Aerobic endospore-forming bacteria isolated from Antarctic soils as producers of bioactive compounds of industrial interest

Spore-forming bacteria are known to produce various enzymes and bioproducts valuable to different industries and to bear the harsh conditions found in the Antarctic environment. However, aerobic or facultative spore-forming bacterial communities found in maritime Antarctic soils yet remain poorly studied. In this study, 80 spore-forming and cold-adapted bacterial strains were isolated from nine different soil samples of King George Island, in maritime Antarctica, and further clustered into amplified ribosomal DNA restriction analysis groups within each soil. Representative strains were then identified as belonging to Bacillus, Rummeliibacillus, Paenibacillus and Sporosarcina by 16S rRNA gene sequencing. The ability to produce extracellular enzymes, antimicrobial substances and biosurfactants was determined in all isolates. The enzymatic activities most frequently found among the isolates were as follows: esterase (45 %), caseinase (30 %), amylase (16.2 %) and gelatinase (15 %). Biosurfactant production was detected in 25 % of the isolates. The growth inhibition of methicillin-resistant Staphylococcus aureus was observed in 13.7 % of the strains tested, but only two strains inhibited the growth of Candida albicans. The isolated spore-forming bacterial species were also compared with the characteristics of the different Antarctic soils sampled based on their physicochemical properties, showing that pH, C and P were the main factors correlated with the distribution of this group of bacteria in the Antarctic soils studied. These Antarctic endospore-forming bacterial strains may have a potential for industrial processes occurring at low temperatures.     

Burkholderia sp. Induces Functional Nodules on the South African Invasive Legume Dipogon lignosus (Phaseoleae) in New Zealand Soils

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678^T which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.     

Nosocomial Bloodstream Candida Infections in a Tertiary-Care Hospital in South Brazil: A 4-Year Survey

The aims of this study were to evaluate the epidemiology of nosocomial candidemia in a tertiary hospital in South Brazil and the in vitro antifungal susceptibility of isolates. Blood strains from 108 patients were identified by PCR-based method. Some 30.5 % of candidemia were caused by Candida tropicalis, 28.7 % were due to Candida albicans, 24.1 % with Candida parapsilosis sensu stricto, 8.3 % with Candida glabrata sensu lato, 1.8 % involved Candida krusei and 6.6 % with other species. Candidemia was more common in intensive care unit settings (66 %). In vitro susceptibility to antifungal drugs was determined by a microdilution method; and new species-specific clinical breakpoints for fluconazole and voriconazole were applied. Overall susceptibility rates were 100 % for itraconazole, 91 % for fluconazole, 98 % for voriconazole and 99 % for amphotericin B. Fluconazole resistance was mostly among C. parapsilosis sensu stricto isolates (26.9 %). Most of the findings reported here agreed with epidemiological features common to other tertiary hospitals in Brazil; but also revealed some peculiarities, such as a high frequency of C. tropicalis associated with candidemia. Besides, high rate of fluconazole resistance among C. parapsilosis stricto sensu isolates was obtained when applying the new species-specific clinical breakpoints.     

Diversity of cultivable halophilic archaea and bacteria from superficial hypersaline sediments of Tunisian solar salterns

Prokaryotes in the superficial sediments are ecologically important microorganisms that are responsible for the decomposition, mineralization and subsequent recycling of organic matter. The aim of this study was to explore the phylogenetic and functional diversity of halophilic archaea and bacteria isolated from the superficial sediments of solar salterns at Sfax, Tunisia. Sixty four strains were isolated from crystallizer (TS18) and non-crystallizer (M1) ponds and submitted to genotypic characterization and evaluation by amplified ribosomal RNA restriction analysis (ARDRA) techniques. Our findings revealed that the archaeal diversity observed for 29 isolates generated five distinct patterns from the non-crystallizer M1 pond, with Halorubrum chaoviator as the most prevalent cultivable species. However, in the TS18 crystallizer pond, ten restriction patterns were observed, with the prevalence of haloarchaea EB27K, a not yet identified genotype. The construction of a neighbour-joining tree of 16S rRNA gene sequences resulted in the division of the potential new species into two major groups, with four strains closely related to the sequence of the unculturable haloarchaeon EB27K and one strain to the recently described Halovenus aranensis strain. The 35 bacterial strains observed in this work were present only in the non-crystallizer pond (M1) and presented two distinct ARDRA patterns. These strains belonged to the γ-proteobacteria subdivision, with members of Salicola marasensis (83 %) being the most predominant species among the isolates. 16S rRNA gene sequencing revealed that Salicola strains displayed different degrees of homogeneity. The results from pulsed field gel electrophoresis assays showed that the Salicola isolates could be clustered in two distinct groups with different genome sizes.     

Anaerobic biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons by a facultative anaerobe Pseudomonas sp. JP1

Polycyclic aromatic hydrocarbons (PAHs) are harmful persistent organic pollutants, while the high-molecular-weight (HMW) PAHs are even more detrimental to the environment and human health. However, microbial anaerobic degradation of HMW PAHs has rarely been reported. One facultative anaerobe Pseudomonas sp. JP1 was isolated from Shantou Bay, Shantou, China, which could degrade a variety of HMW PAHs. After 40 days cultivation with strain JP1, anaerobic biodegradation rate of benzo[a]pyrene (BaP), fluoranthene, and phenanthrene was 30, 47, and 5 %, respectively. Consumption of nitrate as the electron acceptor was confirmed by N-(1-naphthyl) ethylenediamine spectrophotometry. Supplementation of sodium sulfite, maltose, or glycine, and in a salinity of 0–20 ‰ significantly stimulated anaerobic degradation of BaP. Lastly, the anaerobic degradation metabolites of BaP by strain JP1 were investigated using GC/MS, and the degradation pathway was proposed. This study is helpful for further studies on the mechanism of anaerobic biodegradation of PAHs.     

Isolation and characterization of indigenous 2,4-D herbicide degrading bacteria from an agricultural soil in proximity of Sauce Grande River,Argentina

Our objective was to isolate and characterize indigenous bacteria able to use 2,4-D as a sole carbon (C) and energy source from an agricultural soil in the Sauce Grande River basin (Argentina). Culturable-dependant and molecular methods combined were used to identify and characterize putatively dominant indigenous degrading bacteria. Physiological traits, chloride release and biomass production showed the degradative capacity of the isolates obtained and high-performance liquid chromatography (HPLC) was used to corroborate the evidence. Degrading genes (tfdA and tfdB) were detected in all isolates, and their restriction fragment length polymorphisms (RFLP) were analyzed. Altogether, our results suggest that agricultural use of 2,4-D at recommended level leads to selection for a copiotrophic degrading population. The dominant genus able to metabolize 2,4-D in this soil was identified as Cupriavidus by 16S rRNA gene sequencing and the RFLP profiles of all isolates resembled that of Cupriavidus necator JMP134, the model organism for 2,4-D degradation. The strain EMA-G showed a remarkable performance in herbicide degradation (100 % removal in <1 day) in pure culture and is a favorite candidate for future biodegradation experiments.     

Comparison of 16S rRNA gene phylogeny and functional tfdA gene distribution in thirty-one different 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid degraders

31 different bacterial strains isolated using the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, were investigated for their ability to mineralize 2,4-D and the related herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA). Most of the strains mineralize 2,4-D considerably faster than MCPA. Three novel primer sets were developed enabling amplification of full-length coding sequences (CDS) of the three known tfdA gene classes known to be involved in phenoxy acid degradation. 16S rRNA genes were also sequenced; and in order to investigate possible linkage between tfdA gene classes and bacterial species, tfdA and 16S rRNA gene phylogeny was compared. Three distinctly different classes of tfdA genes were observed, with class I tfdA sequences further partitioned into the two sub-classes I-a and I-b based on more subtle differences. Comparison of phylogenies derived from 16S rRNA gene sequences and tfdA gene sequences revealed that most class II tfdA genes were encoded by Burkholderia sp., while class I-a, I-b and III genes were found in a more diverse array of bacteria.     

A comparison of genes involved in sphingan biosynthesis brought up to date

Microbial polysaccharides have a wide range of functional properties and show high relevance in industrial applications. The possibility to create tailor-made polysaccharides by genetic engineering will further enhance the product portfolio and may open new fields of application. Here, we have examined in detail the recently sequenced genome of the welan-producing strain Sphingomonas sp. ATCC 31555 to identify the complete welan cluster and further genes involved in EPS production. The corresponding genes were compared on the nucleotide and amino acid sequence level to the EPS clusters of the described gellan-producing Sphingomonas elodea ATCC 31461, diutan-producing Sphingomonas sp. ATCC 53159, and the S-88-producing Sphingomonas sp. ATCC 31554 strains. We also compared the previously mentioned strains to each other and included the genes upstream of the main cluster in gellan and welan cluster. The cluster organization of Sphingomonas strain S-7 was also compared based on previous hybridization experiments, without nucleotide sequences. We have found that the occurrence of genes in all biosynthesis clusters is connected to the structures of the various produced sphingans. Along these lines, homologous genes responsible for the assembly of the identical repeating unit generally show high sequence identity, whereas genes for putative side chain attachment urf31, urf31.4, and urf34 vary more in distinct areas. Moreover, gene clusters for biosynthesis of diutan, welan, gellan, and S-88 as well as S-7 are similar in general organization but differ in location and arrangement of some genes. Finally, we summarized genetic and mutational engineering approaches toward modified sphingan variants as described in literature.     

Detection of polyketide synthase and nonribosomal peptide synthetase biosynthetic genes from antimicrobial coral-associated actinomycetes

The diversity and properties of actinobacteria, predominant residents in coral holobionts, have been rarely documented. In this study, we aimed to explore the species diversity, antimicrobial activities and biosynthetic potential of culturable actinomycetes within the tissues of the scleractinian corals Porites lutea, Galaxea fascicularis and Acropora millepora from the South China Sea. A total of 70 strains representing 13 families and 15 genera of actinobacteria were isolated. The antimicrobial activity and biosynthetic potential of fifteen representative filamentous actinomycetes were estimated. Crude fermentation extracts of 6 strains exhibited comparable or greater activities against Vibrio alginolyticus than ciprofloxacin. Seven of the 15 actinomycetes strains possess type I polyketide synthases (PKS-I) and/or nonribosomal peptide synthetases (NRPS) genes. Nine tested strains possess type II polyketide synthases (PKS-II). Phylogenetic analysis based on 16S rRNA gene sequences indicated that these PKS and NRPS gene screening positive strains belong to genera Nocardiopsis, Pseudonocardia, Streptomyces, Micromonospora, Amycolatopsis and Prauserella. One PKS-I and four NRPS fragments showed <70 % similarity to their closest relatives, which suggested the novelty of these genes. This study helps uncover the genetic capacity of stony coral-associated actinomycetes to produce bioactive molecules.     

Crystal structure of tRNA m1A58 methyltransferase TrmI from Aquifex aeolicus in complex with S-adenosyl-l-methionine

The N ¹-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m¹A58 methyltransferase TrmI, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi.     

Diversity and antibacterial activities of culturable fungi associated with coral Porites pukoensis

The diversity of coral associated fungi is not enough understood, especially for scleractinian corals. Members of Porites are common and dominant species of scleractinian corals. To date, the fungal communities associated with coral Porites pukoensis have been not reported. In this paper, the diversity and activity of coral associated fungi in P. pukoensis were explored, 23 fungal strains were isolated, belonging to 10 genera and Aspergillus sp. (30.4 %) was predominant fungal genera. The sequence of isolate C1-23 in GenBank was only 90 % similarity to the most closely related sequences. It is concluded that rich fungal symbionts are attached to P. pukoensis, the rate of isolates with antibacterial activity was up to 30 %, particularly some isolates showed stronger bioactivities to gram-negative bacteria. It is included that the diversity of coral associated fungi in P. pukoensis is abundant and its activity is obviously. So the activities of fungi in P. pukoensis were deserved for further study.     

Transformer 2β (Tra2β/SFRS10) positively regulates the progression of NSCLC via promoting cell proliferation

Transformer 2β (Tra2β), a member of the serine/arginine-rich-like protein family, is an important RNA-binding protein involved in alternative splice. Deregulation of Tra2β has been observed in several cancers. However, the detailed role of Tra2β in non-small cell lung cancer (NSCLC) has not been elucidated. In this study, the contribution of Tra2β to NSCLC development was investigated. On histological level, the expression of Tra2β was determined by Western and immunohistochemistry assays. It demonstrated that Tra2β was expressed higher in NSCLC tumor tissues compared with adjacent non-tumor tissues. In addition to confirm the association of Tra2β expression with histological differentiation and clinical stage (p < 0.05), we also confirmed significant positive correlation between the expression level of Tra2β and that of Ki67 (p < 0.05, r = 0.446) by Spearman rank correlation test. Moreover, high expression of Tra2β predicted poor prognosis by Kaplan–Meier survival analysis. And Tra2β among with other clinicopathologic variables was an independent prognostic indicator for patients’ overall survival by multivariate analysis. On cellular level, Tra2β expression was demonstrated to promote proliferation of NSCLC cells through a series of assays, including serum starvation and release assay, Western blot assay and flow cytometry analysis. Moreover, knockdown of Tra2β was confirmed to inhibit proliferation and to induce apoptosis of NSCLC cells through flow cytometry analysis, western analysis, cell counting kit-8 assay and Tunnel assay. Our results indicated that Tra2β was involved in the tumorigenesis of NSCLC and might be a potential therapeutic target of NSCLC.     

Down-Regulation of Adiponectin Receptors in Osteoarthritic Chondrocytes

The objective of this study was to investigate the expression of adiponectin receptors (AdipoR1, R2, and T-cadherin) in both normal subjects and patients with knee osteoarthritis (OA). We used immunofluorescence to assess expression of adiponectin receptors in the chondrocytes of normal subjects (n = 3) and OA patients (n = 3). We also studied mRNA expression of adiponectin receptors in both groups by real-time polymerase chain reaction (real-time PCR). Finally, we utilized Western blotting to confirm the presence of adiponectin receptors. As compared with osteoarthritic chondrocytes, normal chondrocytes showed stronger immunoreactivity for AdipoR1, AdipoR2, and T-cadherin. The expression levels of both AdipoR1 and AdipoR2 mRNA were significantly lower in the osteoarthritic chondrocytes compared with those in the normal chondrocytes, 19 ± 2 and 36 ± 3 % of normal chondrocytes, respectively (P < 0.001). T-cadherin mRNA expression levels of the osteoarthritic chondrocytes were also lower than those in the normal chondrocytes, but not statistical significant (P = 0.072). The expression levels of AdipoR1 and AdipoR2 protein were significantly higher in the normal chondrocytes compared with those in the osteoarthritic chondrocytes (P < 0.001, P < 0.01, respectively). T-cadherin protein expression level of the normal chondrocytes was also higher than those in the osteoarthritic chondrocytes, but the difference is not statistical significant (P = 0.114). Expression of adiponectin receptors protein in normal and osteoarthritic chondrocytes is consistent with its mRNA expression levels. In conclusion, we report for the first time down-regulation of adiponectin receptors (AdipoR1, R2, and T-cadherin) in osteoarthritic chondrocytes. Decreased adiponectin receptors in OA may reduce the tissue sensitivity to adiponectin, thus lost the protection from adiponectin in the progression of OA.     

Plasmid-mediated bioaugmentation of sequencing batch reactors for enhancement of 2,4-dichlorophenoxyacetic acid removal in wastewater using plasmid pJP4

Plasmid-mediated bioaugmentation was demonstrated using sequencing batch reactors (SBRs) for enhancing 2,4-dichlorophenoxyacetic acid (2,4-D) removal by introducing Cupriavidus necator JMP134 and Escherichia coli HB101 harboring 2,4-D-degrading plasmid pJP4. C. necator JMP134(pJP4) can mineralize and grow on 2,4-D, while E. coli HB101(pJP4) cannot assimilate 2,4-D because it lacks the chromosomal genes to degrade the intermediates. The SBR with C. necator JMP134(pJP4) showed 100 % removal against 200 mg/l of 2,4-D just after its introduction, after which 2,4-D removal dropped to 0 % on day 7 with the decline in viability of the introduced strain. The SBR with E. coli HB101(pJP4) showed low 2,4-D removal, i.e., below 10 %, until day 7. Transconjugant strains of Pseudomonas and Achromobacter isolated on day 7 could not grow on 2,4-D. Both SBRs started removing 2,4-D at 100 % after day 16 with the appearance of 2,4-D-degrading transconjugants belonging to Achromobacter, Burkholderia, Cupriavidus, and Pandoraea. After the influent 2,4-D concentration was increased to 500 mg/l on day 65, the SBR with E. coli HB101(pJP4) maintained stable 2,4-D removal of more than 95 %. Although the SBR with C. necator JMP134(pJP4) showed a temporal depression of 2,4-D removal of 65 % on day 76, almost 100 % removal was achieved thereafter. During this period, transconjugants isolated from both SBRs were mainly Achromobacter with high 2,4-D-degrading capability. In conclusion, plasmid-mediated bioaugmentation can enhance the degradation capability of activated sludge regardless of the survival of introduced strains and their 2,4-D degradation capacity.     

Thalassobius aquaeponti sp. nov., an alphaproteobacterium isolated from seawater

A Gram-stain-negative, aerobic, non-motile and rod-shaped or ovoid bacterial strain, GJSW-22^T, which was isolated from seawater at Geoje island in South Korea, was characterized taxonomically. Strain GJSW-22^T was observed to grow optimally at 30 °C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain GJSW-22^T grouped with the type strains of Thalassobius species, forming a stable cluster with the type strain of Thalassobius aestuarii (bootstrap value of 83.2 %). Strain GJSW-22^T exhibited the highest 16S rRNA gene sequence similarity value (98.0 %) to the type strain of T. aestuarii. It exhibited 16S rRNA gene sequence similarity values of 95.6–96.1 % to the type strains of the other Thalassobius species. Strain GJSW-22^T was found to contain Q-10 as the predominant ubiquinone and C_18:1 ω7c and 11-methyl C_18:1 ω7c as the major fatty acids. The major polar lipids of strain GJSW-22^T were identified as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. The DNA G + C content of strain GJSW-22^T is 60.3 mol % and its mean DNA–DNA relatedness value with the type strain of T. aestuarii was 23 %. Differential phenotypic properties, together with the phylogenetic and chemotaxonomic data, confirmed that strain GJSW-22^T is distinct from other Thalassobius species. On the basis of the data presented, strain GJSW-22^T is considered to represent a novel species of the genus Thalassobius, for which the name Thalassobius aquaeponti sp. nov. is proposed. The type strain is GJSW-22^T (=KCTC 42115^T = NBRC 110378^T).     

Investigation of antibacterial activity of Bacillus spp. isolated from the feces of Giant Panda and characterization of their antimicrobial gene distributions

Bacillus group is a prevalent community of Giant Panda’s intestinal flora, and plays a significant role in the field of biological control of pathogens. To understand the diversity of Bacillus group from the Giant Panda intestine and their functions in maintaining the balance of the intestinal microflora of Giant Panda, this study isolated a significant number of strains of Bacillus spp. from the feces of Giant Panda, compared the inhibitory effects of these strains on three common enteric pathogens, investigated the distributions of six universal antimicrobial genes (ituA, hag, tasA, sfp, spaS and mrsA) found within the Bacillus group by PCR, and analyzed the characterization of antimicrobial gene distributions in these strains using statistical methods. The results suggest that 34 strains of Bacillus spp. were isolated which has not previously been detected at such a scale, these Bacillus strains could be classified into five categories as well as an external strain by 16S rRNA; Most of Bacillus strains are able to inhibit enteric pathogens, and the antimicrobial abilities may be correlated to their categories of 16S rRNA; The detection rates of six common antimicrobial genes are between 20.58 %(7/34) and 79.41 %(27/34), and genes distribute in three clusters in these strains. We found that the antimicrobial abilities of Bacillus strains can be one of the mechanisms by which Giant Panda maintains its intestinal microflora balance, and may be correlated to their phylogeny.