Long-Term Storage of Infective Microsporidian Spores in Liquid Nitrogen

Thirty-one species of microsporidia, isolated from insects and stored in liquid nitrogen for up to 25 yr, were infectious when removed from liquid nitrogen. The natural hosts of all of these microsporidia were terrestrial insects, representing six different insect orders: Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, and Orthoptera. All microsporidia from terrestrial insects that were tested survived storage in liquid nitrogen, while Nosema algerae , a microsporidium from aquatic mosquito hosts did not survive freezing in liquid nitrogen. A Nosema species from the alfalfa weevil, Hypera postica , lost some infectivity in a water storage medium after 25 yr in liquid nitrogen. Liquid nitrogen storage of microsporidian spores in 50% and 100% glycerol media reduced loss of infectivity and is recommended for extended storage of microsporidia from terrestrial insect hosts.     

噬菌体治疗细菌性感染及其限制因素

近年来,细菌耐药性已成为抗感染领域面临的严峻问题,临床对一些细菌性感染疾病束手无策。噬菌体疗法是一种通过噬菌体裂解细菌来治疗病原菌感染的治疗手段。噬菌体在抗菌领域表现出显著的优越性,成为目前治疗细菌性感染的研究热点。本文对近年来噬菌体治疗动物和人类病原菌感染、限制其临床应用的因素及解决措施进行综述。     

Modified phages: novel antimicrobial agents to combat infectious diseases

Researchers increasingly believe that microbial, molecular and synthetic biology techniques along with genetic engineering will facilitate the treatment of persistent infectious diseases. However, such therapy has been plagued by the emergence of antibiotic-resistant bacteria, resulting in significant obstacles to treatment. Phage therapy is one promising alternative to antibiotics, especially now that recent modifications to ubiquitous phages have made them more controllable. Additionally, convincing in vitro and in vivo studies of genetically modified lytic phages and engineered non-lytic phages have confirmed the advantages of novel, specific bactericidal agents over antibiotics in some cases. There is still a need for a better understanding of phage therapy, however, before it can be adopted widely.     

Genomic comparison of 93 Bacillus phages reveals 12 clusters, 14 singletons and remarkable diversity

Background

The Bacillus genus of Firmicutes bacteria is ubiquitous in nature and includes one of the best characterized model organisms, B. subtilis, as well as medically significant human pathogens, the most notorious being B. anthracis and B. cereus. As the most abundant living entities on the planet, bacteriophages are known to heavily influence the ecology and evolution of their hosts, including providing virulence factors. Thus, the identification and analysis of Bacillus phages is critical to understanding the evolution of Bacillus species, including pathogenic strains.

Results

Whole genome nucleotide and proteome comparison of the 93 extant Bacillus phages revealed 12 distinct clusters, 28 subclusters and 14 singleton phages. Host analysis of these clusters supports host boundaries at the subcluster level and suggests phages as vectors for genetic transfer within the Bacillus cereus group, with B. anthracis as a distant member of the group. Analysis of the proteins conserved among these phages reveals enormous diversity and the uncharacterized nature of these phages, with a total of 4,922 protein families (phams) of which only 951 (19%) had a predicted function. In addition, 3,058 (62%) of phams were orphams (phams containing a gene product from a single phage). The most populated phams were those encoding proteins involved in DNA metabolism, virion structure and assembly, cell lysis, or host function. These included several genes that may contribute to the pathogenicity of Bacillus strains.

Conclusions

This analysis provides a basis for understanding and characterizing Bacillus phages and other related phages as well as their contributions to the evolution and pathogenicity of Bacillus cereus group bacteria. The presence of sparsely populated clusters, the high ratio of singletons to clusters, and the large number of uncharacterized, conserved proteins confirms the need for more Bacillus phage isolation in order to understand the full extent of their diversity as well as their impact on host evolution.     

Erratum to: genomic comparison of 93 Bacillus phages reveals 12 clusters, 14 singletons and remarkable diversity

Background

The Bacillus genus of Firmicutes bacteria is ubiquitous in nature and includes one of the best characterized model organisms, B. subtilis, as well as medically significant human pathogens, the most notorious being B. anthracis and B. cereus. As the most abundant living entities on the planet, bacteriophages are known to heavily influence the ecology and evolution of their hosts, including providing virulence factors. Thus, the identification and analysis of Bacillus phages is critical to understanding the evolution of Bacillus species, including pathogenic strains.

Results

Whole genome nucleotide and proteome comparison of the 83 extant, fully sequenced Bacillus phages revealed 10 distinct clusters, 24 subclusters and 15 singleton phages. Host analysis of these clusters supports host boundaries at the subcluster level and suggests phages as vectors for genetic transfer within the Bacillus cereus group, with B. anthracis as a distant member. Analysis of the proteins conserved among these phages reveals enormous diversity and the uncharacterized nature of these phages, with a total of 4,442 protein families (phams) of which only 894 (20%) had a predicted function. In addition, 2,583 (58%) of phams were orphams (phams containing a single member). The most populated phams were those encoding proteins involved in DNA metabolism, virion structure and assembly, cell lysis, or host function. These included several genes that may contribute to the pathogenicity of Bacillus strains.

Conclusions

This analysis provides a basis for understanding and characterizing Bacillus and other related phages as well as their contributions to the evolution and pathogenicity of Bacillus cereus group bacteria. The presence of sparsely populated clusters, the high ratio of singletons to clusters, and the large number of uncharacterized, conserved proteins confirms the need for more Bacillus phage isolation in order to understand the full extent of their diversity as well as their impact on host evolution.     

A new method for the preservation of fungus stock cultures by deep-freezing

Recovery of 66 fungus stock cultures including Oomycota, Zygomycota, Ascomycota, Basidiomycota, and mitosporic mycetes were examined after cryopreservation. Almost all the stock cultures remained viable when the mycelia that had grown over the sawdust medium containing 10% glycerol as the cryoprotectant (65% moisture content, W/W) were frozen rapidly at −85°C and then allow to thaw naturally at room temperature. Test stock cultures were preserved for more than 10 years by this preservation method without any programmed precooling and rapid thawing for their cryopreservation. Most of the test fungi could survive for 5 years in medium containing 10% glycerol even after alternate freezing and thawing at intervals of 6 months. When a strain of Flammulina velutipes was tested for mycelial growth rate and productivity of fruit-bodies after cryopreservation for 3 years, the fungus reproduced with its initial capability. These results demonstrate that the sawdust-freezing method using a cryoprotectant is expected to be a reliable and easy preservation method for fungus stock cultures. Received: December 7, 2000 / Accepted: December 19, 2001     

Frozen fungi: cryogenic storage is an effective method to store Fusarium cultures for the long‐term

Microbial culture collections provide a vast amount of genotypic and phenotypic information which are invaluable resources for future advancements in research. For most microbial strains, cryopreservation in the vapour phase above liquid nitrogen provides the most stable and long‐term storage method. However, in the case of fungal microbes, not all are suited for cryogenic storage and few studies have addressed the effectiveness of storage in the vapour phase above liquid nitrogen on a diverse collection of Fusarium species. In this work, a collection of 374 Fusarium strains from the Fungal Genetics Stock Center, including 24 unique species, were duplicated and sent to the National Laboratory for Genetic Resource Preservation for storage in the vapour phase above liquid nitrogen. After 5 years of storage the entire collection was tested for viability and phenotypic stability by using plating, cellular staining assays, assessing the number of viable cells and measuring the rate of growth of each isolate. Additionally, the rate of growth for ～10% of the isolates were compared with the same isolates which had been stored at ?80°C at the Fungal Genetics Stock Center over the same timeframe to determine if cryopreservation in liquid nitrogen vapour provided a comparable method of storage. All National Laboratory for Genetic Resources Preservation isolates grew after being stored at ?165°C for 5 years. In general, the isolates that were stored at ?165°C grew at a faster rate than the isolates stored at ?80°C for the same period. Of the isolates stored at ?165°C, most had greater than 80% cell viability, however, those isolates that had less than 50% cell viability generally also had fewer conidia germinate. These isolates may be at a greater risk for storage over longer times. In conclusion, storage at ?165°C liquid nitrogen provided reliable preservation of a diverse collection of Fusarium spp. over 5 years, and culture viability data indicates that they will remain viable during additional storage for longer periods.     

The relationship between fungal preservation method and secondary metabolite production in Metarhizium anisopliae and Fusarium oxysporum

The effects of preservation regime on secondary metabolite production in two genera of economically important fungi, Metarhizium anisopliae and Fusarium oxysporum, was assessed using thin layer chromatography and high performance liquid chromatography over a 2-year testing period. Five preservation regimes, commonly used in microbial culture collections throughout the world were investigated: continual sub-culture, lyophilization, storage of mycelial plugs in water, storage at –20 °C and cryopreservation with liquid nitrogen. Preservation regime influenced secondary metabolite production in the test fungi. Changes in secondary metabolite profiles occurred after relatively short storage periods in most strains, irrespective of the preservation treatment used. Although no preservation treatment can be guaranteed to provide total stability of secondary metabolite production, cryopreservation was the best of the methods tested. Response to preservation and storage also differed between strains of the same species. Therefore, there is a need to develop new and existing preservation criteria with an emphasis on strain-specific criteria in order to reduce the prospects of instability in secondary metabolite production.     

A decision-based key to determine the most appropriate protocol for the preservation of fungi

The long-term preservation of valuable fungal cultures can be achieved in several ways and the choice of methodology can be problematical. Firstly, there is the decision whether to use a public service culture collection or `in-house' facilities. Secondly, the wide variety of preservation methods available often leads to confusion about which protocol(s) are best suited for specific fungi. No method can be universally applied to all fungi. Some species are notoriously difficult to preserve, whilst other fungi can be preserved by almost any method. A decision-based key has been devised, which uses questions related to fungal characters and user facilities and economics to determine the most appropriate method for long-term preservation of cultures. This key should facilitate the decisions of microbiologists when considering preservation of important fungal cultures.     

Structural protein analysis of the polyvalent staphylococcal bacteriophage 812

Phage 812 is a polyvalent phage with a very broad host range in the genus Staphylococcus, which makes it a suitable candidate for phage therapy of staphylococcal infections. This proteomic study, combining the results of both 1-DE and 2-DE followed by PMF, led to the identification of 24 virion proteins. Twenty new proteins, not yet identified by proteome analysis of closely related staphylococcal phages K and G1 were identified using this approach. Fifteen proteins were assigned unambiguously to the head-tail genome module; the remaining nine proteins are encoded by genes of the left or right arms of the phage genome. As expected, the most abundant proteins in the electrophoretic patterns are the major capsid protein, the major tail sheath protein and proteins identical to ORF 50 and ORF 95 of phage K, although their function is only putative. Identification of these 20 new proteins contributes substantially to a detailed characterization of phage virions, knowledge of which is necessary for rational phage therapy.     

<Emphasis Type="Italic"> Escherichia coli mazEF</Emphasis>-mediated cell death as a defense mechanism that inhibits the spread of phage P1

The Escherichia coli gene pair mazEF is a regulatable chromosomal toxin-antitoxin module: mazF encodes a stable toxin and mazE encodes for a labile antitoxin that overcomes the lethal effect of MazF. Because MazE is labile, inhibition of mazE expression results in cell death. We studied the effect of mazEF on the development of bacteriophage P1 upon thermoinduction of the prophage P1CM c1ts and upon infection with virulent phage particles (P1 _vir ). In several E. coli strains, we showed that the mazEF derivative strains produced significantly more phages than did the parent strain. In addition, upon induction of K38(P1CM c1ts), nearly all of the mazEF mutant cells lysed; in contrast, very few of the parental mazEF + K38 cells underwent lysis. However, most of these cells did not remain viable. Thus, while the mazEF cells die as a result of the lytic action of the phage, most of the mazEF ⁺ cells are killed by a different mechanism, apparently through the action of the chromosomal mazEF system itself. Furthermore, the introduction of lysogens into a growing non-lysogenic culture is lethal to mazEF but not for mazEF ⁺ cultures. Thus, although mazEF action causes individual cells to die, upon phage growth this is generally beneficial to the bacterial culture because it causes P1 phage exclusion from the bacterial population. These results provide additional support for the view that bacterial cultures may share some of the characteristics of multicellular organisms.Communicated by W. Arber     

Use of phages to control Campylobacter spp.

The use of phages to control pathogenic bacteria has been investigated since they were first discovered in the beginning of the 1900s. Over the last century we have slowly gained an in-depth understanding of phage biology including which phage properties are desirable when considering phage as biocontrol agents and which phage characteristics to potentially avoid. Campylobacter infections are amongst the most frequently encountered foodborne bacterial infections around the world. Handling and consumption of raw or undercooked poultry products have been determined to be the main route of transmission. The ability to use phages to target these bacteria has been studied for more than a decade and although we have made progress towards deciphering how best to use phages to control Campylobacter associated with poultry production, there is still much work to be done. This review outlines methods to improve the isolation of these elusive phages, as well as methods to identify desirable characteristics needed for a successful outcome. It also highlights the body of research undertaken so far and what criteria to consider when doing in-vivo studies, especially because some in-vitro studies have not been found to translate into to phage efficacy in-vivo.     

Recovery of fungi after storage for over a quarter of a century

This paper discusses the recovery of a sample of strains originally preserved on silica gel over the period 1970–1973. Fifty-three strains were tested of which 18 recovered, demonstrating survival for more than 20 years. The recovery of 26 of the strains is directly compared with that of replicates from oil storage and freeze-drying. A summary of storage of 421 strains by the silica gel technique is given, reporting survival of 64% for a quarter of a century or more. The technique is ideal for preserving sporulating fungi of the Ascomycota and many species of mitotic fungi for laboratories of limited facilities.     

Post-thaw boar sperm motility is affected by prolonged storage of sperm in liquid nitrogen. A retrospective study

Owing to the quick genetic turnover of the pig industry, most AI-boar sires live 2-3 yr, a period during which for 1-2 yr their semen is extended and used in liquid form for AI. Despite showing low cryosurvival, affecting fertility after AI, boar semen is frozen for easiness of transport overseas and reposition of valuable genetics. For the latter, semen is stored in liquid nitrogen (LN₂, cryostorage) for many years, a controversial practice. Here we studied how length of cryostorage could affect sperm quality. Straws (0.5 mL) frozen using the same cryopreservation protocol at one specific location from AI- sires of proven fertility were stored in LN₂ for up to 8 yr. Post-thaw sperm quality was evaluated after 2, 4 or 8 yr of cryostorage, always compared to early thawing (15 d after freezing). Sperm motility and kinematics were evaluated post-thaw using CASA and sperm viability was cytometrically evaluated using specific fluorophores. Sperm viability was not affected by length of cryostorage, but total and progressive sperm motility were lower (p < 0.01) in sperm samples cryostored for 4 or 8 yr compared to those thawed 15 d after freezing. Cryostorage time affected sperm kinetics, but with greater intensity in the samples cryostored for 4 yr (p < 0.001) than in those for 2 yr (p < 0.01). The fact that the major phenotypic characteristic of boar spermatozoa, motility, is constrained by time of cryostorage should be considered when building cryobanks of pig semen. Attention should be placed on the finding that >2 yr of cryostorage time can be particularly detrimental for the post-thaw motility of some sires, which might require increasing sperm numbers for AI.     

Phage resistance and altered growth habit in a strain of Streptococcus bovis

Bacteriophage (phi Sb01) of Streptococcus bovis, isolated from pooled rumen fluid of cattle, was a small siphovirus of morphotype B1. It contained double-stranded DNA of length 30.9 kb, which was digested by the restriction endonucleases, EcoRI, HindIII, and PvuII. Bacteria which survived phi Sb01 infection (strain 2BAr) grew in long chains (100-200 cells), ultimately forming large clumps of cells. This growth habit was in distinct contrast to that of the parent host strain which grew predominantly in the form of single cells or diplococci. Strain 2BAr was genetically stable, resistant to phi Sb01 attack, and the observed differences in the growth characteristics of the parent strain and 2BAr indicated that cells of 2BAr were more adherent. In the rumen ecosystem, the selection of phage-resistant bacteria with altered growth characteristics may be a factor in modifying bacterial phenotypes, and thus increasing variability among bacteria which are closely related genetically.     

Effects of different storage protocols on cat testis tissue potential for xenografting and recovery of spermatogenesis

The loss of genetic diversity due to premature death of valuable individuals is a significant problem in animal conservation programs, including endangered felids. Testis tissue xenografting has emerged as a system to obtain spermatozoa from dead immature animals, however protocols to store this tissue before xenografting are still lacking. This study focused on testis tissue cryopreservation and storage from the domestic cat (Felis catus) classified as “pre-pubertal” and “pubertal” according to spermatogenesis development. Grafts from testis tissue cryopreserved with DMSO 1.4M, recovered after 10 weeks xenografting, presented seminiferous tubules with no germ cells. On the contrary, testis tissue from pre-pubertal animals preserved in ice-cold medium for 2 to 5 days presented no loss of viability or spermatogenic potential, while the number of grafts of pubertal cat testis tissue with germ cells after 10 weeks of xenografting decreased with increasing storage time. Nevertheless, even grafts from pre-pubertal cat testis tissue presented lower anti-DDX4 and anti-BOULE staining (proteins necessary for the meiosis completion), when compared with adult cat testis. Finally, a strong correlation found between testis weight and xenograft outcome may help choose good candidates for xenografting.     

Successful storage of protocorm-like bodies of hybrid Cymbidium (Orchidaceae) under low temperature conditions

Low temperatures result in lower metabolic cellular activity, thus slowing down cell division and growth. This is advantageous where a plant scientist might seek to store important germplasm without the risks associated with low temperature storage. In this study, two cold temperatures above freezing, namely 4 and 10 °C, were tested to assess for how long PLBs could be preserved without a significant loss in regeneration ability (i.e., the ability to form neo-PLBs). Control treatments were cultured at 25 °C on Teixeira Cymbidium (TC) medium at a 16-h photoperiod at a photosynthetic photon flux density (PPFD) of 45 μmol m⁻² s⁻¹. For the cold treatments, each was replicated in the dark and at low light intensity (12-h photoperiod and a PPFD of 10 μmol m⁻² s⁻¹). All cultures were sub-cultured six times onto fresh medium every 60 days, for approximately 1 year. On the 7th subculture, all neo-PLBs were prepared uniformly and replated onto standard TC medium under light conditions described above for the control. 45 days after the 7th subculture and just before subcultures 1–6, the number of neo-PLBs per half-PLB was measured. The number of neo-PLBs that formed under different treatments depended strongly on the temperature and light conditions with most neo-PLBs forming under control conditions, although that number dropped significantly as the temperature was dropped to 10 °C and then even more to 4 °C, the same trend being observed when explants were cultured and subcultured under dim light, with organogenesis being more strongly negatively influenced in darkness. For all low-temperature treatments, as well as the dimmed light and darkness treatments, the number of neo-PLBs increased significantly when recultured, on the 7th subculture, onto control TC medium under control environmental conditions, almost as high as the control values. In contrast, the control values decreased, with significantly fewer neo-PLBs by the 7th subculture relative to the control, indicating that new PLBs should be induced from shoot cultures at least once a year to maintain their vitality.     

Ecology and evolution of phages encoding anti-CRISPR proteins

CRISPR-Cas are prokaryotic defence systems that provide protection against invasion by mobile genetic elements (MGE), including bacteriophages. MGE can overcome CRISPR-Cas defences by encoding anti-CRISPR (Acr) proteins. These proteins are produced in the early stages of the infection and inhibit the CRISPR-Cas machinery to allow phage replication. While research on Acr has mainly focused on their discovery, structure and mode of action, and their applications in biotechnology, the impact of Acr on the ecology of MGE as well as on the coevolution with their bacterial hosts only begins to be unravelled. In this review, we summarise our current understanding on the distribution of anti-CRISPR genes in MGE, the ecology of phages encoding Acr, and their coevolution with bacterial defence mechanisms. We highlight the need to use more diverse and complex experimental models to better understand the impact of anti-CRISPR in MGE-host interactions.     

Automated classification of tailed bacteriophages according to their neck organization

Background

The genetic diversity observed among bacteriophages remains a major obstacle for the identification of homologs and the comparison of their functional modules. In the structural module, although several classes of homologous proteins contributing to the head and tail structure can be detected, proteins of the head-to-tail connection (or neck) are generally more divergent. Yet, molecular analyses of a few tailed phages belonging to different morphological classes suggested that only a limited number of structural solutions are used in order to produce a functional virion. To challenge this hypothesis and analyze proteins diversity at the virion neck, we developed a specific computational strategy to cope with sequence divergence in phage proteins. We searched for homologs of a set of proteins encoded in the structural module using a phage learning database.

Results

We show that using a combination of iterative profile-profile comparison and gene context analyses, we can identify a set of head, neck and tail proteins in most tailed bacteriophages of our database. Classification of phages based on neck protein sequences delineates 4 Types corresponding to known morphological subfamilies. Further analysis of the most abundant Type 1 yields 10 Clusters characterized by consistent sets of head, neck and tail proteins. We developed Virfam, a webserver that automatically identifies proteins of the phage head-neck-tail module and assign phages to the most closely related cluster of phages. This server was tested against 624 new phages from the NCBI database. 93% of the tailed and unclassified phages could be assigned to our head-neck-tail based categories, thus highlighting the large representativeness of the identified virion architectures. Types and Clusters delineate consistent subgroups of Caudovirales, which correlate with several virion properties.

Conclusions

Our method and webserver have the capacity to automatically classify most tailed phages, detect their structural module, assign a function to a set of their head, neck and tail genes, provide their morphologic subtype and localize these phages within a “head-neck-tail” based classification. It should enable analysis of large sets of phage genomes. In particular, it should contribute to the classification of the abundant unknown viruses found on assembled contigs of metagenomic samples.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1027) contains supplementary material, which is available to authorized users.     

Viability of ectomycorrhizal fungi following cryopreservation

The use of ectomycorrhizal (ECM) fungi in biotechnological processes requires their maintenance over long periods under conditions that maintain their genetic, phenotypic, and physiological stability. Cryopreservation is considered as the most reliable method for long-term storage of most filamentous fungi. However, this technique is not widespread for ECM fungi since many do not survive or exhibit poor recovery after freezing. The aim of this study was to develop an efficient cryopreservation protocol for the long-term storage of ECM fungi. Two cryopreservation protocols were compared. The first protocol was the conventional straw protocol (SP). The mycelium of the ECM isolates was grown in Petri dishes on agar and subsequently collected by punching the mycelium into a sterile straw before cryopreservation. In the second protocol, the cryovial protocol (CP), the mycelium of the ECM isolates was grown directly in cryovials filled with agar and subsequently cryopreserved. The same cryoprotectant solution, freezing, and thawing process, and re-growth conditions were used in both protocols. The survival (positive when at least 60 % of the replicates showed re-growth) was evaluated before and immediately after freezing as well as after 1 week, 1 m, and 6 m of storage at −130 °C. Greater survival rate (80 % for the CP as compared to 25 % for the SP) and faster re-growth (within 10 d for the CP compared to the 4 weeks for the SP) were observed for most isolates with the CP suggesting that the preparation of the cultures prior to freezing had a significant impact on the isolates survival. The suitability of the CP for cryopreservation of ECM fungi was further confirmed on a set of 98 ECM isolates and displayed a survival rate of 88 % of the isolates. Only some isolates belonging to Suillus luteus, Hebeloma crustuliniforme, Paxillus involutus and Thelephora terrestris failed to survive. This suggested that the CP is an adequate method for the ultra-low cryopreservation of a large set of ECM fungi and that further studies are necessary for the more recalcitrant ones.