resulting in significantly higher antioxidant activity than those of other strains harboring pCES-H36-CrtNcM and pCES-H36-CrtNaNc, respectively. This study will promote further engineering of C. glutamicum to increase sesquarterpene productions.

Planococcus salinus sp. nov., a moderately halophilic bacterium isolated from a saline-alkali soil.

A novel aerobic, Gram-stain-positive, motile, moderately halophilic and coccoid bacterial strain, designated LCB217T, was isolated from a saline-alkali soil in north-western China and identified using a polyphasic taxonomic approach. Growth occurred with 3-15 % (w/v) NaCl (optimum 3-5 %), at 10-45 °C (optimum 30 °C) and at pH 7.0-9.0 (optimum pH 9.0). Strain LCB217T contained MK-7 and MK-8 as the predominant menaquinones and anteiso-C15 : 0, iso-C14 : 0 and iso-C16 : 0 as the major fatty acids. The polar lipids from strain LCB217T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, one unidentified phospholipid, one unidentified aminophospholipid and one unidentified lipid. The peptidoglycan type was A4α (l-Lys-d-Glu). Phylogenetic analysis of the 16S rRNA gene sequence showed that strain LCB217T belonged to the genus Planococcus and was closely related to the type strains Planococcus plakortidis AS/ASP6 (II)T (98.2 % similarity), Planococcus maitriensis S1T (97.7 %) and Planococcus salinarum ISL-16T (97.2 %). The G+C content of the genomic DNA was 49.4 mol%. DNA-DNA relatedness values between strain LCB217T andPlanococcusplakortidis AS/ASP6 (II)T, Planococcusmaitriensis S1T andPlanococcussalinarum ISL-16T were 29.5, 38.1 and 39.5 %, respectively. On the basis of the phenotypic, phylogenetic and genomic data, strain LCB217T represents a novel species of the genus Planococcus, for which the name Planococcus salinus sp. nov. is proposed. The type strain is LCB217T (=CGMCC 1.15685T=KCTC 33861T).

Complete genome sequence of Planococcus faecalis AJ003T, the type species of the genus Planococcus and a microbial C30 carotenoid producer.

A novel type strain, Planococcus faecalis AJ003T, isolated from the feces of Antarctic penguins, synthesizes a rare C30 carotenoid, glycosyl-4,4'-diaponeurosporen-4'-ol-4-oic acid. The complete genome of P. faecalis AJ003T comprises a single circular chromosome (3,495,892 bp; 40.9% G + C content). Annotation analysis has revealed 3511 coding DNA sequences and 99 RNAs; seven genes associated with the MEP pathway and five genes involved in the carotenoid pathway have been identified. The functionality and complementation of 4,4'-diapophytoene synthase (CrtM) and two copies of heterologous 4,4'-diapophytoene desaturase (CrtN) involved in carotenoid biosynthesis were analyzed in Escherichia coli.

Planococcus ruber sp. nov., isolated from a polluted farmland soil sample.

A pale-red-pigmented bacterial strain, designated CW1T, was isolated from a polluted soil sample in China and was characterized by a polyphasic taxonomic approach. Strain CW1T was Gram-stain-positive (or variable), coccoid, motile by a single polar flagellum and non-spore-forming. Growth was observed at 15-37 °C, but not at 10 °C or 40 °C, at pH 5.0-9.0 and with 0-5 % NaCl (w/v). Phylogenetic analysis showed that strain CW1T belongs to the genus Planococcus. The 16S rRNA gene sequence similarities between CW1T and the four most closely related type strains, Planococcus antarcticus DSM 14505T, Planococcus halocryophilus DSM 24743T, Planomicrobium soli XN13T and Planomicrobium okeanokoites NBRC 12536T were 97.96, 97.83, 97.83 and 97.82 %, respectively. The whole-cell sugars contained galactose, ribose and glucose. The major respiratory quinone was MK-7 followed by MK-8, and the major fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphoglycolipid. The DNA G+C content was 48.6 mol%. The phylogenetic and chemotaxonomic analyses indicated that strain CW1T belongs unanimously to the genus Planococcus. However, DNA-DNA hybridization showed relatively low relatedness of 21.8 % (DSM 14505T) and 19.6 % (DSM 24743T) with the two most closely related strains of the genus Planococcus. Combined with the genotypic and phenotypic analysis, strain CW1T should represent a novel species in the genus Planococcus, for which the name Planococcus ruber sp. nov. is proposed. The type strain is CW1T (=CCTCC AB 207187T=LMG 24442T).

Complete genome sequence of Planococcus donghaensis JH1T, a pectin-degrading bacterium.

The type strain Planococcus donghaensis JH1T is a psychrotolerant and halotolerant bacterium with starch-degrading ability. Here, we determine the carbon utilization profile of P. donghaensis JH1T and report the first complete genome of the strain. This study revealed the strain's ability to utilize pectin and d-galacturonic acid, and identified genes responsible for degradation of the polysaccharides. The genomic information provided may serve as a fundamental resource for full exploration of the biotechnological potential of P. donghaensis JH1T.

Planococcus versutus sp. nov., isolated from soil.

A taxonomic study was performed on a novel Gram-stain-positive, coccus-shaped, orange-pigmented motile bacterium, designated as strain L10.15T. The organism was isolated from a soil sample collected in Lagoon Island (close to Adelaide Island, western Antarctic Peninsula) using a quorum-quenching enrichment medium. Growth occurred at 4-30 °C, pH 6-11 and at moderately high salinity (0-15 %, w/v, NaCl), with optimal growth at 26 °C, at pH 7-8 and with 6 % (w/v) NaCl. 16S rRNA gene sequence analysis showed that strain L10.15T belonged to the genus Planococcus and was closely related to Planococcus halocryophilus Or1T (99.3 % similarity), Planococcus donghaensis JH1T (99.0 %), Planococcus antarcticus DSM 14505T (98.3 %), Planococcus plakortidis AS/ASP6 (II)T (97.6 %), Planococcus maritimus TF-9T (97.5 %), Planococcus salinarum ISL-6T (97.5 %) and Planococcus kocurii NCIMB 629T (97.5 %). However, the average nucleotide identity-MUMmer analysis showed low genomic relatedness values of 71.1-81.7 % to the type strains of these closely related species of the genus Planococcus. The principal fatty acids were anteiso-C15 : 0, C16 : 1ω7c and anteiso-C17 :  0, and the major menaquinones of strain L10.15T were MK-5 (48 %), MK-6 (6 %) and MK-7 (44 %). Polar lipid analysis revealed the presence of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and aminophospholipid. The DNA G+C content was 39.4 mol%. The phenotypic and genotypic data indicate that strain L10.15T represents a novel species of the genus Planococcus, for which the name Planococcus versutus sp. nov. is proposed. The type strain is L10.15T (=DSM 101994T=KACC 18918T).

De novo assembly of complete genome sequence of Planococcus kocurii ATCC 43650(T), a potential plant growth promoting bacterium.

Planococcus kocurii ATCC 43650(T) is a halotolerant and psychrotolerant bacterium isolated from the skin of a North sea cod. Here, we present the first complete genome and annotation of P. kocurii ATCC 43650(T), identifying its potential as a plant growth promoting bacterium and its capability in the biosynthesis of butanol.

Expression of a functional cold active ß-galactosidase from Planococcus sp-L4 in Pichia pastoris.

Lactase deficiency problem discourages many adults from consuming milk as a major source of micro- and macronutrients. Enzymatic hydrolysis of lactose is an ideal solution for this problem but such processing adds significant costs. In this study, a cold active ß-galactosidase from Planococcus sp-L4 (bgal) was optimized for expression of recombinant "BGalP" in Pichia pastoris. As a result of codon optimization, the codon adaptation index was improved from 0.58 to 0.85 after replacing rare codons. After transformation of two P. pastoris strains (KM71H and GS115), the activity of BGalP enzyme was measured in the culture supernatants using ortho-Nitrophenyl-ß-galactoside (ONPG). Maximal activity was recorded as 3.7U/ml on day 11 in KM71H clone #2 which was 20% higher than the best GS115 clone. Activity measurements under different conditions indicated optimal activity at pH 6.5. It was active at temperatures ranging from 0 to 55°C with deactivation occurring at or above 60°C. Protein analysis of the crude ultra-filtrate showed the enzyme was ∼75kDa and was the major constituent (85%) of the sample. This enzyme have the potential to find utility for the breakdown of lactose in chilled milk and subsequently can be deactivated by pasteurization. The use of BGalP would minimize energy consumption thus decreasing cost and also help to preserve the nutritional elements of the milk.

Planococcus faecalis sp. nov., a carotenoid-producing species isolated from stools of Antarctic penguins.

Taxonomic studies were performed on a novel carotenoid-producing strain, designated AJ003T, isolated from faeces of Antarctic penguins. Cells of strain AJ003T were aerobic, Gram-stain-positive, cocci-shaped and orange. Strain AJ003T was capable of growing in a broad temperature range, including sub-zero growth (below − 20 to 30 °C). 16S rRNA gene sequence analysis revealed that strain AJ003T was closely related to Planococcus halocryophilus Or1T (97.4 % similarity), Planococcus antarcticus DSM 14505T (97.3 %), Planococcus kocurii NCIMB 629T (97.3 %), and Planococcus donghaensis JH1T (97.1 %). The predominant cellular fatty acids were anteiso-C15 : 0, and iso-C16 : 0.MK-7 and MK-8 were the quinones identified, and the major pigment was glycosyl-4,4'-diaponeurosporen-4'-ol-4-oic acid. The major polar lipid was phosphatidylglycerol. DNA­DNA relatedness of strain AJ003T with respect to its closest phylogenetic neighbours was 38.2 ± 0.5 % for Planococcus halocryophilus DSM 24743T, 32.2 ± 0.2 % for Planococcus antarcticus DSM 14505T, 21.0 ± 0.3 % for Planococcus kocurii DSM 20747T and 18.6 ± 1.4 % for Planococcus donghaensis KCTC 13050T. The DNA G+C content of strain AJ003T was 40.0 ± 0.6 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain AJ003T is concluded to represent a novel species of the genus Planococcus, for which the name Planococcus faecalis sp. nov. is proposed. The type strain is AJ003T ( = KCTC 33580T = CECT 8759T).

Planococcus dechangensis sp. nov., a moderately halophilic bacterium isolated from saline and alkaline soils in Dechang Township, Zhaodong City, China.

Strain NEAU-ST10-9(T) is a moderately halophilic, coccoid and non-motile bacterium isolated from saline and alkaline soils in the Dechang Township, Zhaodong City, China. The bacterium was found to be aerobic and Gram-stain positive. It forms orange colonies and grows at NaCl concentrations of 2-10 % (w/v) (optimum, 4 % w/v), at 4-50 °C (optimum, 30 °C) and at pH 6.0-10.0 (optimum, pH 7.0). Phylogenetic analyses based on 16S rRNA gene sequences indicated that it belongs to the genus Planococcus within the family Planococcaceae. The most closely related species was Planococcus maritimus, whose type strain (TF-9(T)) showed gene sequence similarities of 99.1 % for 16S rRNA, 83.7 % for gyrB and 87.0 % for rpoB with those of strain NEAU-ST10-9(T), respectively. DNA-DNA hybridization relatedness values between strain NEAU-ST10-9(T) and type strains P. maritimus DSM 17275(T) , P. rifietoensis DSM 15069(T) , P. plakortidis DSM 23997(T), P. citreus DSM 20549(T), P. maitriensis DSM 15305(T), P. salinarum KCTC 13584(T) and P. columbae DSM 17517(T) were from 55 ± 1 to 32 ± 2 %. The DNA G+C content was found to be 45.2 mol %. The major fatty acids (>5 %) were determined as C15:0 anteiso, C16:1 ω7c alcohol, C17:1 ω9c and C17:0 anteiso. The major menaquinones of strain NEAU-ST10-9(T) were identified as MK-7 and MK-8. The polar lipids were found to contain of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphocholine and two unknown lipids. The genotypic, chemotaxonomic and phenotypic analysis indicated that strain NEAU-ST10-9(T) represents a novel species of the genus Planococcus, for which we proposed the name Planococcus dechangensis sp. nov. The type strain is NEAU-ST10-9(T) (=CGMCC 1.12151(T)=DSM 25871(T)).

Molecular Characterization of a Thermophilic and Salt- and Alkaline-Tolerant Xylanase from Planococcus sp. SL4, a Strain Isolated from the Sediment of a Soda Lake.

To enrich the genetic resource of microbial xylanases with high activity and stability under alkaline conditions, a xylanase gene (xynSL4) was cloned from Planococcus sp. SL4, an alkaline xylanase-producing strain isolated from the sediment of soda lake Dabusu. Deduced XynSL4 consists of a putative signal peptide of 29 residues and a catalytic domain (30-380 residues) of glycosyl hydrolase family 10, and shares the highest identity of 77% with a hypothetical protein from Planomicrobium glaciei CHR43. Phylogenetic analysis indicated that deduced XynSL4 is closely related with thermophilic and alkaline xylanases from Geobacillus and Bacillus species. The gene xynSL4 was expressed heterologously in Escherichia coli and the recombinant enzyme showed some superior properties. Purified recombinant XynSL4 (rXynSL4) was highly active and stable over the neutral and alkaline pH range from 6 to 11, with maximum activity at pH 7 and more than 60% activity at pH 11. It had an apparent temperature optimum of 70°C and retained stable at this temperature in the presence of substrate. rXynSL4 was highly halotolerant, retaining more than 55% activity with 0.25-3.0 M NaCl and was stable at the concentration of NaCl up to 4M. The enzyme activity was significantly enhanced by ß-mercaptoethanol and Ca(2+) but strongly inhibited by heavy-metal ions and SDS. This thermophilic and alkaline- and salt-tolerant enzyme has great potential for basic research and industrial applications.

Characterization of catechol 2,3-dioxygenase from Planococcus sp. strain S5 induced by high phenol concentration.

This study aimed at characterization of a new catechol 2,3-dioxygenase isolated from a Gram-positive bacterium able to utilize phenol as the sole carbon and energy source. Planococcus sp. strain S5 grown on 1 or 2 mM phenol showed activity of both a catechol 1,2- and catechol 2,3-dioxygenase while at a higher concentrations of phenol only catechol 2,3-dioxygenase activity was observed. The enzyme was optimally active at 60°C and pH 8.0. Kinetic studies showed that the K(m) and V(max) of the enzyme were 42.70 µM and 329.96 mU, respectively. The catechol 2,3-dioxygenase showed the following relative meta-cleavage activities for various catechols tested: catechol (100%), 3-methylcatechol (13.67%), 4-methylcatechol (106.33%) and 4-chlorocatechol (203.80%). The high reactivity of this enzyme towards 4-chlorocatechol is different from that observed for other catechol 2,3-dioxygenases. Nucleotide sequencing and homology search revealed that the gene encoding the S5 catechol 2,3-dioxygenase shared the greatest homology with the known genes encoding isoenzymes from Gram-negative Pseudomonas strains.

Degradation of 2,4,6-trichlorophenol by bacteria isolated from secondary sludge of a pulp and paper mill.

Bacterial strains capable of degrading trichlorophenol (2,4,6-TCP) were isolated from the secondary sludge of a pulp and paper mill and were characterized. These isolates were identified as Planococcus rifietoensis (CL4) and Bacillus pumilus (CL5), based on their 16S rRNA sequence analysis. These isolates were able to grow and utilize 2,4,6-TCP as their source of carbon as well as energy. HPLC analysis and stoichometric release of chloride in the medium confirmed the degradation ability of these isolates. Removal efficiency of 2,4,6-TCP by these isolates was discovered to be high. They were able to remove 90% of 2,4,6-TCP when grown at a concentration of 600 mg L(-1). Inoculation of these bacteria completely removed 2,4,6-TCP within 2 weeks from the sludge of the pulp and paper mill when supplemented at the rate of 100 mg L(-1). Absorbable Organic Halogen (AOX) and Extractable Organic Halogen (EOX) were significantly reduced by 63% and 70% respectively from the sludge due to inoculation of these bacteria. These isolates have high potential to remove 2,4,6-TCP and may be used for removal of 2,4,6-TCP from pulp paper mill waste.