Public Microbial Resource Centers: Key Hubs for Findable, Accessible, Interoperable, and Reusable (FAIR) Microorganisms and Genetic Materials.

In the context of open science, the availability of research materials is essential for knowledge accumulation and to maximize the impact of scientific research. In microbiology, microbial domain biological resource centers (mBRCs) have long-standing experience in preserving and distributing authenticated microbial strains and genetic materials (e.g., recombinant plasmids and DNA libraries) to support new discoveries and follow-on studies. These culture collections play a central role in the conservation of microbial biodiversity and have expertise in cultivation, characterization, and taxonomy of microorganisms. Information associated with preserved biological resources is recorded in databases and is accessible through online catalogues. Legal expertise developed by mBRCs guarantees end users the traceability and legality of the acquired material, notably with respect to the Nagoya Protocol. However, awareness of the advantages of depositing biological materials in professional repositories remains low, and the necessity of securing strains and genetic resources for future research must be emphasized. This review describes the unique position of mBRCs in microbiology and molecular biology through their history, evolving roles, expertise, services, challenges, and international collaborations. It also calls for an increased deposit of strains and genetic resources, a responsibility shared by scientists, funding agencies, and publishers. Journal policies requesting a deposit during submission of a manuscript represent one of the measures to make more biological materials available to the broader community, hence fully releasing their potential and improving openness and reproducibility in scientific research.

Organic and mineral imprints in fossil photosynthetic mats of an East Antarctic lake.

Lacustrine microbial mats in Antarctic ice-free oases are considered modern analogues of early microbial ecosystems as their primary production is generally dominated by cyanobacteria, the heterotrophic food chain typically truncated due to extreme environmental conditions, and they are geographically isolated. To better understand early fossilization and mineralization processes in this context, we studied the microstructure and chemistry of organo-mineral associations in a suite of sediments 50-4530 cal. years old from a lake in Skarvsnes, Lützow Holm Bay, East Antarctica. First, we report an exceptional preservation of fossil autotrophs and their biomolecules on millennial timescales. The pigment scytonemin is preserved inside cyanobacterial sheaths. As non-pigmented sheaths are also preserved, scytonemin likely played little role in the preservation of sheath polysaccharides, which have been cross-linked by ether bonds. Coccoids preserved thylakoids and autofluorescence of pigments such as carotenoids. This exceptional preservation of autotrophs in the fossil mats argues for limited biodegradation during and after deposition. Moreover, cell-shaped aggregates preserved sulfur-rich nanoglobules, supporting fossilization of instable intracellular byproducts of chemotrophic or phototrophic S-oxidizers. Second, we report a diversity of micro- to nanostructured CaCO3 precipitates intimately associated with extracellular polymeric substances, cyanobacteria, and/or other prokaryotes. Micro-peloids Type 1 display features that distinguish them from known carbonates crystallized in inorganic conditions: (i) Type 1A are often filled with globular nanocarbonates and/or surrounded by a fibrous fringe, (ii) Type 1B are empty and display ovoid to wrinkled fringes of nanocrystallites that can be radially oriented (fibrous or triangular) or multilayered, and (iii) all show small-size variations. Type 2 rounded carbonates 1-2 µm in diameter occurring inside autofluorescent spheres interpreted as coccoidal bacteria may represent fossils of intracellular calcification. These organo-mineral associations support organically driven nanocarbonate crystallization and stabilization, hence providing potential markers for microbial calcification in ancient rocks.

Genetic diversity and amplification of different clostridial [FeFe] hydrogenases by group-specific degenerate primers.

AIMS: The aim of this study was to explore and characterize the genetic diversity of [FeFe] hydrogenases in a representative set of strains from Clostridium sp. and to reveal the existence of neither yet detected nor characterized [FeFe] hydrogenases in hydrogen-producing strains. METHODS AND RESULTS: The genomes of 57 Clostridium strains (34 different genotypic species), representing six phylogenetic clusters based on their 16S rRNA sequence analysis (cluster I, III, XIa, XIb, XIV and XVIII), were screened for different [FeFe] hydrogenases. Based on the obtained alignments, ten pairs of [FeFe] hydrogenase cluster-specific degenerate primers were newly designed. Ten Clostridium strains were screened by PCRs to assess the specificity of the primers designed and to examine the genetic diversity of [FeFe] hydrogenases. Using this approach, a diversity of hydrogenase genes was discovered in several species previously shown to produce hydrogen in bioreactors: Clostridium sartagoforme, Clostridium felsineum, Clostridium roseum and Clostridium pasteurianum. CONCLUSIONS: The newly designed [FeFe] hydrogenase cluster-specific primers, targeting the cluster-conserved regions, allow for a direct amplification of a specific hydrogenase gene from the species of interest. SIGNIFICANCE AND IMPACT OF THE STUDY: Using this strategy for a screening of different Clostridium ssp. will provide new insights into the diversity of hydrogenase genes and should be a first step to study a complex hydrogen metabolism of this genus.

Genome sequence of the edible cyanobacterium Arthrospira sp. PCC 8005.

We determined the genome sequence of Arthrospira sp. PCC 8005, a cyanobacterial strain of great interest to the European Space Agency for its nutritive value and oxygenic properties in the Micro-Ecological Life Support System Alternative (MELiSSA) biological life support system for long-term manned missions into space.

Cyanobacteria from benthic mats of Antarctic lakes as a source of new bioactivities.

AIMS: To exploit the cyanobacterial diversity of microbial mats growing in the benthic environment of Antarctic lakes for the discovery of novel antibiotic and antitumour activities. METHODS AND RESULTS: In all, 51 Antarctic cyanobacteria isolated from benthic mats were cultivated in the laboratory by optimizing temperature, irradiance and mixing. Productivity was generally very low (

Testing of primers for the study of cyanobacterial molecular diversity by DGGE.

Denaturing Gradient Gel electrophoresis (DGGE) is a PCR-based technique which is widely used in the study of microbial communities. Here, the use of the three specific 16S rRNA cyanobacterial specific primers CYA359F, CYA781R(a) and CYA781R(b) on the assessment of the molecular diversity of cyanobacterial communities is examined. Assignments of the reverse primers CYA781R(a) and CYA781R(b) with cyanobacterial strain sequences showed that the former preferentially targets filamentous cyanobacteria whereas the latter targets unicellular cyanobacteria. The influence of the GC clamp position on the forward or on reverse primer and the use of the two reverse primers separately or in equimolar mixture were investigated. Three environmental samples were subjected to amplification with 6 combinations of primers. The 6 banding patterns as well as the sequences of the bands extracted were analysed and compared. In addition, to assess the effect of the position of the GC clamp, the melting profiles of the sequences of Aphanizomenon flos-aquae PMC9707 and Synechococcus sp. MH305 were determined, with the GC clamp in the 3' or 5' position. Results showed that the use of two separate amplifications allowed a more complete study of the molecular diversity of the cyanobacterial community investigated. Furthermore, similar richness and identical phylogenetic assignments of extracted bands were obtained irrespective of the positioning of the GC clamp.

Amplified rDNA restriction analysis and further genotypic characterisation of metal-resistant soil bacteria and related facultative hydrogenotrophs.

The level of genotypic relationship between czc+ soil bacteria mainly resistant to zinc (but also to various other metals), and related facultative hydrogenotrophs previously assigned to the genera Alcaligenes, Ralstonia, and Burkholderia was evaluated using ARDRA (Amplified Ribosomal DNA Restriction Analysis). The analysis included 44 strains isolated from harsh industrial environments in sediments, soils and wastes with high content of heavy metals. These strains were selected by their ability to grow in the presence of high concentrations of multiple heavy metals and to hybridise with czc or ncc probes. The czc operon confers resistance to cadmium, zinc and cobalt in strain Ralstonia eutropha CH34. The ncc operon confers resistance to nickel, cobalt and cadmium in strain 31A known as Alcaligenes xylosoxidans. The analysis showed a close phylogenetic clustering of the czc+ strains inside the Ralstonia genus despite of their different origins and that the Ralstonia genus contained also the hydrogenotrophs and some catabolic strains assigned to the genus Ralstonia eutropha, strains up to now registrated as CDC IV c-2 strains as well as reference strains belonging to Ralstonia solanacearum and Ralstonia pickettii. The ncc+ strains are phylogenetically less related to each other compared to the czc+ strains. This suggests that the tested czc+ strains and some of the ncc+ strains may be considered as belonging to the genus Ralstonia. Inside this major Ralstonia cluster, a subcluster gathers most of the czc+ isolates maybe giving a clue to define a new species. Besides, from 30 tested strains, 15 metal resistant strains of this subcluster proved to display the unusual mutator phenotype characteristic of the representative strain CH34.

Arthrospira ('Spirulina') strains from four continents are resolved into only two clusters, based on amplified ribosomal DNA restriction analysis of the internally transcribed spacer.

We present the results of a phylogenetic study, based on amplified ribosomal DNA restriction analysis of the rDNA operon, of 37 Arthrospira ('Spirulina') cultivated clonal strains from four continents. In addition, duplicates from different culture collections or markedly different morphotypes of particular strains established as clonal cultures were treated as separate entries, resulting in a total of 51 tested cultures. The strain Spirulina laxissima SAG 256.80 was included as outgroup. The 16S rRNA genes appeared too conserved for discrimination of the strains by amplified ribosomal DNA restriction analysis, and thus the internally transcribed spacer was selected as molecular taxonomic marker. The internally transcribed spacer sequences situated between the 16S and the 23S rRNA were amplified by polymerase chain reaction and yielded amplicons of about 540 bp. Direct use of cells for polymerase chain reaction seemed to inhibit the amplification reaction. This was overcome by the design of a crude lysis protocol and addition of bovine serum albumin in the polymerase chain reaction mix. The amplicons were digested with four restriction enzymes (EcoRV, Hhal, Hinfl, Msel) and the banding patterns obtained were analyzed. Cluster analysis showed the separation of all the strains into two main clusters. No clear relationships could be observed between this division into two clusters and the geographic origin of the strains, or their designation in the culture collections, or their morphology.

Phylogenetic relationships of nonaxenic filamentous cyanobacterial strains based on 16S rRNA sequence analysis.

In order to determine the nearly complete 16S rRNA gene sequences of cyanobacteria originating from nonaxenic cultures, a cyanobacterium-specific oligonucleotide probe was developed to distinguish polymerase chain reaction (PCR) products of the cyanobacterial rRNA operons from those resulting from amplification of contaminating bacteria. Using this screening method the 16S rRNA genes of four nonaxenic filamentous cyanobacterial strains belonging to the genera Leptolyngbya and Oscillatoria were cloned and sequenced. For the genus Leptolyngbya, the 16S rRNA sequence of the axenic strain PCC 73110 was also determined. Phylogenetic trees were constructed based on complete and partial sequences. The results show that the strains Leptolyngbya foveolarum Komárek 1964/112, Leptolyngbya sp. VRUC 135 Albertano 1985/1, and Leptolyngbya boryanum PCC 73110 belong to the same cluster. Strain Oscillatoria cf. corallinae SAG 8.92, which contains the rare photosynthetic pigment CU-phycoerythrin, is not closely related to other CU-phycoerythrin-containing cyanobacteria. Oscillatoria agardhii CYA 18, which is a representative of planktonic Oscillatoria species that form toxic blooms in Norwegian inland waters, has no close relatives in the tree.

An early origin of plastids within the cyanobacterial divergence is suggested by evolutionary trees based on complete 16S rRNA sequences.

It is generally accepted that the plastids arose from a cyanobacterial ancestor, but the exact phylogenetic relationships between cyanobacteria and plastids are still controversial. Most studies based on partial 16S rRNA sequences suggested a relatively late origin of plastids within the cyanobacterial divergence. In order to clarify the exact relationship and divergence order of cyanobacteria and plastids, we studied their phylogeny on the basis of nearly complete 16S rRNA gene sequences. The data set comprised 15 strains of cyanobacteria from different morphological groups, 1 prochlorophyte, and plastids belonging to 8 species of plants and 12 species of diverse algae. This set included three cyanobacterial sequences determined in this study. This is the most comprehensive set of complete cyanobacterial and plastidial 16S rRNA sequences used so far. Phylogenetic trees were constructed using neighbor joining and maximum parsimony, and the reliability of the tree topologies was tested by different methods. Our results suggest an early origin of plastids within the cyanobacterial divergence, preceded only by the divergence of two cyanobacterial genera, Gloeobacter and Pseudanabaena.

Evolutionary affiliation of the marine nitrogen-fixing cyanobacterium Trichodesmium sp. strain NIBB 1067, derived by 16S ribosomal RNA sequence analysis.

The 16S rRNA sequence of Trichodesmium sp. strain NIBB 1067 was determined and used for the construction of a distance tree and bootstrap analysis. The tree shows that, among the available cyanobacterial 16S rRNA sequences, Trichodesmium NIBB 1067 has Oscillatoria PCC 7515 as its closest relative, presenting 94.9% of sequence similarity with the latter strain. This is in contrast to a difference of 9 mol% G+C in mean genomic DNA base composition between the two organisms. Nevertheless, the genotypic heterogeneity presented by a number of strains assigned to the genus Oscillatoria hinders a taxonomic decision on the separate existence of the genera Trichodesmium and Oscillatoria. The sequence of the internal transcribed spacer (ITS) between the 16S and 23S rRNA genes was also determined, as a possible marker to study inter- and intraspecific variability. The ITS contains the genes coding for tRNA(Ile) and tRNA(Ala) and its total length is 547 nucleotides. In six out of eight sequenced clones, there is a duplication of 29 nucleotides, surrounding the 5' end of the tRNA(Ile).

Plasmids for heavy metal resistance in Alcaligenes eutrophus CH34: mechanisms and applications.

Alcaligenes eutrophus CH34 is the main representative of a group of strongly related strains (mostly facultative chemolithotrophs) that are well adapted to environments containing high levels of heavy metals. It harbors the megaplasmids pMOL28 and pMOL30 which carry resistance determinants to Co2+, Ni2+, CrO(4)2-, Hg2+, Tl+, Cd2+, Cu2+ and Zn2+. Among the best characterized determinants are the cnr operon (resistance to Co, Ni) on pMOL28 and the czc operon on pMOL30 (resistance to Co, Cd and Zn). Although the two systems reveal a significant degree of amino acid similarity in the structural genes, the regulation of the operons is different. The resistance mechanism in both cases is based on efflux. The efflux mechanism leads to a pH increase outside of the cytoplasmic membrane. Metals are sequestered from the external medium through the bioprecipitation of metal carbonates formed in the saturated zone around the cell. This latter phenomenon can be exploited in bioreactors designed to remove metals from effluents. The bacteria are immobilized on composite membranes in a continuous tubular membrane reactor (CTMR). The effluent continuously circulates through the intertubular space, while the external surface of the tubes is in contact with the growth medium. Metal crystals are eventually removed by the effluent stream and collected on a glass bead column. The system has been applied to effluents containing Cd, Zn, Co, Ni and Cu. By introducing catabolic plasmids involved in the aerobic degradation of PCBs and 2,4-D into metal-resistant A. eutrophus strains, the application range was widened to include effluents polluted with both organic and inorganic substances. Biosensors have been developed which are based on the fusion of genes induced by metals to a reporter system, the lux operon of Vibrio fischeri. Bacterial luciferases produce light through the oxidation of fatty aldehydes. The gene fusions are useful both for the study of regulatory genes and for the determination of heavy metal concentrations in the environment.

Structure of the 16 S ribosomal RNA of the thermophilic cyanobacterium Chlorogloeopsis HTF ('Mastigocladus laminosus HTF') strain PCC7518, and phylogenetic analysis.

The thermophilic cyanobacterial strain, PCC7518, originally identified as 'Mastigocladus laminosus HTF' does not show branchings or heterocysts. The absence of branchings supports the later assignment to the genus Chlorogloeopsis. The absence of heterocysts may be the result of a mutation because heterocysts were observed in the original isolate. Alternatively, contamination may have happened. To solve this problem, the 16 S rRNA sequence was determined and used to infer a secondary structure model and build distance trees. The trees showed that strain PCC7518 belongs to the cluster of heterocystous species and has most probably lost the ability to produce heterocysts by mutation. It is only distantly related to Chlorogloeopsis fritschii PCC6718.