Isolation, antibacterial characterization, and alternating tangential flow-based preparation of viable cells of Lacticaseibacillus paracasei XLK 401: Potential application in milk preservation.

It is desirable to obtain high levels of viable Lacticaseibacillus paracasei, a widely used food probiotic whose antibacterial activity and potential application in milk remain largely uninvestigated. Here, we isolated and purified the L. paracasei strain XLK 401 from food-grade blueberry ferments and found that it exhibited strong antibacterial activity against both gram-positive and gram-negative foodborne pathogens, including Staphylococcus aureus, Salmonella paratyphi B, Escherichia coli O157, and Shigella flexneri. Then, we applied alternating tangential flow (ATF) technology to produce viable L. paracasei XLK 401 cells and its cell-free supernatant (CFS). Compared with the conventional fed-batch method, 22 h of ATF-based processing markedly increased the number of viable cells of L. paracasei XLK 401 to 12.14 ± 0.13 log cfu/mL. Additionally, the CFS exhibited good thermal stability and pH tolerance, inhibiting biofilm formation in the abovementioned foodborne pathogens. According to liquid chromatography-mass spectrometry analysis, organic acids were the main antibacterial components of XLK 401 CFS, accounting for its inhibition activity. Moreover, the CFS of L. paracasei XLK 401 effectively inhibited the growth of multidrug-resistant gram-positive Staph. aureus and gram-negative E. coli O157 pathogens in milk, and caused a reduction in the pathogenic cell counts by 6 to 7 log cfu/mL compared with untreated control, thus considerably maintaining the safety of milk samples. For the first time to our knowledge, ATF-based technology was employed to obtain viable L. paracasei on a large scale, and its CFS could serve as a broad-spectrum biopreservative for potential application against foodborne pathogens in milk products.

A novel bacteriocin against Staphylococcus aureus from Lactobacillus paracasei isolated from Yunnan traditional fermented yogurt: Purification, antibacterial characterization, and antibiofilm activity.

Staphylococcus aureus and its biofilm have emerged as a significant threat to the safety of dairy products. In recent years, lactic acid bacteria (LAB) bacteriocins have been widely acknowledged as the potential natural antibacterial substance in food biopreservation due to their excellent antibacterial effects. However, few LAB bacteriocins with antibacterial and antibiofilm activity against S. aureus have been reported in dairy products. In the present study, a novel bacteriocin LSX01 of Lactobacillus paracasei LS-6 isolated from a traditional fermented yogurt produced in Yunnan, China, was purified and characterized extensively. The LSX01 possessed a molecular weight of 967.49 Da and an AA sequence of LDQAGISYT. The minimum inhibitory concentration of LSX01 against S. aureus_45 was 16.90 µg/mL, which was close to or lower than the previously reported bacteriocins. The LSX01 exhibited an extensive antimicrobial spectrum against both gram-positive and gram-negative bacteria. Moreover, LSX01 exhibited excellent tolerance to heat and acid-base treatments, and sensitivity to the proteolytic enzymes, such as pepsin and proteinase K. Furthermore, the treatment of S. aureus_45 planktonic cells with LSX01 significantly reduced their metabolic activity and disrupted the cell membrane integrity. Scan electron microscopy results demonstrated that LSX01 induced cytoplasmic content leakage and cell deformation. Additionally, biofilm formation of S. aureus_45 was also significantly inhibited by LSX01. Overall, the results suggested that the novel LAB bacteriocin LSX01 possessed antibacterial activity and antibiofilm activity against S. aureus and, hence, could have potential for improving safety of dairy products.

[Effects of Bupleurum chinense on the diversity of intestinal flora in depressed mice].

Bupleuri Radix, serving as the sovereign medicinal in many antidepressant compound preparations, has been proved effective in treating depression in mice, but its effect on the intestinal flora remains unclear. The present study aimed to investigate the effects of Bupleurum chinense(one of the original materials of Bupleuri Radix) on the behaviors and the diversity of intestinal flora of depressed mice. A depression mouse model was induced by repeated social defeat stress. Specifically, C57 BL/6 J male mice were exposed to the attack from the CD-1 mice. Then, C57 BL/6 J male mice were divided into a depression group and a B. chinense group, with normal saline and B. chinense administered(ig) respectively. Sucrose preference test and tail suspension test were conducted during and after the experiment respectively, to analyze the effects of B. chinense on the behaviors of the depressed mice. The feces were collected after the experiment. The V3-V4 16 S rDNA regions of intestinal flora of mice in each group were sequenced by Ion S5 TMXL for the analysis of the number of operational taxonomic units(OTUs), richness, alpha and beta diversity indexes, and differential phyla and genera. The results indicated that B. chinense could decrease depressive-like behaviors of mice, increase sucrose preference, and shorten the time of immobility in tail suspension test. After B. chinense intervention, the relative abundance of Firmicutes was significantly decreased, while that of Bacteroidetes was increased at the phylum level. At the genus level, the relative abundance of Lactobacillus and Lachnoclostridium decreased(P<0.05), while that of Bacteroides, Alistopes, etc. was elevated(P<0.05). The findings demonstrate that B. chinense can regulate the intestinal flora and improve the depressive-like behaviors of mice with depression.

Comparative transcriptomic analysis of fireflies (Coleoptera: Lampyridae) to explore the molecular adaptations to fresh water.

Aquatic insects are well adapted to freshwater environments, but the molecular basis of these adaptations remains largely unknown. Most firefly species (Coleoptera: Lampyridae) are terrestrial, but the larvae of several species are aquatic. Here, larval and adult transcriptomes from Aquatica leii (freshwater) and Lychnuris praetexta (terrestrial) were generated to test whether the genes associated with metabolic efficiency and morphology have undergone adaptive evolution to fresh water. The aquatic fireflies had a significantly lower ratio of nonsynonymous to synonymous substitutions than the terrestrial insects, indicating a genomewide evolutionary constraint in the aquatic fireflies. We identified 341 fast-evolving genes and 116 positively selected genes in the aquatic fireflies. Of these, 76 genes exhibiting both fast evolution and positive selection were primarily involved in ATP production, energy metabolism and the hypoxia response. We identified 7,271 differentially expressed genes (DEGs) in A. leii (adults versus larvae) and 8,309 DEGs in L. praetexta (adults versus larvae). DEGs specific to the aquatic firefly (n = 1,445) were screened via interspecific comparisons (A. leii versus L. praetexta) and were significantly enriched for genes involved in metabolic efficiency (e.g., ATP production, hypoxia, and immune responses) and certain aspects of morphology (e.g., cuticle chitin, tracheal and compound eye morphology). These results indicate that sequence and expression-level changes in genes associated with both metabolic efficiency and morphological attributes related to the freshwater lifestyle contributed to freshwater adaptation in fireflies. This study provides new insights into the molecular mechanisms of aquatic adaptation in insects.

MMPphg from the thermophilic Meiothermus bacteriophage MMP17 as a potential antimicrobial agent against both Gram-negative and Gram-positive bacteria.

BACKGROUND: New strategies are urgently needed to deal with the growing problem of multidrug-resistant bacterial pathogens. As the natural viruses against bacteria, recently, bacteriophages have received particular attention. Here, we identified and characterized a novel peptidoglycan hydrolase named MMPphg by decoding the complete genome sequence of Meiothermus bacteriophage MMP17, which was isolated in Tengchong hot spring in China and contains a circular genome of 33,172 bp in size and a GC content of 63.4%. FINDINGS: We cloned the MMPphg gene, overproduced and purified the phage lytic protein, which contains a highly conserved M23 metallopeptidase domain and can be activated by Mg2+ and Zn2+. MMPphg is capable of withstanding temperatures up to 70 °C, and preserved more than 80% of its activity after a 30 min treatment between 35 and 65 °C. More interestingly, by disrupting bacterial cells, MMPphg exhibits surprising antimicrobial activity against both Gram-negative and Gram-positive pathogenic bacteria, especially antibiotic-resistant strains such as Escherichia coli O157, Staphylococcus aureus and Klebsiella pneumonia. CONCLUSIONS: In the current age of mounting antibiotic resistance, these results suggest the great potential of MMPphg, the gene product of bacteriophage MMP17, in combating bacterial infections and shed light on bacteriophage-based strategies to develop alternatives to conventional antibiotics for human or veterinary applications.

High cell-density fermentation, expression and purification of bacteriophage lysin TSPphg, a thermostable antimicrobial protein from extremophilic Thermus bacteriophage TSP4.

Recently, high cell-density (HCD) cultivation has become an important tool for production of many microbial products. However, to the best of our knowledge, no study regarding HCD fermentation, overproduction and purification of thermostable bacteriophage lysin has been reported. Here, by employing a glucose-limited fed-batch strategy, we performed high density fermentation of the host Escherichia coli BL21(DE3) cells, compared the efficiency of high pressure homogenization, ultrasonication and thermolysis in bacterial cell disruption after HCD cultivation, and purified TSPphg, a thermostable lysin derived from extremophilic bacteriophage TSP4. On the 20-L scale, the overproduction level of TSPphg was up to 67.8 ± 0.7%. In total, we obtained a broth titer of 3322.8 ± 26 mg/L TSPphg with a purity of 95.5 ± 0.7% from a bacterial cell mass of 86.3 ± 4.9 g/L after 26 h of fermentation. The overall productivity of TSPphg was 127.8 ± 1 mg/L/h. Additionally, the antimicrobial activity of purified TSPphg against both Gram-negative (Escherichia coli O157) and Gram-positive (Staphylococcus aureus) pathogenic bacteria was further confirmed by scanning electron microscope analysis. Summarily, for the first time, we have established a relatively stable and efficient HCD cultivation and purification process for recovery of thermostable lysins from extremophilic Thermus bacteriophages. Our results provide insights into the strategies for time-saving and cost-effective production of antimicrobial proteins to replace or supplement antibiotics in the current age of mounting antibiotic resistance.

Complete genome sequence of MMP7, a novel Meiothermus bacteriophage of the family Myoviridae isolated from a hot spring.

The genome sequence of a novel Meiothermus bacteriophage, named MMP7, which was isolated from Tengchong hot spring in Yunnan Province of China and belongs to the family Myoviridae, was sequenced in this study. To the best of our knowledge, this is the first reported genome sequence of a Meiothermus phage, which has a circular DNA genome of 32,864 bp and a GC content of 64%. The MMP7 genome contains 53 putative protein-encoding genes but no rRNA or tRNA genes, and it exhibits low overall sequence similarity and no significant homology to phage genomes whose sequences are publicly available, suggesting that MMP7 is a novel phage. Consistent with current taxonomic results, whole-genome-based phylogenetic analysis revealed that Meiothermus phage MMP7 has close evolutionary relationship to Thermus phages. Together, our results could be helpful for discovering new thermostable antimicrobial agents and understanding the evolution and genetic diversity of Meiothermus phages in extreme environments.

Identification of circular RNAs and their altered expression under poly(I:C) challenge in key antiviral immune pathways in amphioxus.

Amphioxus is a key model for studying comparative immunity of vertebrates. Circular RNA (circRNA), as RNAs with a circular structure, has received little attention until recently, where several studies have reported that circRNA expression changes are involved in the immune response in animals. However, circRNA and its immune role in amphioxus have not been previously studied. Here, circRNAs in Chinese amphioxus (Branchiostoma belcheri) were sequenced, and 1859 circRNAs were identified using two algorithms (find_circ and CIRI). The analysis of miRNA target sites on circRNAs showed that 332 circRNAs may function as miRNA sponges. Furthermore, we identified circRNAs that were conserved between B. belcheri and vertebrates, tracing the origin of these circRNAs within chordates. Additionally, in combination with several key antiviral immune (poly(I:C), pIC) pathways identified in our previous B. belcheri studies, nine circRNAs potentially involved in these pathways were identified using bioinformatic predictions. Among these nine circRNAs, eight were selected to examine their expression response in B. belcheri challenged by pIC in comparison to control using real-time quantitative PCR. The results showed that four circRNAs were induced as part of the antiviral response against pIC, while expression of two circRNAs was decreased, and the expression levels of the remaining two were not significantly altered after pIC challenge. This work is the first to identify circRNAs and reveal their antiviral role in amphioxus. Therefore, it opens a new window to explore the comparative immunology of circRNAs in chordates and the regulatory roles of circRNAs in antiviral immunity in amphioxus.

MicroRNA profile of immune response in gills of zebrafish (Danio rerio) upon Staphylococcus aureus infection.

The gills of fish are large mucosal surfaces that are very important portals for pathogen entry. Investigations have shown that microRNAs (miRNAs) are key regulators of immune response to bacterial infections in the gills of fish; however, how miRNA expression changes in response to infection by Gram-positive bacteria remains largely unknown. To further investigate the immunological role of miRNAs in fish gills under pathogen stress induced by Gram-positive bacterial infection, this study investigated Staphylococcus aureus (SA)-induced changes in the miRNAs levels in gills of adult zebrafish (Danio rerio). miRNA microarrays were used to analyze expression profiles of known miRNA in the gills of zebrafish in response to SA infection and compared these to uninfected control fish. A total of 30 differentially expressed miRNAs (DEMs) were identified. Target genes likely regulated by DEMs were predicted, and functional enrichment analyses were performed. The results indicated that DEM targets were primarily involved in innate immune processes, apoptosis, defense responses, and antibacterial responses. Pathways involving bacterial infection, innate immunity, metabolic process, disease, and apoptosis were mediated by DEMs. Furthermore, real-time quantitative PCR experiments for nine key SA-responsive DEMs that regulated the "SA infection" pathway validated the accuracy of microarray results. Dynamic variations in gene expression were surveyed in detail for these key SA-responsive DEMs for PBS control and at 6, 12, 24, and 48 h after SA challenge in detail. This study provides novel insight into the mechanisms underlying the miRNA regulation during the SA-induced immune response in zebrafish gills, and provides basic knowledge on the innate immune response against Gram-positive bacterial infection in bony fish.

Long noncoding RNA HOTTIP cooperates with CCCTC-binding factor to coordinate HOXA gene expression.

The spatiotemporal control of HOX gene expression is dependent on positional identity and often correlated to their genomic location within each loci. Maintenance of HOX expression patterns is under complex transcriptional and epigenetic regulation, which is not well understood. Here we demonstrate that HOTTIP, a lincRNA transcribed from the 5' edge of the HOXA locus, physically associates with the CCCTC-binding factor (CTCF) that serves as an insulator by organizing HOXA cluster into disjoint domains, to cooperatively maintain the chromatin modifications of HOXA genes and thus coordinate the transcriptional activation of distal HOXA genes in human foreskin fibroblasts. Our results reveal the functional connection of HOTTIP and CTCF, and shed light on lincRNAs in gene activation and CTCF mediated chromatin organization.

Rapid purification of bacteriophage endolysin TSPphg and its exogenous treatment could act as an alternative bacterial cell disruption method.

Bacteriophage endolysins have long been demonstrated to be effective enzybiotics, and have the potential value in the areas of food, agricultural, and industrial science. Traditionally, extraction of recombinant proteins from bacterium E. coli is achieved by chemical, biological or mechanical disruption methods. Here, we present heat treatment, a simple and highly effective method that differs from the conventional ones, for disruption of E. coli cells to extract recombinant TSPphg, an endolysin originated from thermophilic bacteriophage TSP4. In addition, we found that exogenous TSPphg treatment is able to disrupt E. coli cell and release its intracellular proteins, suggesting its great potentiality to be developed as an alternative bacterial cell disruption method. Moreover, the large scale purification of TSPphg by heat treatment can be carried out directly in fermentation broth in situ without complex downstream processing, which may make it a more applicable approach for commercial scale processes. Our findings shed light on recovery of recombinant thermostable proteins and strategy of bacterial cell disruption.

Comparative transcriptomic analysis provides insights into antibacterial mechanisms of Branchiostoma belcheri under Vibrio parahaemolyticus infection.

Amphioxus, a basal chordate, is widely considered to be an existing proxy of the invertebrate ancestor of vertebrates, and it exhibits susceptibility to various pathogen infections and pathogenic mimic challenges. Here, in order to understand more clearly its antibacterial mechanisms, we analyzed the ribosomal RNA (rRNA)-depleted transcriptome of Chinese amphioxus (Branchiostoma belcheri) infected with Vibrio parahaemolyticus (V. p.) via next-generation deep sequencing technology (RNA-seq). We identified a total of 3214 differentially expressed genes (DEGs) by comparing V. p.-infected and control transcriptome libraries, including 2219 significantly up-regulated and 995 significantly down-regulated DEGs in V. p.-infected amphioxus. The DEGs with the top 10 most dramatic expression fold changes after V. p. infection, as well as 53 immune-related DEGs (IRDs) belonging to four primary categories of innate immunity were analyzed further. Through gene ontology (GO) and pathway enrichment analysis, DEGs were found to be primarily related to immune processes, apoptosis, catabolic and metabolic processes, binding and enzyme activity, while pathways involving bacterial infection, immune signaling, immune response, cancer, and apoptosis were overrepresented. We validated the RNA-seq results by detecting the expression levels of 10 IRDs using qRT-PCR, and we surveyed the dynamic variation in gene expression for these IRDs at 0, 6, 12, 24, and 48 h after V. p. TREATMENT: Subsequently, according to the RNA-seq results, the presence of a primitive Toll-like receptor (TLR)-mediated antibacterial immune signaling pathway was predicted in B. belcheri. This study provides valuable information regarding antibacterial immunity for further research into the evolution of immunity in vertebrates and broadens our understanding of the innate immune response against bacterial invasion in amphioxus.

Isolation and characterization of two lytic cold-active bacteriophages infecting Pseudomonas fluorescens from the Napahai plateau wetland.

As the "kidneys of the Earth", wetlands play important roles as biodiversity reservoirs, in water purification, and in flood control. In this study, 2 lytic cold-active bacteriophages, named VW-6S and VW-6B, infecting Pseudomonas fluorescens W-6 cells from the Napahai plateau wetland in China were isolated and characterized. Electron microscopy showed that both VW-6S and VW-6B had an icosahedral head (66.7 and 61.1 nm, respectively) and a long tail (8.3 nm width × 233.3 nm length and 11.1 nm width × 166.7 nm length, respectively). The bacteriophages VW-6S and VW-6B were classified as Siphoviridae and had an approximate genome size of 30-40 kb. The latent and burst periods of VW-6S were 60 and 30 min, whereas those of VW-6B were 30 and 30 min, respectively. The optimal pH values for the bacteriophages VW-6S and VW-6B were 8.0 and 10.0, respectively, and their activity decreased rapidly at temperatures higher than 60 °C. These cold-active bacteriophages provide good materials for further study of cold-adaptation mechanisms and interaction with the host P. fluorescens.

A novel malic enzyme gene, Mime2, from Mortierella isabellina M6-22 contributes to lipid accumulation.

OBJECTIVE: This study was aimed at cloning and characterizing a novel malic enzyme (ME) gene of Mortierella isabellina M6-22 and identifying its relation with lipid accumulation. METHODS: Mime2 was cloned from strain M6-22. Plasmid pET32aMIME2 was constructed to express ME of MIME2 in Escherichia coli BL21. After purification, the optimal pH and temperature of MIME2, as well as Km and Vmax for NADP+ were determined. The effects of EDTA or metal ions (Mn2+, Mg2+, Co2+, Cu2+, Ca2+, or Zn2+) on the enzymatic activity of MIME2 were evaluated. Besides, plasmid pRHMIME2 was created to express MIME2 in Rhodosporidium kratochvilovae YM25235, and its cell lipid content was measured by the acid-heating method. The optimal pH and temperature of MIME2 are 5.8 and 30 °C, respectively. RESULTS: The act ivity of MIME2 was significantly increased by Mg2+, Ca2+, or Mn2+ at 0.5 mM but inhibited by Cu2+ or Zn2+ (p < 0.05). The optimal enzymatic activity of MIME2 is 177.46 U/mg, and the Km and Vmax for NADP+ are 0.703 mM and 156.25 µg/min, respectively. Besides, Mime2 transformation significantly increased the cell lipid content in strain YM25235 (3.15 ± 0.24 vs. 2.17 ± 0.31 g/L, p < 0.01). CONCLUSIONS: The novel ME gene Mime2 isolated from strain M6-22 contributes to lipid accumulation in strain YM25235.

Complete genome sequence of the lytic cold-active Pseudomonas fluorescens bacteriophage VSW-3 from Napahai plateau wetland.

The lytic cold-active bacteriophage VSW-3, belonging to the Podoviridae family and infecting the host Pseudomonas fluorescens SW-3, was isolated from the Napahai plateau wetland in China. With the development of sequencing technology, the study of Pseudomonas genomic diversity has increased; however, knowledge of cold-active phages infecting Pseudomonas is limited. The newly sequenced phage VSW-3 was classified based on virion morphology by transmission electron microscope. Sequence analysis revealed that the genome size was 40,556 bp with an overall GC content of 57.54 % and 46 open reading frames. The genome was organized into several modules containing genes for packaging, structural proteins, replication/transcription, and phage lysis. The sequence contained 45 potential promoters, 3 transcription terminators, and yet no tRNAs. This is the first report of cold-active Pseudomonas fluorescens bacteriophage genome sequencing.

Isolation and characterization of wetland VSW-3, a novel lytic cold-active bacteriophage of Pseudomonas fluorescens.

Wetlands are often called the "kidneys of the Earth" and contribute substantially to environmental improvement. Pseudomonas fluorescens is a major contaminant of milk products and causes the spoilage of refrigerated foods and fresh poultry. In this study, we isolated and characterized a lytic cold-active bacteriophage named VSW-3 together with P. fluorescens SW-3 cells from the Napahai wetland in China. Electron microscopy showed that VSW-3 had an icosahedral head (56 nm) and a tapering tail (20 nm × 12 nm) and a genome size of approximate 40 kb. On the basis of the top-scoring hits in the BLASTP analysis, VSW-3 showed a high degree of module similarity to the Pseudomonas phages Andromeda and Bf7. The latent and burst periods were 45 and 20 min, respectively, with an average burst size of 90 phage particles per infected cell. The pH and thermal stability of VSW-3 were also explored. The optimal pH was found to be 7.0 and the activity decreased rapidly when the temperature exceeded 60 °C. VSW-3 is a cold-active bacteriophage, hence, it is important to research its ability to prevent product contamination caused by P. fluorescens and to characterize its relationship with its host P. fluorescens in the future.

Proteomic analysis of Mortierella isabellina M6-22 during cold stress.

We aimed to gain a better understanding of cold adaption in Mortierella isabellina M6-22 by using proteomics approaches. The temperature range and optimal temperature for M6-22 growth were investigated, and composition changes in fatty acids were analyzed. Accompanied with the 2-D gel electrophoresis, MALDI-TOF/TOF-MS analysis was conducted to characterize alterations in protein profiling in M6-22 cultured at 30 °C for 24 h and 15 °C for another 24 h when compared with those cultured at 30 °C for 48 h. Gene Ontology (GO) cluster analysis was finally conducted for successfully identified proteins. M6-22 cells could grow well at temperatures ranging from 15 to 30 °C. As temperature decreased from 30 to 15 °C, LA and GLA significantly increased from 11.63 to 17.85 % and from 9.12 to 13.19 %, respectively, while oleic acid significantly decreased from 47.25 to 36.53 %. Proteomics analyses revealed 111 differentially expressed protein spots, among which 5 unique proteins (A38, A40, A47, A49 and A58), 29 up-regulated proteins and 10 down-regulated proteins were identified by MALDI-TOF/TOF-MS. GO enrichment analysis demonstrated that these proteins mainly involved in glycolytic pathway (A34 and A50), electron transport (A28), ATP production (A35 and B39) and protein modification (A38). A total of 44 differentially expressed proteins have been successfully identified in M. isabellina M6-22 cultured at 15 °C. These proteins may play important roles in cold adaption via regulation of ATP synthesis, activation of cold-adaptive proteins, degradation of needless protein, accumulation of PUFAs, etc.

Complete genome sequence of the cold-active bacteriophage VMY22 from Bacillus cereus.

The cold-active bacteriophage VMY22, belonging to the Podoviridae family, was isolated from Mingyong Glacier in China. Sequence analysis revealed that the genome is 18,609 bp long, with an overall G + C content of 36.4 mol%, and 25 open reading frames (ORFs). The sequence contains 46 potential promoters, 6 transcription terminators, and no tRNAs. Most of the ORFs show a high degree of similarity to B103 (NC_004165). Two noteworthy findings were made. First, one of the predicted proteins, ORF 19, shows high sequence similarity to the bacteriocin biosynthesis protein from Bacillus cereus. From this information, we propose that the VMY22 phage is at an intermediate phase in its coevolution with its bacterial host. Second, seven of the hypothetical proteins appear to be unique to this cold-active B. cereus phage (i.e., not found in temperate-active B. cereus phages). These observations add to our current knowledge about the coevolution of bacteriophages and their hosts. The identification of a novel group of gene and protein structures and functions will lead to a better understanding of cold-adaptation mechanisms in bacteria and their bacteriophages.

Isolation and Characterization of the Lytic Cold-Active Bacteriophage MYSP06 from the Mingyong Glacier in China.

As unique ecological systems, glaciers are characterized by low temperatures and low nutrient levels, which allow them to be considered as "living fossils" for the purpose of researching the evolution of life and the environmental evolution of the earth. Glaciers are also natural microbial "reservoirs". In this work, a lytic cold-active bacteriophage designated MYSP06 was isolated from Janthinobacterium sp. MYB06 from the Mingyong Glacier in China, and its major characteristics were determined. Electron microscopy revealed that bacteriophage MYSP06 had an isometric head (74 nm) and a long tail (10 nm in width, 210 nm in length). It was classified as a Siphoviridae with an approximate genome size of 65­70 kb. A one-step growth curve revealed that the latent and burst periods were 95 and 65 min, respectively, with an average burst size of 16 bacteriophage particles per infected cell. The bacteriophage particles (100 %) adsorbed to the host cells within 10 min after infection. Moreover, the pH value and thermal stability of bacteriophage MYSP06 were also investigated. The maximum stability of the bacteriophage was observed at the optimal pH 7.0, and the bacteriophage became completely unstable at the extremely alkaline pH 11.0; however, it was comparatively stable at the acidic alkaline pH 6.0. As MYSP06 is a cold-active bacteriophage with a lower production temperature, its characterization and its relationship with its host Janthinobacterium sp. MYB06 deserve further study.

Identification and characterization of a novel bifunctional Δ(12)/Δ(15)-fatty acid desaturase gene from Rhodosporidium kratochvilovae.

OBJECTIVES: To elucidate the biosynthesis pathway of linoleic acid and α-linolenic acid in Rhodosporidium kratochvilovae YM25235 and investigate the correlation of polyunsaturated fatty acids with its cold adaptation. RESULTS: A 1341 bp cDNA sequence, designated as RKD12, putatively encoding a Δ(12)-desaturase was isolated from YM25235. Sequence analysis indicated that this sequence comprised a complete ORF encoding 446 amino acids of 50.6 kDa. The encoded amino acid sequence shared higher similarity to known fungal Δ(12)-desaturases that are characteristic of three conserved histidine-rich motifs. RKD12 was further transformed into Saccharomyces cerevisiae INVScl for functional characterization. Fatty acid analysis showed the yeast transformants accumulated two new fatty acids: linoleic acid and α-linolenic acid. Furthermore, mRNA expression level of RKD12 and the content of linoleic acid and α-linolenic acid were increased significantly with the culture temperature downshift from 30 to 15 °C, which might be helpful for the cold adaptation of YM25235. CONCLUSION: RKD12 is a novel bifunctional ∆(12)/∆(15)-desaturase gene, and the increased RKD12 mRNA expression level and PUFAs content at low temperature might be helpful for the cold adaptation of YM25235.