High altitude adaptation of the schizothoracine fishes (Cyprinidae) revealed by the mitochondrial genome analyses

The schizothoracine fishes, also known as “mountain carps” are widely distributed in the Qinghai-Tibetan Plateau and its peripheral regions. Although they provide a prime example of high altitude adaptation, the phylogenetic relationships and the divergence times among these carp lineages are still controversial. Moreover, the genetic basis for high altitude adaptation is also poorly understood. In this study, we determined the mitochondrial genomes from two species of the schizothoracine fishes, representing a “morphologically primitive” clade and “morphologically specialized” clade, respectively. The phylogenetic tree and the divergence times were estimated within the evolutionary framework of the entire order Cypriniformes. Our results indicate a polyphylyetic relationship of the schizothoracine fishes and suggest two independent migration events into the Qinghai-Tibetan Plateau: one by the “morphologically primitive” clade in the Late Miocene and another by the “morphologically specialized” clade in the Eocene. Rapid speciation events of each clade from the Late Miocene to the Pliocene correspond to the timing of the geologic acceleration of the Qinghai-Tibetan Plateau. Interestingly, we found evidence for positive selection acting on the protein coding genes in the mitochondrial genomes of the “morphologically specialized” clade, implying a possible genetic basis for high altitude adaptation in this derived lineage of cypriniform fishes.     

Interrelationships of the 11 gasterosteiform families (sticklebacks, pipefishes, and their relatives): a new perspective based on whole mitogenome sequences from 75 higher teleosts

The fishes currently recognized as members of the order Gasterosteiformes (sticklebacks, pipefishes, and their relatives) number 278 species, classified into two suborders (Gasterosteoidei and Syngnathoidei), 11 families and 71 genera. Members of this group exhibit unique appearances, many of which are derived from armored bodies with bony plates in various forms. Although recent molecular phylogenetic studies have repeatedly questioned the monophyly of this order, none of the studies examined all of the representative families and the phylogenetic reality of the group has remained unclear. In this study, we examined whole mitochondrial genome (mitogenome) sequences from 13 gasterosteiform species representing all 11 families in the order, and subjected them to partitioned maximum likelihood and Bayesian analyses, with additional data from other percomorphs and outgroups (75 mitogenome sequences considered overall, including 10 newly determined). The resultant phylogenies indicated explicitly that previously recognized members of Gasterosteiformes had diverged basally within the Percomorpha into three different clades with the following subgroups: Syngnathoidei, Gasterosteoidei (minus Indostomidae), and Indostomidae. Monophyly of the order Gasterosteiformes and any combinations of the three subgroups were confidently rejected by statistical tests. Syngnathoidei (together with Dactylopteroidei) formed a monophyletic group, a sister-group relationship between Gasterosteoidei (minus Indostomidae) and Zoarcoidei was reconfirmed and Indostomidae was nested within the Synbranchiformes, rendering the latter group paraphyletic. Our study demonstrates a new perspective of gasterosteiform phylogeny, which will provide fundamental information for future studies of phylogeny, systematics, and evolution.     

Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes

Environmental changes, such as changes in the coastal topography due to Eurasian plate movements, climate oscillation during the Pleistocene, and alteration of ocean currents, have complicated the geographical structure of marine species and deepened their divergence between populations. As two widely distributed species of crustacean (Oratosquilla oratoria and Eriocheir japonica), weak differences were expected due to their high dispersal potential of planktonic larvae with ocean currents. However, results showed a significant genetic divergence between north of China and south of China in the study. In addition, the estimated north–south divergence time (27–30.5 Myr) of mantis shrimp was near the time of the Himalayan movement, and the China–Japan clade divergence time (10.5–11.9 Myr) of mitten crabs was also coincident with the time of the opening of the Sea of Japan. Thus, we hypothesized that environmental changes in the coastal topography contributed to the marine species divergence. Furthermore, based on phylogenetic analysis, network analysis and haplotype distribution, we surmised that mitten crabs originated from a population with the oldest haplotype (H6) and then divided into the north and south populations due to the recent Eurasian plate movements and ocean currents. And lineage of Japan originated from the north population for the opening of the Sea of Japan. While O. oratoria was guessed to originate from two separate populations in the China Sea. The results of “star-like” network, negative values in neutral test, and Tajima's D statistics of two marine species supported a recent rapid population expansion event after the Pleistocene glaciations.     

Mitogenomic evaluation of the unique facial nerve pattern as a phylogenetic marker within the percifom fishes (Teleostei: Percomorpha)

Percomorpha has been described as the “(unresolved) bush at the top” of the teleostean phylogenies and its intrarelationships are intrinsically difficult to solve because of its huge diversity (>15,000 spp.) and ill-defined higher taxa. Patterns of facial nerves, such as those of the ramus lateralis accessorius (RLA), have been considered as one of the candidate characters to delimit a monophyletic group within the percomorphs. Six families of the suborder Percoidei (Arripidae, Dichistiidae, Kyphosidae, Terapontidae, Kuhliidae, and Oplegnathidae) and suborder Stromateoidei (including six families) share the unique pattern 10 of RLA and it has been suggested that those fishes form a monophyletic group across the two perciform suborders. To evaluate the usefulness of the RLA pattern 10 as a phylogenetic marker within the percomorphs, we newly determined whole mitochondrial genome (mitogenome) sequences for the 13 species having RLA pattern 10 and their putatively, closely-related species (5 spp.). Unambiguously aligned sequences (14,263 bp) from those 18 species plus 50 percomrphs and two outgroups (total 70 species) were subjected to partitioned maximum likelihood and Bayesian analyses. The resulting trees clearly indicated that there were at least two independent origins of the unique facial nerve pattern: one in a common ancestor of Kyphosidae, Terapontidae, Kuhliidae, and Oplegnathidae and another one in that of the percoid Arripidae and Stromateoidei. Thus further detailed anatomical studies are needed to clarify the homology of this character between the two lineages. It should be noted that the latter two taxa (Arripidae and Stromateoidei) formed an unexpected, highly-supported monophyletic group together with Scombridae and possibly Chiasmodontidae and Bramidae, all lacking RLA pattern 10 (the former two are members of other perciform suborders Scombroidei and Trachinoidei, respectively). This novel, trans-subordinal clade has never been suggested by any morphological studies, although they share a common ecological characteristic, dwelling in the pelagic realm and often associated with long-distance migrations.     

Characterisation of physiological and immunological differences between Pacific oysters (Crassostrea gigas) genetically selected for high or low survival to summer mortalities and fed different rations under controlled conditions

Within the framework of a national scientific program named “MORtalités ESTivales de l'huître creuse Crassostrea gigas” (MOREST), a family-based experiment was developed to study the genetic basis of resistance to summer mortality in the Pacific oyster, Crassostrea gigas. As part of the MOREST project, the second generation of three resistant families and two susceptible families were chosen and pooled into two respective groups: “R” and “S”. These two groups of oysters were conditioned for 6 months on two food levels (4% and 12% of oyster soft-tissue dry weight in algal dry weight per day) with a temperature gradient that mimicked the Marennes-Oléron natural cycle during the oyster reproductive period. Oyster mortality remained low for the first two months, but then rapidly increased in July when seawater temperature reached 19 °C and above. Mortality was higher in “S” oysters than in “R” oysters, and also higher in oysters fed the 12% diet than those fed 4%, resulting in a decreasing, relative order in cumulative mortality as follows; 12% “S” > 12% “R” > 4% “S” > 4% “R”. Although the observed mortality rates were lower than those previously observed in the field, the mortality differential between “R” and “S” oysters was similar. Gonadal development, estimated by tissue lipid content, followed a relative order yielding a direct, positive relationship between reproductive effort and mortality as we reported precedently by quantitative histology. Regarding hemocyte parameters, one of the most striking observations was that reactive oxygen species (ROS) production was significantly higher in “S” oysters than in “R” oysters in May and June, regardless of food level. The absence of known environmental stress under these experimental conditions suggests that the ROS increase in “S” oyster could be related to their higher reproductive activity. Finally, a higher increase in hyalinocyte counts was observed for”S” oysters, compared to “R” oysters, in July, just before mortality. Taken together, our results suggest an association of genetically based resistance to summer mortality, reproductive strategy and hemocyte parameters.     

Feeding ecology of invasive lionfish (<Emphasis Type="Italic">Pterois volitans</Emphasis>) in the Bahamian archipelago

Feeding ecology of the lionfish (Pterois volitans), an invasive species in the Western North Atlantic, was examined by collecting stomach content data from fishes taken throughout the Bahamian archipelago. Three relative metrics of prey quantity, including percent number, percent frequency, and percent volume, were used to compare three indices of dietary importance. Lionfish largely prey upon teleosts (78% volume) and crustaceans (14% volume). Twenty-one families and 41 species of teleosts were represented in the diet of lionfish; the top 10 families of dietary importance were Gobiidae, Labridae, Grammatidae, Apogonidae, Pomacentridae, Serranidae, Blenniidae, Atherinidae, Mullidae, and Monacanthidae. The proportional importance of crustaceans in the diet was inversely related to size with the largest lionfish preying almost exclusively on teleosts. Lionfish were found to be diurnal feeders with the highest predation occurring in the morning (08:00–11:00).     

Phylogeny and biogeography of cichlid fishes (Teleostei: Perciformes: Cichlidae)

Family level molecular phylogenetic analyses of cichlid fishes have generally suffered from a limited number of characters and/or poor taxonomic sampling across one or more major geographic assemblage, and therefore have not provided a robust test of early intrafamilial diversification. Herein we use both nuclear and mitochondrial nucleotide characters and direct optimization to reconstruct a phylogeny for cichlid fishes. Representatives of major cichlid lineages across all geographic assemblages are included, as well as nearly twice the number of characters as any prior family‐level study. In a strict consensus of 81 equally most‐parsimonious hypotheses, based on the simultaneous analysis of 2222 aligned nucleotide characters from two mitochondrial and two nuclear genes, four major subfamilial lineages are recovered with strong support. Etroplinae, endemic to Madagascar (Paretroplus) and southern Asia (Etroplus), is recovered as the sister taxon to the remainder of Cichlidae. Although the South Asian cichlids are monophyletic, the Malagasy plus South Asian lineages are not. The remaining Malagasy lineage, Ptychochrominae, is monophyletic and is recovered as the sister group to a clade comprising the African and Neotropical cichlids. The African (Pseudocrenilabrinae) and Neotropical (Cichlinae) lineages are each monophyletic in this reconstruction. The use of multiple molecular markers, from both mitochondrial and nuclear genes, results in a phylogeny that in general exhibits strong support, notably for early diversification events within Cichlidae. Results further indicate that Labroidei is not monophyletic, and that the sister group to Cichlidae may comprise a large and diverse assemblage of percomorph lineages. This hypothesis may at least partly explain why morphological studies that have attempted to place Cichlidae within Percomorpha, or that have tested cichlid monophyly using only “labroid” lineages, have met with only limited success. © The Willi Hennig Society 2004.     

Codon sextets with leading role of serine create “ideal” symmetry classification scheme of the genetic code

The standard classification scheme of the genetic code is organized for alphabetic ordering of nucleotides. Here we introduce the new, “ideal” classification scheme in compact form, for the first time generated by codon sextets encoding Ser, Arg and Leu amino acids. The new scheme creates the known purine/pyrimidine, codon–anticodon, and amino/keto type symmetries and a novel A + U rich/C + G rich symmetry. This scheme is built from “leading” and “nonleading” groups of 32 codons each. In the ensuing 4 × 16 scheme, based on trinucleotide quadruplets, Ser has a central role as initial generator. Six codons encoding Ser and six encoding Arg extend continuously along a linear array in the “leading” group, and together with four of six Leu codons uniquely define construction of the “leading” group. The remaining two Leu codons enable construction of the “nonleading” group. The “ideal” genetic code suggests the evolution of genetic code with serine as an initiator.     

Marinifilum flexuosum sp. nov., a new Bacteroidetes isolated from coastal Mediterranean Sea water and emended description of the genus Marinifilum Na et al., 2009

A facultatively anaerobe, moderately halophilic, Gram-negative, filamentous, non motile and unpigmented bacterium, designated M30^T, was isolated from coastal Mediterranean Sea water in Valencia, Spain. Phylogenetic analysis based on 16S rRNA sequences placed this strain in the phylum “Bacteroidetes” with Marinifilum fragile JC2469^T as its closest relative with 97% sequence similarity. Average nucleotide identity (ANI) values between both strains were far below the 95% threshold value for species delineation (about 89% using BLAST and about 90% using MUMmer). A comprehensive polyphasic study, including morphological, biochemical, physiological, chemotaxonomic and phylogenetic data, confirmed the independent species status of strain M30^T within the genus Marinifilum, for which the name Marinifilum flexuosum sp. nov. is proposed. The type strain of Marinifilum flexuosum is M30^T (=CECT 7448^T = DSM 21950^T).     

Crystal structure and characterization of coiled-coil domain of the transient receptor potential channel PKD2L1

The cation-permeable channel PKD2L1 forms a homomeric assembly as well as heteromeric associations with both PKD1 and PKD1L3, with the cytoplasmic regulatory domain (CRD) of PKD2L1 often playing a role in assembly and/or function. Our previous work indicated that the isolated PKD2L1 CRD assembles as a trimer in a manner dependent on the presence of a proposed oligomerization domain. Herein we describe the 2.7 Å crystal structure of a segment containing the PKD2L1 oligomerization domain which indicates that trimerization is driven by the β-branched residues at the first and fourth positions of a heptad repeat (commonly referred to as “a” and “d”) and by a conserved R-h-x-x-h-E salt bridge motif that is largely unique to parallel trimeric coiled coils. Further analysis of the PKD2L1 CRD indicates that trimeric association is sufficiently strong that no other species are present in solution in an analytical ultracentrifugation experiment at the lowest measurable concentration of 750 nM. Conversely, mutation of the “a” and “d” residues leads to formation of an exclusively monomeric species, independent of concentration. Although both monomeric and WT CRDs are stable in solution and bind calcium with 0.9 μM affinity, circular dichroism studies reveal that the monomer loses 25% more α-helical content than WT when stripped of this ligand, suggesting that the CRD structure is stabilized by trimerization in the ligand-free state. This stability could play a role in the function of the full-length complex, indicating that trimerization may be important for both homo- and possibly heteromeric assemblies of PKD2L1.     

Mutations in Desmin's Carboxy-Terminal “Tail” Domain Severely Modify Filament and Network Mechanics

Inherited mutations in the gene coding for the intermediate filament protein desmin have been demonstrated to cause severe skeletal and cardiac myopathies. Unexpectedly, some of the mutated desmins, in particular those carrying single amino acid alterations in the non-α-helical carboxy-terminal domain (“tail”), have been demonstrated to form apparently normal filaments both in vitro and in transfected cells. Thus, it is not clear if filament properties are affected by these mutations at all. For this reason, we performed oscillatory shear experiments with six different desmin “tail” mutants in order to characterize the mesh size of filament networks and their strain stiffening properties. Moreover, we have carried out high-frequency oscillatory squeeze flow measurements to determine the bending stiffness of the respective filaments, characterized by the persistence length l_p. Interestingly, mesh size was not altered for the mutant filament networks, except for the mutant DesR454W, which apparently did not form proper filament networks. Also, the values for bending stiffness were in the same range for both the “tail” mutants (l_p = 1.0-2.0 μm) and the wild-type desmin (l_p = 1.1 ± 0.5 μm). However, most investigated desmin mutants exhibited a distinct reduction in strain stiffening compared to wild-type desmin and promoted nonaffine network deformation. Therefore, we conclude that the mutated amino acids affect intrafilamentous architecture and colloidal interactions along the filament in such a way that the response to applied strain is significantly altered.In order to explore the importance of the “tail” domain as such for filament network properties, we employed a “tail”-truncated desmin. Under standard conditions, it formed extended regular filaments, but failed to generate strain stiffening. Hence, these data strongly indicate that the “tail” domain is responsible for attractive filament-filament interactions. Moreover, these types of interactions may also be relevant to the network properties of the desmin cytoskeleton in patient muscle.     

Multilocus sequence analysis of putative Vibrio mediterranei strains and description of Vibrio thalassae sp. nov.

A multilocus sequence analysis based on partial gyrB, mreB, rpoD and pyrH genes was undertaken with 61 putative Vibrio mediterranei/V. shilonii strains from different hosts (mussels, oysters, clams, coral, fish and plankton) or habitat (seawater and sediment) and geographical origins (Mediterranean, Atlantic and Pacific). A consistent grouping was obtained with individual and concatenated gene sequences, and the clade, comprising 54 strains, was split into three subclades by all methods: subclade A (40 strains, including AK1, the former type strain of Vibrio shilonii), subclade B (8 strains) corresponding to the species V. mediterranei, and subclade C (six strains) representing a new species, V. thalassae sp. nov., with strain MD16^T (=CECT 8203^T = KCTC 32373^T) as the proposed type strain.     

The complete mitochondrial genome sequence of the world's largest fish,the whale shark (Rhincodon typus), and its comparison with those of related shark species

The whale shark (Rhincodon typus) is the largest extant species of fish, belonging to the order Orectolobiformes. It is listed as a “vulnerable” species on the International Union for Conservation of Nature (IUCN)'s Red List of Threatened Species, which makes it an important species for conservation efforts. We report here the first complete sequence of the mitochondrial genome (mitogenome) of the whale shark obtained by next-generation sequencing methods. The assembled mitogenome is a 16,875 bp circle, comprising of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. We also performed comparative analysis of the whale shark mitogenome to the available mitogenome sequences of 17 other shark species, four from the order Orectolobiformes, five from Lamniformes and eight from Carcharhiniformes. The nucleotide composition, number and arrangement of the genes in whale shark mitogenome are the same as found in the mitogenomes of the other members of the order Orectolobiformes and its closest orders Lamniformes and Carcharhiniformes, although the whale shark mitogenome had a slightly longer control region. The availability of mitogenome sequence of whale shark will aid studies of molecular systematics, biogeography, genetic differentiation, and conservation genetics in this species.     

“Protoisochores” in certain archaeal species are formed by replication-associated mutational pressure

This report shows that isochore-like structures can be found not only in warm-blooded animals, some reptiles, fishes and yeast, but also in certain archaeal species. In perfectly shaped isochore-like structures (in “protoisochores”) from Sulfolobus acidocaldarius and Thermofilum pendens genomes the difference in 3GC levels between genes from different “protoisochores” is about 30%. In these archaeal species GC-poor “protoisochores” are situated near the origin of replication, while GC-rich “protoisochores” are situated near the terminus of replication. There is a strong linear dependence between position of a gene and its 3GC level in S. acidocaldarius (an average difference in 3GC per 100?000 base pairs is equal to 3.6%). Detailed analyses of nucleotide usage biases in genes from leading and lagging strands led us to the suggestion that 3GC in genes situated near terminus of replication grows due to higher rates of thymine oxidation producing T to C transitions in lagging strands.     

Repeatability of clades as a criterion of reliability: a case study for molecular phylogeny of Acanthomorpha (Teleostei) with larger number of taxa

Although much progress has been made recently in teleostean phylogeny, relationships among the main lineages of the higher teleosts (Acanthomorpha), containing more than 60% of all fish species, remain poorly defined. This study represents the most extensive taxonomic sampling effort to date to collect new molecular characters for phylogenetic analysis of acanthomorph fishes. We compiled and analyzed three independent data sets, including: (i) mitochondrial ribosomal fragments from 12S and 16s (814bp for 97 taxa); (ii) nuclear ribosomal 28S sequences (847bp for 74 taxa); and (iii) a nuclear protein-coding gene, rhodopsin (759bp for 86 taxa). Detailed analyses were conducted on each data set separately and the principle of taxonomic congruence without consensus trees was used to assess confidence in the results as follows. Repeatability of clades from separate analyses was considered the primary criterion to establish reliability, rather than bootstrap proportions from a single combined (total evidence) data matrix. The new and reliable clades emerging from this study of the acanthomorph radiation were: Gadiformes (cods) with Zeioids (dories); Beloniformes (needlefishes) with Atheriniformes (silversides); blenioids (blennies) with Gobiesocoidei (clingfishes); Channoidei (snakeheads) with Anabantoidei (climbing gouramies); Mastacembeloidei (spiny eels) with Synbranchioidei (swamp-eels); the last two pairs of taxa grouping together, Syngnathoidei (aulostomids, macroramphosids) with Dactylopteridae (flying gurnards); Scombroidei (mackerels) plus Stromatoidei plus Chiasmodontidae; Ammodytidae (sand lances) with Cheimarrhichthyidae (torrentfish); Zoarcoidei (eelpouts) with Cottoidei; Percidae (perches) with Notothenioidei (Antarctic fishes); and a clade grouping Carangidae (jacks), Echeneidae (remoras), Sphyraenidae (barracudas), Menidae (moonfish), Polynemidae (threadfins), Centropomidae (snooks), and Pleuronectiformes (flatfishes).     

Examining Polyglutamine Peptide Length: A Connection between Collapsed Conformations and Increased Aggregation

Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, of which the best known is Huntington's. Expansion of the polyglutamine domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. The age of onset of disease is inversely correlated with the length of the polyglutamine domain; this correlation motivates an examination of the role of the length of the domain on aggregation. In this investigation, peptides containing 8 to 24 glutamines were synthesized, and their conformational and aggregation properties were examined. All peptides lacked secondary structure. Fluorescence resonance energy transfer studies revealed that the peptides became increasingly collapsed as the number of glutamine residues increased. The effective persistence length was estimated to decrease from ∼ 11 to ∼ 7 Å as the number of glutamines increased from 8 to 24. A comparison of our data with theoretical results suggests that phosphate-buffered saline is a good solvent for Q8 and Q12, a theta solvent for Q16, and a poor solvent for Q20 and Q24. By dynamic light scattering, we observed that Q16, Q20, and Q24, but not Q8 or Q12, immediately formed soluble aggregates upon dilution into phosphate-buffered saline at 37 °C. Thus, Q16 stands at the transition point between good and poor solvent and between stable and aggregation-prone peptide. Examination of aggregates by transmission electron microscopy, along with kinetic assays for sedimentation, provided evidence indicating that soluble aggregates mature into sedimentable aggregates. Together, the data support a mechanism of aggregation in which monomer collapse is accompanied by formation of soluble oligomers; these soluble species lack regular secondary structure but appear morphologically similar to the sedimentable aggregates into which they eventually mature.     

Interrelationships of Atherinomorpha (medakas, flyingfishes, killifishes, silversides, and their relatives): The first evidence based on whole mitogenome sequences

Series Atherinomorpha, with its plentiful number of species and highly diversified ecological and morphological characters, is the most successful fish group at the surface layer of the ocean and many freshwater habitats, comprising 1552 species classified into three orders, six suborders, 21 families, and 193 genera. The group includes one of the most important research model organisms, the medaka (Oryzias latipes), together with diverse fishes with morphological, physiological, and ecological specializations, such as highly developed pectoral fins to glide, self-fertilization, and live-bearing. In this study, we examined the whole mitochondrial genomes (mitogenomes) from 17 species representing all of the three orders and six suborders within Atherinomorpha, with data from 70 additional percomorph species as ingroups, and two non-percomorph outgroup species. We subjected the unambiguously aligned mitogenome sequences to partitioned maximum likelihood and Bayesian phylogenetic analyses. The resulting phylogenies recovered a monophyletic Atherinomorpha within the Percomorpha, and demonstrated its phylogenetic affinity to the percomorph fishes (including cichlids) spawning demersal eggs with filaments. This study, further, provided the first molecular evidence for the monophyly of the respective atherinomorph orders (Atheriniformes, Beloniformes, and Cyprinodontiformes) with high posterior probabilities and mostly high bootstrap values, providing an important basis for the future studies on the phylogeny and evolution of this diverse group.     

Inhibition of proton-transfer steps in transhydrogenase by transition metal ions

Transhydrogenase couples proton translocation across a bacterial or mitochondrial membrane to the redox reaction between NAD(H) and NADP(H). Purified intact transhydrogenase from Escherichia coli was prepared, and its His tag removed. The forward and reverse transhydrogenation reactions catalysed by the enzyme were inhibited by certain metal ions but a “cyclic reaction” was stimulated. Of metal ions tested they were effective in the order Pb²⁺ > Cu²⁺ > Zn²⁺ = Cd²⁺ > Ni²⁺ > Co²⁺. The results suggest that the metal ions affect transhydrogenase by binding to a site in the proton-transfer pathway. Attenuated total-reflectance Fourier-transform infrared difference spectroscopy indicated the involvement of His and Asp/Glu residues in the Zn²⁺-binding site(s). A mutant in which βHis91 in the membrane-spanning domain of transhydrogenase was replaced by Lys had enzyme activities resembling those of wild-type enzyme treated with Zn²⁺. Effects of the metal ion on the mutant were much diminished but still evident. Signals in Zn²⁺-induced FTIR difference spectra of the βHis91Lys mutant were also attributable to changes in His and Asp/Glu residues but were much smaller than those in wild-type spectra. The results support the view that βHis91 and nearby Asp or Glu residues participate in the proton-transfer pathway of transhydrogenase.