Phylogenetic diversity of actinomycetes cultured from coastal multipond solar saltern in Tuticorin, India

ABSTRACT: BACKGROUND: Hypersaline solar salterns are extreme environments in many tropical and subtropical regions throughout the world. In India, there are several coastal solar salterns along with the coastal line of the Bay of Bengal and Arabian Sea and inland solar salterns around Sambhar saltlake, from which sodium chloride is obtained for human consumption and industrial needs. Studies on characterization of such coastal and inland solar salterns are scarce and both the bacterial and archaeal diversity of these extreme saline environment remains poorly understood. Moreover, there are no reports on exclusive diversity of actinomycetes inhabiting Indian solar salterns. RESULTS: Soil sediments were collected from both concentrator and crystallizer ponds of solar salterns and subjected to detailed physico-chemical analysis. Actinomycetes were selectively isolated by employing selective processing methods and agar media. A total of 12 representatives were selected from the 69 actinomycete isolates obtained from the saltern soil samples, using Amplified Ribosomal DNA Restriction Analysis. Sequencing and analysis of 16S rDNA from chosen representative isolates displayed the presence of members affiliated to actinobacterial genera: Streptomyces, Micromonospora, Nocardia, Nocardiopsis, Saccharopolyspora and Nonomuraea. The genus Streptomyces was found to be the dominant among the isolates. Furthermore, rare actinomycete genus Nonomuraea was isolated for the first time from Indian solar salterns. CONCLUSIONS: To the best of our knowledge, this study constitutes the first characterization of actinomycete diversity centred on solar salterns located in the eastern coastal region of India. Furthermore, this is the very first report of isolation of Nonomuraea species from solar salterns and also from India. As actinomycetes encompass recurrently foremost sources of biotechnologically important member of the microbial communities, the actinomycetes retrieved from the Indian saltern soil samples laid the platform to search for novel biotechnologically significant bioactive substances.     

Cloning,sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum

A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC^- Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.     

High-Level Expression of the Antimicrobial Peptide Plectasin in Escherichia coli

Plectasin is a defensin-like antimicrobial peptide isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin showed marked antibacterial activity in vitro against Gram-positive bacteria, especially Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin could kill the sensitive strain as efficaciously as vancomycin and penicillin and without cytotoxic effects on mammalian cell viability. In order to establish a bacterium-based plectasin production system, in the present study, the coding sequence of plectasin was optimized, and then cloned into pET32a (+) vector and expressed as a thioredoxin (Trx) fusion protein in Escherichia coli. The soluble fusion protein collected from the supernatant of the cell lysate was separated by Ni²⁺-chelating affinity chromatography. The purified protein was then cleaved by Factor Xa protease to release mature plectasin. Final purification was achieved by Ni²⁺-chelating chromatography again. The recombinant plectasin exhibited the same antimicrobial activity as reported previously. This is the first study to describe the expression of plectasin in E. coli expression system, and these works might provide a significant foundation for the following production or study of plectasin, and contribute to the development and evolution of novel antimicrobial drugs in clinical applications.     

Role of carcinoscorpin, a haemolymph lectin of horseshoe crabCarcinoscorpius rotundacauda as humoral factor

This study demonstrated that a marine Indian horseshoe crab,Carcinoscorpious rotundacauda showed higher self defence in an experimental infection upon the induction of its circulatory lectin, carcinoscorpin. It resisted an infection with 10⁷ liveEscherichia coli per crab when the circulatory carcinoscorpin was 8–16-fold higher after administering 2-ketodeoxyoctonate (Kdo) into the live crab. The naive control with its natural level of circulatory lectin could tolerate a maximum infective dose of 10⁶ liveE. coli per crab. Bacterial killing and phagocytic uptake in association with the isolated crab amoebocytes in anex vivo system was considerably higher for the lectin opsonizedE. coli compared to unopsonized samples. Carcinoscorpin is thus functionally an opsonin in the defence of the primitive marine arthropod,C. rotundacauda, like vertebrate antibody, a humoral factor involved in the defence of the host. The natural capacity for defending an infection with 10⁶ liveE. coli per crab suggested that the crabs in the natural habitat hardly face such an infection and is possibly one of the reasons for its survival over millions of years as a living fossil.     

Comparison of the sequences of the nagE operons from Klebsiella pneumoniae and Escherichia coli K12: enhanced variability of the enzyme IIN-acetylglucosamine in regions connecting functional domains.

Summary The nagE operon, encoding the enzyme II specific for N-acetylglucosamine (EII^Nag), and adjacent DNA from the chromosome of Klebsiella pneumoniae were sequenced and compared with the corresponding sequence from Escherichia colt K12. The deduced EII^Nag sequences differ in 72 out of 651 amino acids, the K. pneumoniae sequence being three residues longer. The amino acid differences were distributed unevenly, and were most frequent in regions connecting the three functional domains of the protein. In the nagE-nagB intergenic region, two promoter, two operator, and one CAP consensus sequence with regulatory functions were highly conserved. The nag structural genes from both species were very similar (83% DNA similarity; 89% amino acid similarity) except for frequent AT to GC exchanges in the wobble base of codons in K. pneumoniae DNA relative to the E. coli DNA.     

<Emphasis Type="Italic">Pontibacter rhizosphera</Emphasis> sp. nov., isolated from rhizosphere soil of an Indian medicinal plant <Emphasis Type="Italic">Nerium indicum</Emphasis>

A gram-negative, motile, straight to curved rod shaped, pink pigmented bacterium was isolated from a soil sample collected from the rhizosphere of an Indian medicinal plant, Nerium indicum (Chuvanna arali) and subjected to a detailed polyphasic taxonomic study. The strain, designated as IMTB-1969^T, matched with most of the phenotypic and chemotaxonomic properties of the genus Pontibacter and represents a novel species. The major fatty acids of the strain were monounsaturated iso/anteiso branched C17 fatty acids (45.1%) and iso-C15:0 (16.5%). MK-7 was the predominant isoprenoid quinone. According to 16S rRNA gene sequence analysis, strain IMTB-1969^T was indicated to belonged to the phylum Bacteroidetes and further phylogenetic analysis revealed that the strain IMTB-1969^T belongs to the family Cytophagaceae and genus Pontibacter. The highest 16S rRNA gene sequence similarity was with Pontibacter korlensis CCTCC AB 206081^T (97.2%) and lower sequence similarity was observed with other species in the genus Pontibacter (95.9–94.0%). DNA–DNA relatedness study of the strain IMTB-1969^T confirmed that it represents a novel species. The G+C content of the genomic DNA was 52.2 (±0.5) mol%. The results of physiological and biochemical tests allowed the genotypic and phenotypic distinction of strain IMTB-1969^T from its closest phylogenetic relatives. The strain IMTB-1969^T should be classified as novel species of the genus Pontibacter, for which the name Pontibacter rhizosphera sp. nov. is proposed. The type strain is IMTB-1969^T (=MTCC 10673^T = DSM 24399^T).     

Expression of a Recombinant Molt-Inhibiting Hormone of the Kuruma Prawn Penaeus japonicus in Escherichia coli

The crustacean molt-inhibiting hormone (MIH) suppresses ecdysteroid synthesis by the Y-organ. The MIH of the kuruma prawn Penaeus japonicus has recently been isolated and its cDNA cloned. In this study, we expressed the MIH in Escherichia coli to obtain a large quantity of this hormone with biological activity. The MIH cDNA was processed and ligated into an expression plasmid. E. coli was transformed with this plasmid, and then the recombinant MIH (r-MIH) was expressed. The r-MIH was put through the refolding reaction and was purified by reverse-phase HPLC. N-terminal amino acid sequence and time-of-flight mass spectral analyses supported the idea that the r-MIH had the entire sequence. By in vitro bioassay using the Y-organ of the crayfish, the r-MIH was found to be comparable to natural MIH in inhibiting ecdysteroid synthesis.     

<Emphasis Type="Italic">Salinicoccus kekensis</Emphasis> sp. nov., a novel alkaliphile and moderate halophile isolated from Keke Salt Lake in Qinghai,China

A novel alkaliphilic and moderate halophilic bacterium, designated strain K164^T, was isolated from Keke Salt Lake in Qinghai, China. The strain grew with 2.0–20.0% (w/v) NaCl, at 4–50°C and pH 6.5–11.5, with an optimum of 8% (w/v) NaCl, 37°C and pH 10, respectively. The predominant respiratory quinone was menaquinone 6 (MK-6) and the major polar lipid was phosphatidylethanolamine. The major cellular fatty acids were anteiso-C_15:0 and iso-C_15:0. The genomic DNA G+C content was 50.16 mol. Phylogenetic analysis based on the full-length 16S rRNA gene sequence revealed that strain K164^T was a member of the genus Salinicoccus. Strain K164^T showed the highest similarity (98.4%) with Salinicoccus alkaliphilus AS 1.2691^T and below 97% similarity with other recognized members of the genus in 16S rRNA gene sequence. Level of DNA–DNA relatedness between strain K164^T and Salinicoccus alkaliphilus AS 1.2691^T was 20.1%. On the basis of its phenotypic characteristics and the level of DNA–DNA hybridization, strain K164^T is considered to represent a novel species of the genus Salinicoccus, for which the name Salinicoccus kekensis sp. nov. is proposed. The type strain is K164^T (=CGMCC 1.10337^T = DSM 23173^T).     

Isolation and functional expression of the bop gene from Halobiforma lacisalsi

A novel bop gene was described from Halobiforma lacisalsi strain AJ5^T, an extremely halophilic archaeon isolated from Ayakekum Lake, China. Following six rounds of PCR amplification based on the conserved fragment of the bop gene, the complete sequence of the bop gene, including the 5′ and 3′ flanking regions of the conserved fragment, was obtained by the ligation-mediated PCR amplification (LPA) approach. The data presented provide us with further insight into the distribution of bop-like genes in the family Halobacteriaceae. This is the first example of a bop-like gene in halophiles found in the high-pH environment. Alignment and hydropathy analysis of the deduced amino acid sequence identified the conserved functional sites as well as some variations compared with other bacterio-opsins. Molecular phylogenetic analysis revealed the position of the bacterio-opsin of strain AJ5, which is closest to that of Haloterrigena sp. arg-4 with 85% identity. In the presence of all-trans retinal, recombinant Escherichia coli cells expressing the gene turned dark purple. The purple membrane from the recombinant E. coli showed maximal absorption at 540 nm.     

Extremely halophilic Archaea from Tuz Lake,Turkey, and the adjacent Kaldirim and Kayacik salterns

Tuz Lake is a hypersaline lake located in Central Anatolia, Turkey. The lake and its salterns, Kaldirim and Kayacik, are the major sources of solar salt for industrial applications in Turkey, especially in the food and leather industries. Use of the crude solar salt often results in microbial deterioration of the products. We therefore initiated a thorough characterization of the microbial communities in Tuz Lake and its adjacent salterns, and we present here the results of investigations on diversity of extremely halophilic Archaea. Twenty-seven colonies of aerobic red or pink Archaea (family Halobacteriaceae) were selected according to colony shape, size, consistency and pigmentation, and characterized according to their phenotypic characteristics, polar lipid contents, and antibiotic sensitivities. Furthermore, 16S rRNA genes of the isolates were screened by DGGE analysis and partially sequenced. Phylogenetic analysis showed that most isolates belonged to the genera Haloarcula, Halorubrum and Halobacterium. Haloarcula was found to be dominant both in Tuz Lake and in the saltern samples. Halorubrum species were isolated from Tuz Lake and from the Kaldirim saltern, and Halobacterium species were recovered from Tuz Lake and from the Kayacik saltern. All strains showed various activities of hydrolytic enzymes (proteases, amylases, cellulases, and others), activities which are responsible for the detrimental effects of the crude salt in food and leather products.     

Plasmids expressing the Herpes simplex virus thymidine kinase gene in mammalian and bacterial cells

Summary Two plasmids containing either the complete thymidine kinase gene of Herpes simplex virus type I (pSK2) or the gene without the remote control sequence (pSK1) just behind the lac promoter and the first codons of the lacZ gene were constructed. Both plasmids efficiently transform mouse Ltk^- cells as well as E. coli tk^- cells to the Tk⁺ phenotype and are well suited for plasmid rescue from transformed mouse cells by direct functional selection for tk expression using a tk ^- mutant of E. coli C600.     

Diversity of microorganisms in solar salterns of Tamil Nadu,India

In this study, the diversity of prokaryotes inhabiting crystallizer ponds of three solar salterns, located along Bengal Bay in Tamil Nadu, India was examined. Unlike other salterns studied the Tamil Nadu salterns are fed by hypersaline spring water mixed with seawater and led to the ponds from bore wells. In addition, prokaryotic community development is restricted as salterns operate only during the arid part of the year. Both culture-based and culture-independent polymerase chain reaction 16S rRNA molecular phylogenetic approaches were employed. Representatives of the family Halobacteriaceae dominated in cultivable portion of diversity encountered with members of genera Haloferax, Halorubrum, Haloarcula, Halobacterium and Halogeometricum recovered in pure culture. In contrast, members of Bacteria were recovered from only one sampling site and were represented by members of genera Salinibacter, Cytophaga and Marinococcus. Based on culture-independent sampling, the predominant members of the haloarchaeal crystallizer community belonged to the genus Natrinema.     

<Emphasis Type="Italic">Kazachstania wufongensis</Emphasis> sp. nov., an ascosporogenous yeast isolated from soil in Taiwan

A new yeast species, Kazachstania wufongensis, is proposed in this paper based on six strains isolated from soil in Taiwan. The species may produce one to four ellipsoidal ascospores in each ascus, directly transformed from diploid cells. Genus assignment and distinction of the species from other recognized species of Kazachstania is based on morphological and physiological characteristics, and on phylogenetic analysis of nucleotide sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene. Sequence analysis of the D1/D2 domains of the LSU rRNA gene reveals that K. wufongensis is a member of the Kazachstania exigua complex, and its phylogenetically closest relatives are K. exigua, K. barnettii, K. bulderi, and K. turicensis. The species can be further differentiated from the other phylogenetically related species based on internal transcribed spacer sequence and electrophoretic karyotype. Therefore, the new species Kazachstania wufongensis sp. nov. is proposed. The type strain of this new species, which was isolated from forest soil in Wufong, Hsinchu, Taiwan, is FN21S03^T (=CBS 10886^T = BCRC 23138^T).     

Actinoplanes capillaceus sp. nov., a new species of the genus Actinoplanes

Two motile actinomycete strains, K95–5561^T and K95–5562, were isolated from a soil sample collected at Sayama City, Saitama Prefecture, Japan. They produced bell shaped spore vesicles (sporangia) with hairy surfaces on substrate hyphae. When released into water, the sporangiospores became motile by a tuft of polar flagella. The chemotaxonomic and morphological characteristics together with 16S rRNA gene sequence data indicated that the two isolates belonged to the genus Actinoplanes. The two strains were assigned to a single species on the basis of phenotypic, notably cultural, morphological and physiological characteristics, and DNA-DNA pairing data. The two strains were distinguished from representatives of all validly described species of Actinoplanes using a combination of genotypic and phenotypic properties. It is, therefore, proposed that strains K95–5561 and K95–5562 be recognized as a new species of the genus Actinoplanes with the name Actinoplanes capillaceus sp. nov. The type strain of the species is strain K95–5561^T (=JCM 10268^T =IFO 16408^T). The invalidly proposed species `Ampullariella cylindrica', `Ampullariella pekinensis' and `Ampullariella pilifera' were assigned to Actinoplanes capillaceus on the basis of genotypic and phenotypic data.     

Preliminary Study on High-level Expression of Tandem-Arranged Tachyplesin-Encoding Gene in Bacillus subtilis Wb800 and its Antibacterial Activity

To produce tachyplesin, an antimicrobial peptide, by a stable and efficient gene engineering approach, cDNAs containing single tachyplesin gene sequence (tac)¹ and tandem repeat of tachyplesin gene sequence (tac2) were respectively developed by annealing two synthesized complementary single-stranded DNAs and constructed into pSBPTQ shuttle vector under the control of the SacB.p.s promoter. The vectors containing the target gene sequence were then transformed into Bacillus subtilis WB800, respectively. Both expression of tac and tac2 were induced by 2% sucrose. The fermentation supernatant was purified by regenerated cellulose membrane tubing (MWCO 2000) and the secreted TAC² and TAC2 were about 5 and 10 mg/l of supernatant, respectively. The antimicrobial activities of TAC and TAC2 were measured by the size of bacteriostatic circle of the fermentation supernatants against Escherichia coli K88. Ultrastructural alteration of E. coli K88 and Salmonella typhimurium was observed under scanning electron microscope and transmission electron microscopy. The results showed that in comparison with TAC, TAC2 was expressed at a higher level and also indicating strong antimicrobial activity both in vitro and in vivo. ¹ tac is tachyplesin gene; tac2 is a tandem repeat tachyplesin gene.²TAC is the expression product of tac; TAC2 is the expression product of tac2.     

The influence of biofilm structure and total interaction energy on Escherichia coli retention by Pseudomonas aeruginosa biofilm

The retention of a surrogate pathogenic bacterium, Escherichia coli^T , in Pseudomonas aeruginosa biofilms (with various EPS excreting capacities) was investigated using a laboratory flow cell system. The structural characteristics of the biofilm, as well as the quantity of E. coli^T retained in the biofilm, were assessed using confocal laser scanning microscopy coupled with image analysis. In addition, the total interaction energy between E. coli^T and the P. aeruginosa biofilm was computed with the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, which provided an additional context to explain the pathogen interaction in aquatic biofilms. The correlations between the quantity of detained E. coli^T cells and the structural characteristics of the biofilm were analysed and the results indicated that the heterogeneity of the biofilm could create a quiescent zone leading to temporary retention of E. coli^T within the biofilm. Overall, this study provided insights toward understanding the retention of pathogenic bacteria in environmental biofilms.     

Physiological and Biochemical Role of Brassinosteroids and Their Structure-Activity Relationship in the Green Alga Chlorella vulgaris Beijerinck (Chlorophyceae)

This paper studies the influence of the 7-oxalactone type of brassinosteroids (BRs) and 6-ketone upon the biological activity of the alga Chlorella vulgaris (Chlorophyceae). The results of the study indicate significant differences in the growth and metabolism of C. vulgaris cells caused by the different chemical structures of the BRs used. The most significant differences in the stimulation of the growth of the biomass and metabolites contained in it were caused by structural differences in the B ring of BRs. It was found that in C. vulgaris 7-oxalactone type of BRs [brassinolide (BL) and its derivatives] are more active than 6-ketone type of BRs [castasterone (CS) and its derivatives]. It was found that BRs used within the range of concentration of 10⁻¹² to 10⁻⁸ m stimulate two- to threefold the growth and division of C. vulgaris cells. The most stimulating influence upon the number of the algal cells and the phosphorus, chlorophyll, and monosaccharides contained in the alga, as well as the intensity of the photosynthesis, and sugar and glycolate excretion was demonstrated by BL at a concentration 10⁻⁸ m in the 36th h of cultivation. HomoCS was characterized by the lowest biological activity. In turn, after the 48th h an inhibition of the rate of growth and development of the alga takes place. In the range from 10⁻⁷ to 10⁻⁶ m the inhibition of growth and development of the alga was manifested by BRs. During the further toxic activity of BRs the cells of C. vulgaris undergo complete degradation. In turn, in concentrations lower than 10⁻¹² m, BRs do not exert any biologically significant influence upon C. vulgaris cells. On the basis of the study, the biological activity of BRs was arranged in the following order: BL > 24-epiBL > homoBL > CS > 24-epiCS > homoCS. Received July 21, 1997; accepted April 7, 1998     

Pigmentiphaga soli sp. nov., a bacterium isolated from soil

Strain BS12^T, a Gram-negative motile bacterium, was isolated from soil in South Korea and characterized to determine its taxonomic position. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the strain belonged to the family Alcaligenaceae in the class Betaproteobacteria. The highest degree of sequence similarities of strain BS12^T were found with Pigmentiphaga litoralis JSM 061001^T (98.3%), Pigmentiphaga daeguensis K110^T (98.2%), and Pigmentiphaga kullae K24^T (98.1%). Chemotaxonomic data revealed that strain BS12^T possessed ubiquinone-8, which is common in the family Alcaligenaceae, and the predominant fatty acids were C_16:0, C_17:0 cyclo, summed feature 3 (C_16:1 ω6c/ω7c), and summed feature 8 (C_18:1 ω6c/ω7c). The major polar lipids of strain BS12^T were phosphatidylethanolamine and phosphatidylglycerol. Based on these data, BS12^T (=KCTC 23577^T =JCM 17666^T =KEMB 9004-082^T) should be classified as a type strain of a novel species, for which the name Pigmentiphaga soli sp. nov. is proposed.     

Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis

Background  

Laccases have huge potential for biotechnological applications due to their broad substrate spectrum and wide range of reactions they are able to catalyze. These include, for example, the formation and degradation of dimers, oligomers, polymers, and ring cleavage as well as oxidation of aromatic compounds. Potential applications of laccases include detoxification of industrial effluents, decolorization of textile dyes and the synthesis of natural products by, for instance, dimerization of phenolic acids. We have recently published a report on the cloning and characterization of a CotA Bacillus licheniformis laccase, an enzyme that catalyzes dimerization of phenolic acids. However, the broad application of this laccase is limited by its low expression level of 26 mg l^-1 that was achieved in Escherichia coli. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of CotA.