Contribution of Next-Generation Sequencing to Aquatic and Fish Virology.

The recent technological advances in nucleic acid sequencing, called next-generation sequencing (NGS), have revolutionized the field of genomics and have also influenced viral research. Aquatic viruses, and especially those infecting fish, have also greatly benefited from NGS technologies, which provide a huge amount of molecular information at a low cost in a relatively short period of time. Here, we review the use of the current high-throughput sequencing platforms with a special focus on the associated challenges (regarding sample preparation and bioinformatics) in their applications to the field of aquatic virology, especially for: (i) discovering novel viruses that may be associated with fish mortalities, (ii) elucidating the mechanisms of pathogenesis, and finally (iii) studying the molecular epidemiology of these pathogens.

Rapid detection and identification of nontuberculous mycobacterial pathogens in fish by using high-resolution melting analysis.

Mycobacterial infections in fish are commonly referred to as piscine mycobacteriosis, irrespectively of the specific identity of the causal organism. They usually cause a chronic disease and sometimes may result in high mortalities and severe economic losses. Nearly 20 species of Mycobacterium have been reported to infect fish. Among them, Mycobacterium marinum, M. fortuitum, and M. chelonae are generally considered the major agents responsible for fish mycobacteriosis. As no quick and inexpensive diagnostic test exists, we tested the potential of high-resolution melting analysis (HRMA) to rapidly identify and differentiate several Mycobacterium species involved in fish infections. By analyzing both the melting temperature and melting profile of the 16S-23S rRNA internal transcribed spacer (ITS), we were able to discriminate 12 different species simultaneously. Sensitivity tests conducted on purified M. marinum and M. fortuitum DNA revealed a limit of detection of 10 genome equivalents per reaction. The primers used in this procedure did not lead to any amplification signal with 16 control non-Mycobacterium species, thereby demonstrating their specificity for the genus Mycobacterium.

Microarrays in virology: basic concepts and applications for virus detection.

For a few decades, the introduction and development of molecular methods in microbiology have shaped the detection and characterization of pathogens. Although serological and, more punctually, viral culture methods remain basic tools for viral diagnosis, molecular advances based on qPCR have brought a number of novel advantages, in terms of speed, specificity and costs. On the other hand, microarrays have demonstrated their own advantages by increasing drastically the capabilities of detection and characterization of a large range of viruses in a unique step. Nowadays, several microarray-based platforms exist that can be classified in different families according to the type of matrix (solid or liquid), the size and density of probes, the method used for visualizing hybridization results with the target and finally relative costs. The aims of this review will be to overview (i) basic concepts of the different technologies used and to enlighten differences, advantages and drawbacks of each type of platform and (ii) the applications in virology for the detection and characterization of viral agents.

Structural characterization of ISCR8, ISCR22, and ISCR23, subgroups of IS91-like insertion elements.

Analysis of ISCR8 (ISPps1) revealed that this group of insertion elements has to be subdivided into three subgroups: ISCR8, ISCR22, and ISCR23. The distinction of three subgroups is supported by phylogenetic analysis of the transposase open reading frames (ORFs). Comparison of over 20 complete and partial ISCR8/22/23 elements identified oriIS candidate sequences for all groups and a terIS candidate sequence for ISCR8. The oriIS sequences, their distance to the transposase ORFs, and the sequence of this intervening region are group specific, further supporting the definition of two new ISCR elements. ISCR8/22/23 have a very broad host range, including Gram-positive and Gram-negative bacteria, among which are several (opportunistic) pathogens. The IS often resides on plasmids or in the vicinity of other mobile elements and is mostly associated with genes for the degradation of halo- or nitro-aromatics. However, in one case ISCR8 was found in the neighborhood of an antibiotic resistance determinant in Klebsiella pneumoniae. ISCR8 resembles other IS91 family elements in mediating genetic rearrangements by homologous recombination between two copies. In Delftia acidovorans this led to the loss of the genes encoding dichlorprop cleavage. In conclusion, this study shows that ISCR8 could be a fully functional and active member of the IS91 family of insertion elements.

Identification of brevibacteriaceae by multilocus sequence typing and comparative genomic hybridization analyses.

Multilocus sequence typing with nine selected genes is shown to be a promising new tool for accurate identifications of Brevibacteriaceae at the species level. A developed microarray also allows intraspecific diversity investigations of Brevibacterium aurantiacum showing that 13% to 15% of the genes of strain ATCC 9174 were absent or divergent in strain BL2 or ATCC 9175.

Microbial interactions within a cheese microbial community.

The interactions that occur during the ripening of smear cheeses are not well understood. Yeast-yeast interactions and yeast-bacterium interactions were investigated within a microbial community composed of three yeasts and six bacteria found in cheese. The growth dynamics of this community was precisely described during the ripening of a model cheese, and the Lotka-Volterra model was used to evaluate species interactions. Subsequently, the effects on ecosystem functioning of yeast omissions in the microbial community were evaluated. It was found both in the Lotka-Volterra model and in the omission study that negative interactions occurred between yeasts. Yarrowia lipolytica inhibited mycelial expansion of Geotrichum candidum, whereas Y. lipolytica and G. candidum inhibited Debaryomyces hansenii cell viability during the stationary phase. However, the mechanisms involved in these interactions remain unclear. It was also shown that yeast-bacterium interactions played a significant role in the establishment of this multispecies ecosystem on the cheese surface. Yeasts were key species in bacterial development, but their influences on the bacteria differed. It appeared that the growth of Arthrobacter arilaitensis or Hafnia alvei relied less on a specific yeast function because these species dominated the bacterial flora, regardless of which yeasts were present in the ecosystem. For other bacteria, such as Leucobacter sp. or Brevibacterium aurantiacum, growth relied on a specific yeast, i.e., G. candidum. Furthermore, B. aurantiacum, Corynebacterium casei, and Staphylococcus xylosus showed reduced colonization capacities in comparison with the other bacteria in this model cheese. Bacterium-bacterium interactions could not be clearly identified.

Spore-forming bacteria responsible for food spoilage.

This review explores the main spore-forming bacteria involved in the spoilage of various processed foods. Bakery products are specifically spoiled by Bacillus species, the dominant one being Bacillus amyloliquefaciens, while different Clostridium species classically contaminate refrigerated vacuum-packed meats. These two genera have also been isolated from milk products, even when milk is pasteurized, sterilized, dehydrated or fermented, according to heat treatment and storage temperature. Finally, the most heat-resistant microorganisms are isolated in low-acid canned foods, the three predominant species being Geobacillus stearothermophilus, Moorella thermoacetica and Thermoanaerobacterium spp.

Manual and expert annotation of the nearly complete genome sequence of Staphylococcus sciuri strain ATCC 29059: A reference for the oxidase-positive staphylococci that supports the atypical phenotypic features of the species group.

Staphylococcus sciuri is considered to be one of the most ancestral species in the natural history of the Staphylococcus genus that consists of 48 validly described species. It belongs to the basal group of oxidase-positive and novobiocin-resistant staphylococci that diverged from macrococci approximately 250 million years ago. Contrary to other groups, the S. sciuri species group has not developed host-specific colonization strategies. Genome analysis of S. sciuri ATCC 29059 provides here the first genetic basis for atypical traits that would support the switch between the free-living style and the infective state in animals and humans. From among the most remarkable features, it was noticed in this extensive study that there were a number of phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS), almost twice as many as any other staphylococci, and the co-occurrence of mevalonate and non-mevalonate pathways for isoprenoid synthesis. The sequenced strain was devoid of the main virulence factors present in Staphylococcus aureus, although it exhibited numerous heme and iron acquisition systems, as well as crt and aldH genes necessary for gold pigment synthesis. The sensing and signaling networks, exemplified by a large and typical repertoire of two-component regulatory systems and a complete panel of master regulators, such as agr, rex, mgrA, rot, sarA and sarR genes, depict the background in which S. aureus virulence genes were later acquired. An additional sigma factor, a distinct set of electron transducer elements and many gene operons similar to those found in Bacillus spp. would constitute the most visible remnant links with Bacillaceae organisms.

Genomic diversity and evolution within the species Streptococcus agalactiae.

Streptococcus agalactiae is a leading cause of invasive infections in neonates, and responsible for bovine mastitis. It is also a commensal bacterium adapted to asymptomatic colonization of the mammalian gut and of the genitourinary tract. Here, we report the analysis of a collection of 75 strains of human and animal origin by using serotyping, multilocus sequence typing, whole genome DNA-array hybridizations and sequence comparison of putatively virulence-associated loci. Although the most variable parts of the genome are the previously predicted genomic islands, significant genetic variations were present in the genome backbone. Evolution within genes encoding surface and secreted proteins and those involved in the biosynthesis of different capsular types is mainly due to recombination events leading to the replacement of a locus of several genes or to the allelic exchange of the internal part of a gene. These two processes, which led to a broad diversity of surface protein patterns, are probably involved in the diversity of interactions with the host and its immune system. According to gene content comparisons and phylogeny, recent gene replacements by horizontal gene transfer may occur but are rare events. Although specific gene patterns, with respect to the origin of the strains and the epidemiological characteristics, were not identified, we show that the recently described hypervirulent ST-17 lineage is a homogeneous group. The study highlights for the first time that this lineage contains a specific and conserved set of surface proteins, probably accounting for its high capacity to cause infections in newborns.

LadR, a new PadR-related transcriptional regulator from Listeria monocytogenes, negatively regulates the expression of the multidrug efflux pump MdrL.

The Listeria monocytogenes genome encodes putative multidrug efflux transporters but only the MdrL transporter has been partially characterized in the wild-type LO28 strain. Here, we show in the LO28 strain, that the expression of MdrL is repressed at the transcriptional level, under standard growth conditions, by the product of a new gene ladR (lmo1408), and the expression of MdrL is induced in the presence of rhodamine. Phylogenetic analysis in related firmicutes shows that LadR, conserved in all sequenced Listeria genomes, forms an independent group from the large and diverse PadR transcriptional regulator family (PF03551). This is the first report of a bacterial multidrug transporter controlled by a member of the PadR family.

Ralstonia metallidurans, a bacterium specifically adapted to toxic metals: towards a catalogue of metal-responsive genes.

Ralstonia metallidurans, formerly known as Alcaligenes eutrophus and thereafter as Ralstonia eutropha, is a beta-Proteobacterium colonizing industrial sediments, soils or wastes with a high content of heavy metals. The type strain CH34 carries two large plasmids (pMOL28 and pMOL30) bearing a variety of genes for metal resistance. A chronological overview describes the progress made in the knowledge of the plasmid-borne metal resistance mechanisms, the genetics of R. metallidurans CH34 and its taxonomy, and the applications of this strain in the fields of environmental remediation and microbial ecology. Recently, the sequence draft of the genome of R. metallidurans has become available. This allowed a comparison of these preliminary data with the published genome data of the plant pathogen Ralstonia solanacearum, which harbors a megaplasmid (of 2.1 Mb) carrying some metal resistance genes that are similar to those found in R. metallidurans CH34. In addition, a first inventory of metal resistance genes and operons across these two organisms could be made. This inventory, which partly relied on the use of proteomic approaches, revealed the presence of numerous loci not only on the large plasmids pMOL28 and pMOL30 but also on the chromosome. It suggests that metal-resistant Ralstonia, through evolution, are particularly well adapted to the harsh environments typically created by extreme anthropogenic situations or biotopes.

Targeted genomic enrichment and sequencing of CyHV-3 from carp tissues confirms low nucleotide diversity and mixed genotype infections.

Koi herpesvirus disease (KHVD) is an emerging disease that causes mass mortality in koi and common carp, Cyprinus carpio L. Its causative agent is Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV). Although data on the pathogenesis of this deadly virus is relatively abundant in the literature, still little is known about its genomic diversity and about the molecular mechanisms that lead to such a high virulence. In this context, we developed a new strategy for sequencing full-length CyHV-3 genomes directly from infected fish tissues. Total genomic DNA extracted from carp gill tissue was specifically enriched with CyHV-3 sequences through hybridization to a set of nearly 2 million overlapping probes designed to cover the entire genome length, using KHV-J sequence (GenBank accession number AP008984) as reference. Applied to 7 CyHV-3 specimens from Poland and Indonesia, this targeted genomic enrichment enabled recovery of the full genomes with >99.9% reference coverage. The enrichment rate was directly correlated to the estimated number of viral copies contained in the DNA extracts used for library preparation, which varied between ∼5000 and ∼2×107. The average sequencing depth was >200 for all samples, thus allowing the search for variants with high confidence. Sequence analyses highlighted a significant proportion of intra-specimen sequence heterogeneity, suggesting the presence of mixed infections in all investigated fish. They also showed that inter-specimen genetic diversity at the genome scale was very low (>99.95% of sequence identity). By enabling full genome comparisons directly from infected fish tissues, this new method will be valuable to trace outbreaks rapidly and at a reasonable cost, and in turn to understand the transmission routes of CyHV-3.

Molecular Detection of the Three Major Pathogenic Vibrio Species from Seafood Products and Sediments in Tunisia Using Real-Time PCR.

Vibrio spp. have emerged as a serious threat to human health worldwide. V. parahaemolyticus , V. cholerae , and V. vulnificus pose a considerable public health risk in Tunisia because they cause sporadic and epidemic foodborne infections associated with the consumption of raw or undercooked contaminated seafood. More recently, toxR-positive V. alginolyticus was also reported to be a potential source of contaminated seafood. A total of 247 samples, including 113 fishes ( Labrus viridis , Penaeus kerathurus , Diplodus annularis , Diplodus sparaillon , Scorparna porcus , Sarpa salpa , Dentex dentex , Scorparna scrofa , Sardinella aurita , Trachurus trachurus , Synodus saurus , Pagellus erythrinus , and Metapenaeus monoceros ), 83 clams ( Ruditapes decussatus species), 30 seawater samples, and 21 sediment samples were analyzed using traditional culture methods (ISO/TS 21872-1; International Organization for Standardization 2007) and a conventional PCR method for Vibrio spp. IDENTIFICATION: A rapid, sensitive, and highly reproducible real-time PCR assay was developed to detect the three major Vibrio spp. pathogenic for humans in Tunisian seafood products and sediments. A conventional culture method found 102 (41.3%) of 247 analyzed samples positive for Vibrio spp.; a conventional PCR method found 126 (51%) of the 247 samples positive. Real-time PCR assay found 126 (51.1%) samples positive; V. alginolyticus toxR was the most common, found in 99 (78.57%) of samples, followed by V. parahaemolyticus in 26 (20.63%) and V. cholerae in 1 (0.7%). All culture-positive samples were PCR positive. However, 24 samples that were positive by conventional PCR and real-time PCR were culture negative. Our findings indicate that retail seafood is commonly contaminated with Vibrio spp. and presents a potential risk to human health in Tunisia. These data also indicate that real-time PCR can provide sensitive species-specific detection of Vibrio spp. in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.

A probable link between the DedA protein and resistance to selenite.

To understand the molecular events involved in the reduction of selenite to non-toxic elemental selenium, 4000 clones of Ralstonia metallidurans CH34 were produced by random Tn5 transposon integration mutagenesis. Eight mutants were able to resist up to 15 mM selenite while the MIC for the wild-type strain was estimated as 4-6 mM selenite. The identification of the disrupted genes was carried out by Southern blot analysis and inverse PCR. The three resistant mutants containing only one insertion were further characterized. Tn5 disrupted a gene that encoded a protein which was closely related to proteins of the DedA family. This family represents a group of integral membrane proteins with completely unknown functions. Phenotypic characterization of the dedA mutants and selenite consumption experiments strongly suggest that DedA is involved in the uptake of selenite.

Fungal diversity in adult date palm (Phoenix dactylifera L.) revealed by culture-dependent and culture-independent approaches.

Endophytic flora plays a vital role in the colonization and survival of host plants, especially in harsh environments, such as arid regions. This flora may, however, contain pathogenic species responsible for various troublesome host diseases. The present study is aimed at investigating the diversity of both cultivable and non-cultivable endophytic fungal floras in the internal tissues (roots and leaves) of Tunisian date palm trees (Phoenix dactylifera). Accordingly, 13 isolates from both root and leaf samples, exhibiting distinct colony morphology, were selected from potato dextrose agar (PDA) medium and identified by a sequence match search wherein their 18S-28S internal transcribed spacer (ITS) sequences were compared to those available in public databases. These findings revealed that the cultivable root and leaf isolates fell into two groups, namely Nectriaceae and Pleosporaceae. Additionally, total DNA from palm roots and leaves was further extracted and ITS fragments were amplified. Restriction fragment length polymorphism (RFLP) analysis of the ITS from 200 fungal clones (leaves: 100; roots: 100) using HaeIII restriction enzyme revealed 13 distinct patterns that were further sequenced and led to the identification of Alternaria, Cladosporium, Davidiella (Cladosporium teleomorph), Pythium, Curvularia, and uncharacterized fungal endophytes. Both approaches confirmed that while the roots were predominantly colonized by Fusaria (members of the Nectriaceae family), the leaves were essentially colonized by Alternaria (members of the Pleosporaceae family). Overall, the findings of the present study constitute, to the authors' knowledge, the first extensive report on the diversity of endophytic fungal flora associated with date palm trees (P. dactylifera).

Application of high-density DNA resequencing microarray for detection and characterization of botulinum neurotoxin-producing clostridia.

BACKGROUND: Clostridium botulinum and related clostridia express extremely potent toxins known as botulinum neurotoxins (BoNTs) that cause severe, potentially lethal intoxications in humans. These BoNT-producing bacteria are categorized in seven major toxinotypes (A through G) and several subtypes. The high diversity in nucleotide sequence and genetic organization of the gene cluster encoding the BoNT components poses a great challenge for the screening and characterization of BoNT-producing strains. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we designed and evaluated the performances of a resequencing microarray (RMA), the PathogenId v2.0, combined with an automated data approach for the simultaneous detection and characterization of BoNT-producing clostridia. The unique design of the PathogenID v2.0 array allows the simultaneous detection and characterization of 48 sequences targeting the BoNT gene cluster components. This approach allowed successful identification and typing of representative strains of the different toxinotypes and subtypes, as well as the neurotoxin-producing C. botulinum strain in a naturally contaminated food sample. Moreover, the method allowed fine characterization of the different neurotoxin gene cluster components of all studied strains, including genomic regions exhibiting up to 24.65% divergence with the sequences tiled on the arrays. CONCLUSIONS/SIGNIFICANCE: The severity of the disease demands rapid and accurate means for performing risk assessments of BoNT-producing clostridia and for tracing potentials sources of contamination in outbreak situations. The RMA approach constitutes an essential higher echelon component in a diagnostics and surveillance pipeline. In addition, it is an important asset to characterise potential outbreak related strains, but also environment isolates, in order to obtain a better picture of the molecular epidemiology of BoNT-producing clostridia.

An efficient DNA isolation protocol for filamentous cyanobacteria of the genus Arthrospira.

Thanks to their photosynthetic and nutritive properties, cyanobacteria of the Arthrospira genus are of interest as food supplements, as efficient oxygen producing life support system organisms for manned space flight, and for the production of biofuels. Despite these potential valuable applications, full genome sequences and genetic information in general on Arthrospira remain scarce. This is mainly due to the difficulty to extract sufficient high molecular weight nucleic acids from these filamentous cyanobacteria. In this article, an efficient and reproducible DNA extraction procedure for cyanobacteria of the genus Arthrospira was developed. The method is based on the combination of a soft mechanical lysis with enzymatic disruption of the cell wall. The comparison with other extraction protocols clearly indicates that this optimised method allows the recovery of a larger amount of DNA. Furthermore, the extracted DNA presents a high molecular weight, a reduced degradation and an excellent overall quality. It can be directly used for molecular biology purposes such as PCR, and clone library construction.

The arthrobacter arilaitensis Re117 genome sequence reveals its genetic adaptation to the surface of cheese.

Arthrobacter arilaitensis is one of the major bacterial species found at the surface of cheeses, especially in smear-ripened cheeses, where it contributes to the typical colour, flavour and texture properties of the final product. The A. arilaitensis Re117 genome is composed of a 3,859,257 bp chromosome and two plasmids of 50,407 and 8,528 bp. The chromosome shares large regions of synteny with the chromosomes of three environmental Arthrobacter strains for which genome sequences are available: A. aurescens TC1, A. chlorophenolicus A6 and Arthrobacter sp. FB24. In contrast however, 4.92% of the A. arilaitensis chromosome is composed of ISs elements, a portion that is at least 15 fold higher than for the other Arthrobacter strains. Comparative genomic analyses reveal an extensive loss of genes associated with catabolic activities, presumably as a result of adaptation to the properties of the cheese surface habitat. Like the environmental Arthrobacter strains, A. arilaitensis Re117 is well-equipped with enzymes required for the catabolism of major carbon substrates present at cheese surfaces such as fatty acids, amino acids and lactic acid. However, A. arilaitensis has several specificities which seem to be linked to its adaptation to its particular niche. These include the ability to catabolize D-galactonate, a high number of glycine betaine and related osmolyte transporters, two siderophore biosynthesis gene clusters and a high number of Fe(3+)/siderophore transport systems. In model cheese experiments, addition of small amounts of iron strongly stimulated the growth of A. arilaitensis, indicating that cheese is a highly iron-restricted medium. We suggest that there is a strong selective pressure at the surface of cheese for strains with efficient iron acquisition and salt-tolerance systems together with abilities to catabolize substrates such as lactic acid, lipids and amino acids.

The complete genome sequence of Cupriavidus metallidurans strain CH34, a master survivalist in harsh and anthropogenic environments.

Many bacteria in the environment have adapted to the presence of toxic heavy metals. Over the last 30 years, this heavy metal tolerance was the subject of extensive research. The bacterium Cupriavidus metallidurans strain CH34, originally isolated by us in 1976 from a metal processing factory, is considered a major model organism in this field because it withstands milli-molar range concentrations of over 20 different heavy metal ions. This tolerance is mostly achieved by rapid ion efflux but also by metal-complexation and -reduction. We present here the full genome sequence of strain CH34 and the manual annotation of all its genes. The genome of C. metallidurans CH34 is composed of two large circular chromosomes CHR1 and CHR2 of, respectively, 3,928,089 bp and 2,580,084 bp, and two megaplasmids pMOL28 and pMOL30 of, respectively, 171,459 bp and 233,720 bp in size. At least 25 loci for heavy-metal resistance (HMR) are distributed over the four replicons. Approximately 67% of the 6,717 coding sequences (CDSs) present in the CH34 genome could be assigned a putative function, and 9.1% (611 genes) appear to be unique to this strain. One out of five proteins is associated with either transport or transcription while the relay of environmental stimuli is governed by more than 600 signal transduction systems. The CH34 genome is most similar to the genomes of other Cupriavidus strains by correspondence between the respective CHR1 replicons but also displays similarity to the genomes of more distantly related species as a result of gene transfer and through the presence of large genomic islands. The presence of at least 57 IS elements and 19 transposons and the ability to take in and express foreign genes indicates a very dynamic and complex genome shaped by evolutionary forces. The genome data show that C. metallidurans CH34 is particularly well equipped to live in extreme conditions and anthropogenic environments that are rich in metals.

Molecular structure and metal-binding properties of the periplasmic CopK protein expressed in Cupriavidus metallidurans CH34 during copper challenge.

The copK gene is localized on the pMOL30 plasmid of Cupriavidus metallidurans CH34 within the complex cop cluster of genes, for which 21 genes have been identified. The expression of the corresponding periplasmic CopK protein is strongly upregulated in the presence of copper, leading to a high periplasmic accumulation. The structure and metal-binding properties of CopK were investigated by NMR and mass spectrometry. The protein is dimeric in the apo state with a dissociation constant in the range of 10(-5) M estimated from analytical ultracentrifugation. Mass spectrometry revealed that CopK has two high-affinity Cu(I)-binding sites per monomer with different Cu(I) affinities. Binding of Cu(II) was observed but appeared to be non-specific. The solution structure of apo-CopK revealed an all-beta fold formed of two beta-sheets in perpendicular orientation with an unstructured C-terminal tail. The dimer interface is formed by the surface of the C-terminal beta-sheet. Binding of the first Cu(I)-ion induces a major structural modification involving dissociation of the dimeric apo-protein. Backbone chemical shifts determined for the 1Cu(I)-bound form confirm the conservation of the N-terminal beta-sheet, while the last strand of the C-terminal sheet appears in slow conformational exchange. We hypothesize that the partial disruption of the C-terminal beta-sheet is related to dimer dissociation. NH-exchange data acquired on the apo-protein are consistent with a lower thermodynamic stability of the C-terminal sheet. CopK contains seven methionine residues, five of which appear highly conserved. Chemical shift data suggest implication of two or three methionines (Met54, Met38, Met28) in the first Cu(I) site. Addition of a second Cu(I) ion further increases protein plasticity. Comparison of the structural and metal-binding properties of CopK with other periplasmic copper-binding proteins reveals two conserved features within these functionally related proteins: the all-beta fold and the methionine-rich Cu(I)-binding site.