Natural Populations of Chickpea Rhizobia Evaluated by Antibiotic Resistance Profiles and Molecular Methods

The aims of this study were to investigate the hypothesis that intrinsic antibiotic resistance (IAR) profiles of chickpea rhizobia are correlated with the isolates site of origin, and to compare the discriminating power of IAR profiles with molecular approaches in rhizobial strain identification and differentiation. Rhizobial diversity from five Portuguese soils was assessed by IAR profiles and molecular methods [16S rDNA restriction fragment length polymorphism (RFLP) analysis, direct amplified polymorphic DNA (DAPD) fingerprinting, and SDS–PAGE analysis of protein profiles]. For each analysis, a dendrogram was generated using the software BioNumerics. All three molecular methods generated analogous clustering of the isolates, supporting previous results on 16S rDNA sequence-based phylogeny. Clusters obtained with IAR profile are similar to the species groups generated with the molecular methods used. IAR groups do not correlate significantly with the geographic origin of the isolates. These results may indicate a chromosomal location of antibiotic resistance genes, and suggest that IAR is species related. DAPD and IAR profiles proved to be the most discriminating approaches in strain differentiation and can be used as fast methods to screen diversity in new isolates.     

Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

Amplified fragment length polymorphism data provide a poor solution to the Littorina littorea puzzle

The status of Littorina littorea as an exotic species in North America has been debated in the biological literature for over a century. In recent decades, molecular data have been used to supplement historical and archaeological evidence that has suggested that the snail may have persisted in North America well before European settlers arrived. However, these earlier molecular studies have since been questioned due to incomplete sampling of both the geographical range of L. littorea and the genome of the species itself. Here we consider an amplified fragment length polymorphism screen of the nuclear genome, which provides a large number of independent dominant markers that can be used in resolving this historical puzzle. Although these data appear to refute earlier allozyme studies of L. littorea as indicating long-term divergence between European and American populations, the data are also problematic in that they include clear artefacts. We discuss these artefacts and suggest future approaches to definitively resolving the debate over L. littorea's introduction to North America.     

Rapid identification of Leishmania species from formalin-fixed biopsy samples by polymorphism-specific polymerase chain reaction

The precise identification and classification of Leishmania species is important for public health surveillance since different species cause different clinical features of the disease. A highly specific polymerase chain reaction (PCR) panel was developed to enable the identification of the five major Leishmania species that cause New World cutaneous leishmaniases. The primers used for this panel were designed to distinguish the polymorphism in sequences of commonly amplified DNA bands of the parasites produced by arbitrarily primed PCR. These polymorphism-specific PCR diagnoses were performed with formalin-fixed biopsy specimens of the leishmanial lesions from four patients in Ecuador and one hamster skin lesion, and these lesions were determined to be caused by Leishmania (Viannia) panamensis, L. (Leishmania) mexicana, and L. (L.) amazonensis. The PCR panel may offer an important and practical approach to the standardized identification of Leishmania species in field examinations.     

ITS/CHS1靶位序列分析结合SRAP分子标记技术鉴定Nannizzia incurvata临床分离株

以转录间隔区（internal transcribed spacer,ITS）和几丁质合成酶1（chitinase synthase 1,CHS1）为鉴定靶位,结合种系进化分析将2007年至2016年分离自头癣或面癣患儿的12株石膏样小孢子菌复合体成员（现被归入Nannizzia菌属）鉴定到种水平,其中5株为Nannizzia gypsea（Microsporum gypseum）,另7株为Nannizzia incurvata（Microsporum incurvatum）。在此基础上,通过序列相关扩增多态性（SRAP）分子标记进一步证实二者在DNA多位点存在扩增多态性。本研究证实在我国湖北地区存在Nannizzia incurvata,为我国开展Nannizzia菌属的分子流行病学研究提供了实验室依据和支持。     

Molecular evolution of the MLO gene family in Oryza sativa and their functional divergence

The Leishmania genome project has identified new genes at a rapid rate. The 32.8-megabase haploid genome of Leishmania major (Friedlin strain) is published and the comparative analysis of genome sequences of two other species, Leishmania infantum and Leishmanai braziliensis has been done. The haploid genome of Leishmania major (Friedlin strain) has around 8272 protein-coding genes, of which only 36% can be ascribed a putative function. Out of these open reading frames around 910 Leishmania major genes have no orthologs in the other two Tritryp genomes. These “Leishmania -restricted” genes hold a potential as novel drug targets and potential vaccine candidates. Open reading frame, ORFF, is a single copy gene located on the chromosome 35 as a part of the multigene LD1 locus. Indirect immunofluorescence study and creation of ORFF-GFP fusion showed that ORFF is localized in the DNA containing compartments of Leishmania donovani, the nucleus and the kinetoplast. In order to characterize ORFF gene of L. donovani, we have created ORFF over-expressors and single allele deletion mutants by homologous replacement strategy. ORFF is likely to be an important gene for the parasite growth since results from over-expression studies and characterization of ORFF heterozygous knockout mutants reveal marked alterations in the cell cycle phenotype compared to the wild-type parasites. Flowcytometry based cell cycle analysis showed selective increase in the DNA synthetic phase of the ORFF over-expressors and a subversion of the same in heterozygous knockouts of ORFF suggesting its potential role in cell cycle progression.     

禄丰古猿蝴蝶种下第四前臼齿釉质-齿质交界面的三维几何形态

禄丰古猿蝴蝶种 (Lufengpithecus hudienensis) 也称蝴蝶古猿,是重要的早期人科成员,化石产自云南元谋盆地竹棚-小河及雷老两个地点群,其年代为中新世晚期。上世纪八、九十年代的发掘工作共获得幼年个体颅骨1具、残上颌骨10件、残下颌骨17件及1500多枚单个牙齿。受限于当时的技术条件,蝴蝶古猿牙齿内部结构及三维形态一直未有报道和对比研究。本文首次使用高精度CT配合三维几何形态测量方法,对6枚产自小河地点的蝴蝶古猿下颌第四前臼齿的釉质-齿质交界面形态进行了观察和对比,对比材料包括步氏巨猿、猩猩（化石）、大猩猩、黑猩猩及现代人。多变量分析显示,蝴蝶古猿釉质-齿质交界面几何形态接近于本文所涉及的大型猿类对比标本,但并没有表现出与某一特定类群的相似性;咬合面轮廓狭长,前凹尺寸明显小于后凹;整体形态介于齿质尖较高的大猩猩和齿质尖较低、釉质-齿质交界面形态扁平的巨猿、猩猩和黑猩猩之间。本文所观察到的类群之间的异同可能与趋同演化有关,也需要更多数据的进一步验证。将釉质-齿质交界面的三维几何形态和其他牙齿内部结构的信息（如釉质厚度及其三维分布规律等）综合,有助于进一步讨论蝴蝶古猿的分类学、系统发育和食性。     

Utility of the polymerase chain reaction-restriction fragment length polymorphisms of the intergenic spacer region of the rDNA for characterizing Gibberella fujikuroi isolates

In the present report, a total of thirty-one isolates of Gibberella fujikuroi (Sawada) Wollenw. species complex of Fusarium (section Liseola) morphologically classified as F. moniliforme according to the taxonomy of Nelson, Toussoun and Marasas (1983) were analyzed for their ability to produce fumonisin B₁ and fumonisin B₂ by an optimized liquid chromatographic method. They were isolated from three hosts (Zea mays, Musa sapientum and Pinus pinea). The results indicate that M. sapientum is a preferential host for G. fujikuroi isolates with low or null capacity for producing fumonisins, while isolates from Z. mays and P. pinea are generally high fumonisin producers.

The molecular characterization of isolates was carried out in parallel using an optimized, simple and low-cost method for isolating DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the rDNA intergenic spacer (IGS) region. The haplotypes obtained with Hha I enzyme and combinations of Hha I, EcoR I, Alu I, Pst I and Xho I enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin B₁ and B₂ producing capacity. IGS region restriction patterns showed no relationship to isolate geographical origin. This is the first report on this method's capacity to detect polymorphism permitting discrimination between G. fujikuroi isolates from different hosts and with different toxigenic profiles.     

Lactobacillus harbinensis sp. nov., consisted of strains isolated from traditional fermented vegetables 'Suan cai' in Harbin, Northeastern China and Lactobacillus perolens DSM 12745

Taxonomical analysis of two genetically distinguished Lactobacillus strains isolated from traditional Chinese fermented vegetables ‘Suan cai’ was performed. They formed l-lactate from glucose, were facultatively heterofermentative, and had a DNA G+C content of 53–54 mol%. They fermented d- and l-arabinose. They produced lactate, ethanol and acetate from gluconate at a molar ratio of 1.1:0.4:0.7. Phylogenetic analysis of 16S rDNA revealed that the two strains were closely related to L. perolens. DNA–DNA hybridization analysis revealed that the two strains were different from L. perolens type strain DSM 12744 and formed a separate cluster with L. perolens DSM 12745. G+C molar content of DNA of the former is 51%, whereas those of the latter strains were in the range of 53–54%. Based on the results, we propose that the new species be named L. harbinensis sp. nov. and that L. perolens DSM 12745 be reclassified as L. harbinensis DSM 12745. The type strain of L. harbinensis DSM 16991^T (=AHU 1762^T=SBT 10908^T).     

Genetic differentiation of populations of the copepod sea louse Lepeophtheirus salmonis (Krøyer) ectoparasitic on wild and farmed salmonids around the coasts of Scotland: Evidence from RAPD markers

Sea trout are the sea-going migratory form of the freshwater brown trout (Salmo trutta L.) and since 1989 there have been marked declines in their stocks on the west coasts both of Scotland and Ireland. Various factors have been attributed as possible causal agents in these stock declines, including fresh water acidification, overfishing, climatic fluctuations, habitat degradation and sea lice parasitic burdens. The putative impact of infestations of sea trout by the ectoparasitic copepod sea louse, Lepeophtheirus salmonis (Krøyer), has featured prominently in the controversy, especially with regard to the role of inshore commercial salmon farms as a possible source of infestation of wild salmonids by sea lice. This study focused on the population genetics of L. salmonis around the coasts of Scotland: We sampled fish from wild and cultured stocks and included salmon (Salmo salar L.), rainbow trout (Oncorhynchus mykiss Walbaum) and sea trout as host species. Analyses of allozyme variation of sea lice were confined to data for two polymorphic loci (Fum, Got-2) and conformed to our initial expectation — that the inclusion of a planktonic larval phase in the life cycle of the copepod, in addition to the high mobility of the host fish, would enhance gene flow and preclude genetic differentiation of L. salmonis populations as a result of random drift alone. DNA polymorphism was quantified by means of PCR and RAPD analysis. Six primers were screened for 16 samples (from wild and farmed salmon, wild sea trout and farmed rainbow trout) — including the east, north and west coasts of Scotland — and the data analyzed by AMOVA (Analysis of Molecular Variance). In contrast to the allozyme results, the RAPD analysis showed striking patterns of genetic differentiation around the coasts of Scotland. The overall pattern was one of genetic homogeneity of L. salmonis populations sampled from wild salmon and sea trout. All of the L. salmonis samples taken from farmed salmon and rainbow trout did, however, show highly significant levels of genetic differentiation, both between wild and farmed salmonids and among the various farms themselves. Evidence of high levels of small-scale spatial or temporal heterogeneity of RAPD marker band frequencies was shown for the one farm from which repeat samples (July and November, 1995) were analysed. Samples of sea lice taken from west coast wild sea trout subjected to RAPD analysis also revealed the occurrence of putative “farm markers” in some individual parasites, indicating that they had possibly originated from salmon farms.     

马铃薯甲虫气味结合蛋白的序列和基因表达谱分析

【目的】昆虫气味结合蛋白(OBPs)在昆虫嗅觉行为中发挥着重要作用。马铃薯甲虫Leptinotarsa decemlineata是马铃薯上一种最主要的毁灭性害虫。为阐明该虫嗅觉识别分子机制,本研究对马铃薯甲虫26个OBP基因序列特征及组织表达谱进行研究。【方法】基于马铃薯甲虫触角转录组测序数据,利用生物信息学方法及qRT-PCR技术,分别对马铃薯甲虫26个LdecOBPs (LdecOBP1-LdecOBP26)的系统进化及基因的组织表达谱进行分析。【结果】除LdecOBP26基因外,其余25个LdecOBPs基因序列均具有完整的开放阅读框,编码120~255个氨基酸残基,预测的蛋白分子量为13.66~29.38 kD,等电点为4.12~8.42,它们属于两个亚家族,其中13个为Classical-C OBPs, 12个为Minus-C OBPs。除LdecOBP3和LdecOBP26外,其他24个LdecOBPs的N端均由16~23个氨基酸组成的信号肽序列。不同的OBPs亚家族均具有各自典型保守的Cys残基。LdecOBPs之间高度分化,氨基酸序列一致性在3.20%~41.91%。系统进化树分析表明,LdecOBPs与沙葱萤叶甲Galeruca daurica的GdauOBPs亲缘关系最近。基因表达谱分析显示,26个LdecOBPs基因在马铃薯甲虫的不同组织中表达,其中有12个LdecOBPs基因(LdecOBP2, LdecOBP4, LdecOBP6, LdecOBP9, LdecOBP10, LdecOBP12, LdecOBP13, LdecOBP16, LdecOBP20-22和LdecOBP24)在触角中高表达,2个LdecOBPs基因(LdecOBP5和LdecOBP17)在足中高表达,其他12个LdecOBPs基因(LdecOBP1, LdecOBP3, LdecOBP7, LdecOBP8, LdecOBP11, LdecOBP14, LdecOBP15, LdecOBP18, LdecOBP19, LdecOBP23, LdecOBP25和LdecOBP26)在触角、头(去除触角)、胸、腹、足和翅这些组织中均表达。【结论】本研究结果为进一步研究马铃薯甲虫嗅觉识别分子机制奠定了基础。     

Megaplasmids in Thermus oshimai isolates from two widely separated geographical areas: restriction fragment profiling and DNA homology

Megaplasmid DNA was detected in ten isolates belonging to the recently described thermophilic eubacterial species Thermus oshimai and isolated from hot springs in Portugal (eight isolates) and Iceland (two isolates). The estimated size of the large plasmids purified from T. oshimai SPS-18 from S. Pedro do Sul, Portugal, and from isolate JK-91 from Hveragerdhi-Hengill, Iceland, was 214 and 275 kb, respectively. No sequence homologous to isolate SPS-18 megaplasmid is present in chromosomal DNA as indicated by Southern hybridization analysis. Overall examination of the HindIII fragment profiles of megaplasmid DNAs purified from isolates from the same geographical area gave similar but not always identical restriction profiles on agarose gels. Restriction fragment length polymorphism (RFLP) was higher for megaplasmids present in isolates purified from the Portuguese and Icelandic isolates than for megaplasmids from the same hot spring. Megaplasmid RFLP correlated with previous results obtained on the polymorphism of macrorestriction patterns of whole genomic DNA and with the RFLP of co-resident small plasmid DNA that was found in one half of the isolates examined. The 16-kb HindIII–HindIII fragment from isolate SPS-18 megaplasmid showed DNA–DNA homology with restriction fragments of similar size generated by the large plasmids present in all the other isolates, even in those from hot springs of widely separated geographical areas. This suggests a high degree of sequence conservation in T. oshimai megaplasmids. Received: 12 May 1997 / Accepted: 17 July 1997     

The phylogeny and systematics of the endemic Ethiopian Lophuromys flavopunctatus species complex based upon random amplified polymorphic DNA (RAPD) analysis

Random amplified polymorphic DNA (RAPD) analysis was performed to clarify systematics and phylogenetic relationships within the Ethiopian endemic representatives of Lophuromys flavopunctatus species complex. Data were analysed by both phenetic (UPGMA) and phylogenetic (neighbor-joining (NJ) and maximum parsimony) procedures. NJ and maximum parsimony analyses yielded identical phylogenetic trees that demonstrate the basal position of L. melanonyx with L. brevicaudus splitting next and sister-group relationship for L. brunneus–L. chrysopus. This phylogenetic pattern is congruent with inferences from allozymes for the considered species suggesting early divergence of Afroalpine species and recent origin of forest taxa. Thus, the results demonstrate that RAPD-PCR might be a useful technique for phylogenetic analysis at the species levels in vertebrates. Controversial taxonomy of L. brevicaudus, L. brunneus and L. chrysopus is clarified, with their specific ranks confirmed on the basis of tree topology and genetic distances.     

Genetic analysis of Helicobacter pylori strain populations colonizing the stomach at different times postinfection

Genetic diversity of the human gastric pathogen Helicobacter pylori in an individual host has been observed; whether this diversity represents diversification of a founding strain or a mixed infection with distinct strain populations is not clear. To examine this issue, we analyzed multiple single-colony isolates from two to four separate stomach biopsies of eight adult and four pediatric patients from a high-incidence Mexican population. Eleven of the 12 patients contained isolates with identical random amplified polymorphic DNA, amplified fragment length polymorphism, and vacA allele molecular footprints, whereas a single adult patient had two distinct profiles. Comparative genomic hybridization using whole-genome microarrays (array CGH) revealed variation in 24 to 67 genes in isolates from patients with similar molecular footprints. The one patient with distinct profiles contained two strain populations differing at 113 gene loci, including the cag pathogenicity island virulence genes. The two strain populations in this single host had different spatial distributions in the stomach and exhibited very limited genetic exchange. The total genetic divergence and pairwise genetic divergence between isolates from adults and isolates from children were not statistically different. We also analyzed isolates obtained 15 and 90 days after experimental infection of humans and found no evidence of genetic divergence, indicating that transmission to a new host does not induce rapid genetic changes in the bacterial population in the human stomach. Our data suggest that humans are infected with a population of closely related strains that vary at a small number of gene loci, that this population of strains may already be present when an infection is acquired, and that even during superinfection genetic exchange among distinct strains is rare.     

Taxonomic revision of Lepisorus (J. Sm.) Ching sect. Lepisorus (Polypodiaceae) from China

The taxonomy of Lepisorus (J. Sm.) Ching sect. Lepisorus in China was revised based on herbarium specimen examinations, field observations, and microscopic study of rhizome scales, soral paraphyses, leaf epidermis and spores. As a result nine species were recognized: Lepisorus macrosphaerus (Baker) Ching, L. asterolepis (Baker) Ching, L. marginatus Ching, L. kuchenensis (Y. C. Wu) Ching, L. megasorus(C. Chr.) Ching, L. kawakamii (Hayata) Tagawa, L. subsessilis Ching & Y. X. Lin, L. affinis Ching and L. nudus (Hook.) Ching. Lepisorus kawakamii (Hayata) Tagawa was reinstated; L. gyirongensis Ching & S. K. Wu and L. longus Ching were reduced to synonyms of L. nudus and L. affinis respectively. The subdivision of L. macrosphaerusis was not accepted. Rhizome scales and paraphyses are the most useful characters for species delimitation as well as for infrageneric classification. Characteristics of the leaf epidermis and spore ornamentation are usually stable and thus of great significance in understanding the relationships among groups within the genus.     

薰衣草叶片对低温胁迫的生理与分子响应机制

薰衣草(Lavandula angustifolia)作为名贵的芳香植物, 其生长、繁育、品质和产量均受低温影响。前期研究已获得1个耐低温薰衣草品种。该研究将对其处理的温度从20°C降至0°C, 揭示薰衣草响应冷胁迫的生理及分子调控机制, 同时结合薰衣草的细胞质膜透性、可溶性糖和蛋白质含量及抗氧化酶活性等生理变化。采用转录组学和生物信息学方法挖掘分析相关耐寒基因, 并探讨外施水杨酸缓解-10°C冻胁迫的可行性。研究发现7个编码脂肪酸去饱和酶和转移酶的基因(LaFADs)、3个参与合成可溶性糖的基因(LaBAM1和LaSS2)、19个编码胚胎晚期丰富蛋白的基因(LaLEAs)及7个编码过氧化物酶的基因(LaPODs), 这些基因在低温胁迫下均上调表达, 指导薰衣草合成并积累保护物质, 维持膜稳定性以应对胁迫。此外, 150 mg·L^-1水杨酸预处理能有效缓解植株冻害, 可作为低温保护剂。该研究丰富了薰衣草重要抗逆基因家族的遗传背景, 为后续分子遗传学功能分析和定向品种改良奠定基础。     

Biodiversity of Saccharomyces cerevisiae isolated from a survey of pito production sites in various parts of Ghana

Biodiversity among Saccharomyces cerevisiae predominating the spontaneous fermentation of Dagarti pito in Ghana was assessed. Two hundred and forty-nine isolates obtained from samples of dried yeast taken from commercial pito production sites in eight geographical regions of Ghana were characterized phenotypically by colony and cell morphology as well as carbohydrate assimilation profiling. Yeast populations ranged between 10⁶ and 10⁸ cfug⁻¹. Ninety-nine percent of the isolates (247) investigated showed macro-and micro morphological characteristics typical of S. cerevisiae. Of these, 72% (179) had assimilation profiles similar to S. cerevisiae while 28% (68) had assimilation profiles atypical of S. cerevisiae or any other member of the Saccharomyces sensu stricto complex. Amplification of the region spanning the two intergenic transcribed spacers (ITS) and the 5.8S ribosomal gene (ITS1-5.8S rDNA-ITS2), followed by restriction analysis, as well as determination of chromosome length polymorphism by pulsed field gel electrophoresis (PFGE) of 25 representative isolates strongly indicated that all belonged to S. cerevisiae, notwithstanding the phenotypic differences. Sequencing of the mitochondrial cytochrome-c oxidase II gene (COX 2) and the actin-encoding gene (ACT1) of four isolates, confirmed their close relatedness to S. cerevisiae, particularly to the type strain CBS1171 (98.7%), as well as other members of the Saccharomyces sensu stricto complex. Twenty isolates selected from eight geographical regions of Ghana and investigated for their technological properties, showed different patterns of growth and flocculation but otherwise similar technological characteristica. Most of the isolates produced pito having sensory attributes, which compared favourably with commercially produced pito.     

Rb transport in Leishmania infantum promastigotes under various in vitro culture conditions

The survival of Leishmania, which encounter drastic changes of environment during their life-cycle, requires regulation and control of ionic concentrations within the cell. We analysed the influence of growth stage, ionic composition of the medium, heat and acidic stress on ⁸⁶Rb⁺ influx in L. infantum promastigetes. Proliferating promastigotes exibited faster and higher ⁸⁶Rb⁺ uptake than stationary cells. Cl⁻ anion did not have any effect, but in the presence of physiological concentration of HCO₃⁻, ⁸⁶Rb⁺ uptake was significantly increased. This enhancing effect was only partially inhibited by N,N′-dicyclohexylcarbodiimide (DCCD), a blocker of ion-translocating ATPases. ⁸⁶Rb⁺ influx was abolished by N-ethylmaleimide (NEM), indicating a major contribution of plasma membrane transporters. Heat shock and acidic shock notably decreased ⁸⁶Rb⁺ influx. Our data provide indirect evidence that an energy-dependent system which brings K⁺ in, such as K⁺/H⁺-ATPase evidenced by Jiang et al. (1994), is active in Leishmania in different environments. Mechanism(s) other than ion-translocating ATPase occur, at least in the presence of HCO₃⁻, and their contribution to K⁺ pathways varies in different environmental conditions.