Ecophysiological and Phylogenetic Studies of Nevskia ramosa in Pure Culture

During the last 100 years, the neuston bacterium Nevskia ramosa has been described several times. This bacterium forms conspicuous rosette-like microcolonies at the air-water interface. In this study, pure cultures of Nevskia ramosa were obtained for the first time, from a bog lake (strain Soe1, DSMZ 11499^T) and a freshwater ditch (strain OL1, DSMZ 11500). The isolates showed special adaptations to life in the epineuston. They formed hydrophobic surface films with a dull appearance. N. ramosa is sensitive to UV radiation but revealed a very effective photorepair mechanism. Exposure to light at a wavelength of 350 nm after UV treatment raised the number of surviving cells by several orders of magnitude. The isolates grew with a broad range of organic substrates. Surface films were formed only in the absence of combined nitrogen; however, nitrogenase activity was not detected. It appears that during growth at the air-water interface the cells benefit from trapping ammonia from the air. The G+C content of the DNA was 67.8 and 69.0 mol% for strains Soe1 and OL1, respectively. The slight difference was confirmed by enterobacterial repetitive intergenic consensus PCR. The 16S rRNA sequences revealed 99.2% similarity. Thus, both isolates belong to the same species. The phylogenetic analysis indicated that Nevskia is a member of the gamma-subclass Proteobacteria that has no known close relatives.Some morphologically conspicuous bacteria were observed in the 19th century but still have not been isolated in pure culture. In 1892, Famintzin (7) described Nevskia ramosa from the water surface of an aquarium in the botanical garden of St. Petersburg, Russia. The typical microcolonies consist of flat rosettes with a bush-like appearance on the water surface. The rosettes are colonies of dichotomously branched slime stalks with rod-shaped, slightly bent cells in the tips. The cells contain refractile globules, which were presumed to be ethereal oil (7), sulfur globules (12), or fat droplets (3). The slime stalks consist of polysaccharides (3) and sometimes appear to contain iron and aluminum encrustations (11).Enrichments of Nevskia-like cells in lake water supplied with lactate were described by Babenzien (1–4). He observed the following life cycle of N. ramosa. Young motile cells develop submersed, then adsorb to the water surface, lose the polar flagellum, and form a hyaline slime stalk on the concave side of the cell. When a cell multiplies by binary fission, branching of the stalk occurs. The resulting flat rosette can reach a size of 70 μm in diameter.Since pure cultures have not been available, little is known about the physiology, phylogeny, and ecology of Nevskia. It was assumed that Nevskia is oligocarbophilic (14). Tests with the nitrification inhibitor nitrapyrin gave no indications that the cells oxidize ammonia (16). N. ramosa was assumed to be related to the stalk-forming genera Caulobacter and Gallionella or to the sulfur-oxidizing Thiobacterium. In Bergey’s manual (4) N. ramosa was affiliated with the budding and/or appendaged bacteria.In addition to its conspicuous morphology, the typical habitat of N. ramosa prompted us to initiate the present investigation. The water-air interface is a very special environment, characterized by high surface tension and a relatively high hydrophobicity. Organic compounds and various typical bacteria are enriched in this zone. The living community is called the neuston (18, 21). Depending on whether they adsorb to the underside or the top of the water surface, organisms belong to the hyponeuston or epineuston, respectively. This habitat requires special adaptations with respect to adsorption, substrate uptake, and UV tolerance.In our study we have isolated N. ramosa in pure culture and carried out ecophysiological and phylogenetic characterizations. We found several adaptions to life in the epineuston in this interesting bacterium.     

F-Actin Dynamics in Neurospora crassa

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.Actins are highly conserved proteins found in all eukaryotes and have an enormous variety of cellular roles. The monomeric form (globular actin, or G-actin) can self-assemble, with the aid of numerous actin-binding proteins (ABPs), into microfilaments (filamentous actin, or F-actin), which, together with microtubules, form the two major components of the fungal cytoskeleton. Numerous pharmacological and genetic studies of fungi have demonstrated crucial roles for F-actin in cell polarity, exocytosis, endocytosis, cytokinesis, and organelle movement (6, 7, 20, 34, 35, 51, 52, 59). Phalloidin staining, immunofluorescent labeling, and fluorescent-protein (FP)-based live-cell imaging have revealed three distinct subpopulations of F-actin-containing structures in fungi: patches, cables, and rings (1, 14, 28, 34, 60, 63, 64). Actin patches are associated with the plasma membrane and represent an accumulation of F-actin around endocytic vesicles (3, 26, 57). Actin cables are bundles of actin filaments stabilized with cross-linking proteins, such as tropomyosins and fimbrin, and are assembled by formins at sites of active growth, where they form tracks for myosin V-dependent polarized secretion and organelle transport (10, 16, 17, 27, 38, 47, 48). Cables, unlike patches, are absolutely required for polarized growth in the budding yeast Saccharomyces cerevisiae (34, 38). Contractile actomyosin rings are essential for cytokinesis in budding yeast, whereas in filamentous fungi, actin rings are less well studied but are known to be involved in septum formation (20, 28, 34, 39, 40).Actin cables and patches have been particularly well studied in budding yeast. However, there are likely to be important differences between F-actin architecture and dynamics in budding yeast and those in filamentous fungi, as budding yeasts display only a short period of polarized growth during bud formation, which is followed by isotropic growth over the bud surface (10). Sustained polarized growth during hyphal morphogenesis is a defining feature of filamentous fungi (21), making them attractive models for studying the roles of the actin cytoskeleton in cell polarization, tip growth, and organelle transport.In Neurospora crassa and other filamentous fungi, disruption of the actin cytoskeleton leads to rapid tip swelling, which indicates perturbation of polarized tip growth, demonstrating a critical role for F-actin in targeted secretion to particular sites on the plasma membrane (7, 22, 29, 56). Immunofluorescence studies of N. crassa have shown that F-actin localizes to hyphal tips as “clouds” and “plaques” (7, 54, 59). However, immunolabeling has failed to reveal actin cables in N. crassa and offers limited insights into F-actin dynamics. Live-cell imaging of F-actin architecture and dynamics has not been accomplished in N. crassa, yet it is expected to yield key insights into cell polarization, tip growth, and intracellular transport.We took advantage of a recently developed live-cell imaging probe for F-actin called Lifeact (43). Lifeact is a 17-amino-acid peptide derived from the N terminus of the budding yeast actin-binding protein Abp140 (5, 63) and has recently been demonstrated to be a universal live-cell imaging marker for F-actin in eukaryotes (43). Here, we report the successful application of fluorescent Lifeact fusion constructs for live-cell imaging of F-actin in N. crassa. We constructed two synthetic genes consisting of Lifeact fused to “synthetic” green fluorescent protein (sGFP) (S65T) (henceforth termed GFP) (12) or red fluorescent protein (TagRFP) (33) and expressed these constructs in various N. crassa strains. In all strain backgrounds, fluorescent Lifeact constructs clearly labeled actin patches, cables, and rings and revealed a direct association of F-actin structures with sites of cell polarization and active tip growth. Our results demonstrate the efficacy of Lifeact as a nontoxic live-cell imaging probe in N. crassa.     

Comparison of Gas Chromatography and Mineralization Experiments for Measuring Loss of Selected Polychlorinated Biphenyl Congeners in Cultures of White Rot Fungi

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than Phanerochaete chrysosporium ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4′-dichlorobiphenyl, 2,4′,5-trichlorobiphenyl (2,4′,5-TCB), 2,2′,4,4′-tetrachlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl. The congener tested for mineralization was 2,4′,5-[U-¹⁴C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of Bjerkandera adusta (UAMH 8258 and UAMH 7308), one strain of Pleurotus ostreatus (UAMH 7964), and Trametes versicolor UAMH 8272 gave the highest biodegradation and mineralization. P. chrysosporium ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of P. chrysosporium, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower, ≤11%, because it measures a complete oxidation of at least part of the congener molecule but the results were more consistent and therefore more reliable in assessment of PCB biodegradation.

Polychlorinated biphenyls (PCBs) are produced by chlorination of biphenyl, resulting in up to 209 different congeners. Commercial mixtures range from light oily fluids to waxes, and their physical properties make them useful as heat transfer fluids, hydraulic fluids, solvent extenders, plasticizers, flame retardants, organic diluents, and dielectric fluids (1, 21). Approximately 24 million lb are in the North American environment (19). The stability and hydrophobic nature of these compounds make them a persistent environmental hazard.To date, bacterial transformations have been the main focus of PCB degradation research. Aerobic bacteria use a biphenyl-induced dioxygenase enzyme system to attack less-chlorinated congeners (mono- to hexachlorobiphenyls) (1, 5, 7, 8, 22). Although more-chlorinated congeners are recalcitrant to aerobic bacterial degradation, microorganisms in anaerobic river sediments reductively dechlorinate these compounds, mainly removing the meta and para chlorines (1, 6, 10, 33, 34).The degradation of PCBs by white rot fungi has been known since 1985 (11, 18). Many fungi have been tested for their ability to degrade PCBs, including the white rot fungi Coriolus versicolor (18), Coriolopsis polysona (41), Funalia gallica (18), Hirneola nigricans (35), Lentinus edodes (35), Phanerochaete chrysosporium (3, 11, 14, 17, 18, 35, 39, 41–43), Phlebia brevispora (18), Pleurotus ostreatus (35, 43), Poria cinerescens (18), Px strain (possibly Lentinus tigrinus) (35), and Trametes versicolor (41, 43). There have also been studies of PCB metabolism by ectomycorrhizal fungi (17) and other fungi such as Aspergillus flavus (32), Aspergillus niger (15), Aureobasidium pullulans (18), Candida boidinii (35), Candida lipolytica (35), Cunninghamella elegans (16), and Saccharomyces cerevisiae (18, 38). The mechanism of PCB biodegradation has not been definitively determined for any fungi. White rot fungi produce several nonspecific extracellular enzymes which have been the subject of extensive research. These nonspecific peroxidases are normally involved in lignin degradation but can oxidize a wide range of aromatic compounds including polycyclic aromatic hydrocarbons (37). Two peroxidases, lignin peroxidase (LiP) and Mn peroxidase (MnP), are secreted into the environment of the fungus under conditions of nitrogen limitation in P. chrysosporium (23, 25, 27, 29) but are not stress related in fungi such as Bjerkandera adusta or T. versicolor (12, 30).Two approaches have been used to determine the biodegradability of PCBs by fungi: (i) loss of the parent congener analyzed by gas chromatography (GC) (17, 32, 35, 42, 43) and (ii) mineralization experiments in which the ¹⁴C of the universally labeled ¹⁴C parent congener is recovered as ¹⁴CO₂ (11, 14, 18, 39, 41). In the first method, the loss of a peak on a chromatogram makes it difficult to decide whether the PCB is being partly degraded, mineralized, adsorbed to the fungal biomass, or bound to glassware, soil particles, or wood chips. Even when experiments with killed-cell and abiotic controls are performed, the extraction efficiency and standard error can make data difficult to interpret. For example, recoveries can range anywhere from 40 to 100% depending on the congener used and the fungus being investigated (17). On the other hand, recovery of significant amounts of ¹⁴CO₂ from the cultures incubated with a ¹⁴C substrate provides definitive proof of fungal metabolism. There appears to be only one report relating data from these two techniques (18), and in that study, [U-¹⁴C]Aroclor 1254, rather than an individual congener, was used.In this study, we examined the ability of 12 white rot fungal strains to metabolize selected PCB congeners to determine which strains were the most active degraders. Included in this group was P. chrysosporium ATCC 24725, a strain used extensively in PCB studies (3, 14, 18, 35, 39, 41–43). Six PCB congeners were selected to give a range of chlorine substitutions and therefore a range of potential biodegradability which was monitored by GC. One of the chosen congeners was ¹⁴C labeled and used in studies to compare the results from a mineralization method with those from the GC method.     

A Saccharomyces cerevisiae Model Reveals In Vivo Functional Impairment of the Ogden Syndrome N-Terminal Acetyltransferase NAA10 Ser37Pro Mutant

N-terminal acetylation (Nt-acetylation) occurs on the majority of eukaryotic proteins and is catalyzed by N-terminal acetyltransferases (NATs). Nt-acetylation is increasingly recognized as a vital modification with functional implications ranging from protein degradation to protein localization. Although early genetic studies in yeast demonstrated that NAT-deletion strains displayed a variety of phenotypes, only recently, the first human genetic disorder caused by a mutation in a NAT gene was reported; boys diagnosed with the X-linked Ogden syndrome harbor a p.Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of the NatA complex, and suffer from global developmental delays and lethality during infancy. Here, we describe a Saccharomyces cerevisiae model developed by introducing the human wild-type or mutant NatA complex into yeast lacking NatA (NatA-Δ). The wild-type human NatA complex phenotypically complemented the NatA-Δ strain, whereas only a partial rescue was observed for the Ogden mutant NatA complex suggesting that hNaa10 S37P is only partially functional in vivo. Immunoprecipitation experiments revealed a reduced subunit complexation for the mutant hNatA S37P next to a reduced in vitro catalytic activity. We performed quantitative Nt-acetylome analyses on a control yeast strain (yNatA), a yeast NatA deletion strain (yNatA-Δ), a yeast NatA deletion strain expressing wild-type human NatA (hNatA), and a yeast NatA deletion strain expressing mutant human NatA (hNatA S37P). Interestingly, a generally reduced degree of Nt-acetylation was observed among a large group of NatA substrates in the yeast expressing mutant hNatA as compared with yeast expressing wild-type hNatA. Combined, these data provide strong support for the functional impairment of hNaa10 S37P in vivo and suggest that reduced Nt-acetylation of one or more target substrates contributes to the pathogenesis of the Ogden syndrome. Comparative analysis between human and yeast NatA also provided new insights into the co-evolution of the NatA complexes and their substrates. For instance, (Met-)Ala- N termini are more prevalent in the human proteome as compared with the yeast proteome, and hNatA displays a preference toward these N termini as compared with yNatA.Up to 85% of soluble eukaryotic proteins carry an N-terminal acetyl group at their N terminus, which is the result of a co-translational protein modification referred to as N-terminal protein acetylation (Nt-acetylation) or N^α-acetylation (1). This presumed irreversible protein modification is catalyzed by a specific category of the GCN5-related N-acetyltransferase domain containing superfamily of acetyltransferases; the ribosome associated N-terminal acetyltransferases or NATs¹ (2). NATs catalyze the acetyl transfer from acetyl coenzyme A (Ac-CoA) to a primary α-amine of the first amino acid residue of a nascent protein chain. In eukaryotes, NATs are composed of at least one catalytic subunit and mainly target different substrate N termini based on their N-terminal sequences (3).To date, five human NATs hNatA, hNatB, and hNatC; constituting the major human NAT complexes, and hNatD and hNatF have been identified and their substrate specificity characterized (1, 4–8). In addition, a putative hNatE complex has been described (9–10). Except for NatF, which is only expressed in higher eukaryotes (1), the substrate specificity profiles of the NatA-E complexes seem to be conserved among eukaryotes (5–9, 11–13).Contrary to the original assumption that Nt-acetylation protected proteins from degradation (14), it was more recently demonstrated that this modification creates specific degradation signals (termed Ac/N-degrons) in cellular proteins, thereby diversifying this original view substantially. These degrons target at least some Nt-acetylated proteins for the conditional degradation by a novel branch of the N-end rule pathway, an ubiquitin-dependent proteolytic system (15–16). In addition, numerous reports implicate Nt-acetylation in cellular differentiation, survival, metabolism, and proliferation, thereby linking it to cancer (17–18). As such, Nt-acetylation is now linked to a whole range of molecular implications including protein destabilization and degradation by the Nt-acetylation dependent recruitment of ubiquitin ligases (15–16), protein translocation (19), membrane attachment (20), and protein complex formation (21).Among all characterized NATs, NatA displays the broadest substrate specificity profile and thus represents the primary NAT in terms of substrate N termini as it is responsible for the Nt-acetylation of the methionine aminopeptidase (MetAP) iMet-processed serine, threonine, alanine, glycine, and valine starting N termini (3). The human NatA complex is composed of two essential subunits; the catalytic subunit hNaa10 (hARD1) and the regulatory subunit hNaa15 (NATH/hNAT1) (4). Deregulations of hNaa10 and/or NatA expression have been linked to various signaling molecules including hypoxia inducible factor-1α, DNA methyltransferase1/E-cadherin, β-catenin/cyclin D1, and Bcl-xL, showing its involvement in hypoxia, tumorigenesis, cell cycle progression, and apoptosis (17, 22–26).Recently, the first structures of NATs and a NAT-complex were solved, providing a molecular understanding of the sequence specific Nt-acetylation of protein N termini (27–30). Structural analyses of noncomplexed Naa10 and NatA from Schizosaccharomyces pombe reveal an allosteric modulator function of Naa15 in steering Naa10 specificity and provide a rational for the distinctive substrate specificity profiles observed when assaying non-complexed versus complexed Naa10 (10, 27), with both forms co-existing in cells (10). In particular, three essential catalytic Naa10 residues were found to be incorrectly positioned in non-complexed Naa10, while these shift into the active site in Naa15-complexed Naa10, thereby permitting canonical NatA-mediated Nt-acetylation. Interestingly, noncomplexed Naa10 was shown to efficiently Nt-acetylate glutamate and aspartate starting N termini, whereas poorly acetylating canonical NatA type N termini (10). The study of Liszczak et al. further showed that NatA substrate binding specificity was coupled to the catalytic mechanism being used (27). More specifically, an essential glutamate residue (Glu24 in the protein accession {"type":"entrez-protein","attrs":{"text":"Q9UTI3","term_id":"74625957","term_text":"Q9UTI3"}}Q9UTI3 (Swiss-Prot)) involved in catalysis, precludes methionine from entering the specificity pocket, whereas cognate NatA substrate N-terminal residues can easily be accommodated. Interestingly, and in contrast to NatA, both wild-type Naa10 and Glu24 mutated Naa10 (Naa10 E24A) were still capable of Nt-acetylating acidic amino acid starting N termini, most likely because of the substrate side-chain carboxyl moiety acting as a functional replacement group in the process of catalysis, whereas essentially no activity could be observed when probing a cognate NatA substrate (27).Early yeast studies demonstrated that strains with mutated or deleted NAT genes were viable, but displayed a number of different phenotypes (31). For NatA, the first phenotypes described were defects in sporulation, mating, and entry into stationary phase when NAA10 (ARD1) was mutated (32). Four years later, the overlapping phenotypes of NAA10 and NAA15 (NAT1) mutant strains, revealed, along with other data, that Naa10 and Naa15 are in fact components of the NatA acetyltransferase complex (33–34). As compared with NatA phenotypes, NatB phenotypes are more severe, including slow growth and defects in mitochondrial inheritance (35–36). NatC subunits were initially found to be essential for propagation of the l-A dsRNA virus, and further for growth on nonfermentable carbon sources (37–39). The first reports implicating NAT gene point mutations in human genetic disorders only recently emerged. More specifically, two different point mutations in the X-linked NAA10 gene were both found to cause developmental delays and were linked to the Ogden syndrome (S37P) (40) and intellectual disability (R116W) (41), highlighting the essential importance of NATs and protein Nt-acetylation in biology and disease. Further, in Caenorhabditis elegans (42), Drosophila melanogaster (43), and Trypanosoma brucei (44), Naa10 was proven to be essential and, strengthened by the observed detrimental effects of NAA10 mutations (40–41), the NAA10 gene function is also believed to be essential in human.Ogden syndrome boys harboring the p.Ser37Pro variant in the gene encoding Naa10 are characterized by craniofacial abnormalities, failure to thrive, developmental delay, hypotonia, cardiac arrhythmias, cryptorchidism, and an aged appearance, ultimately resulting in mortality during infancy (40). Although this mutation was shown to significantly impair Naa10 catalytic activity in vitro, we here assessed the influence and functional in vitro and in vivo consequences of this mutation on NatA complex formation and NatA activity in a yeast model. By phenotypic screening in yeast, we show that hNaa10 S37P displays a significantly impaired functionality in vivo. Further, using immunoprecipitation, we show that the human Naa10-Naa15 complex formation is negatively affected by the S37P mutation, and that immunoprecipitated hNatA S37P also displays a reduced in vitro catalytic activity as compared with wild-type hNatA. Finally, quantitative Nt-acetylome analyses suggest that reduced Nt-acetylation of one or more target substrates contributes to the pathogenesis of the Ogden syndrome.     

Rescue of a Trafficking Defective Human Pacemaker Channel via a Novel Mechanism: ROLES OF Src, Fyn, AND Yes TYROSINE KINASES*

Therapeutic strategies such as using channel blockers and reducing culture temperature have been used to rescue some long QT-associated voltage-gated potassium Kv trafficking defective mutant channels. A hyperpolarization-activated cyclic nucleotide-gated HCN4 pacemaker channel mutant (D553N) has been recently found in a patient associated with cardiac arrhythmias including long QT. D553N showed the defective trafficking to the cell surface, leading to little ionic current expression (loss-of-function). We show in this report that enhanced tyrosine phosphorylation mediated by Src, Fyn, and Yes kinases was able to restore the surface expression of D553N for normal current expression. Src or Yes, but not Fyn, significantly increased the current density and surface expression of D553N. Fyn accelerated the activation kinetics of the rescued D553N. Co-expression of D553N with Yes exhibited the slowest activation kinetics of D553N. Src, Fyn, and Yes significantly enhanced the tyrosine phosphorylation of D553N. A combination of Src, Fyn, and Yes rescued the current expression and the gating of D553N comparable with those of wild-type HCN4. In conclusion, we demonstrate a novel mechanism using three endogenous Src kinases to rescue a trafficking defective HCN4 mutant channel (D553N) by enhancing the tyrosine phosphorylation of the mutant channel protein.Defective trafficking leading to the reduced surface expression of ion channels is one of the mechanisms responsible for a loss-of-function of the ion channel on the plasma membrane (1). Several methods have been developed to rescue the voltage-gated potassium Kv trafficking defective channels: reducing the culture temperature, applying the channel blockers, altering the molar ratio of glycerol, and using the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (2–6).Hyperpolarizing-activated cyclic nucleotide-gated (HCN)³ pacemaker channels generate time- and voltage-dependent inward currents, named I_h in neurons or I_f in the heart (7). They are important in various cell functions including excitability, synapse transmission, and rhythmic activity (7). The most well studied regulation of I_f is its response to autonomic stimulation. β-Adrenergic receptor activation increases and acetylcholine receptor activation decreases the intracellular cAMP levels, which in turn increases/decreases I_f by binding to the cyclic nucleotide-binding domain of the HCN channels, respectively (7). Other important mechanisms for the modulation of I_f/HCN channels have recently been found including β-subunit (8), lipids (9, 10), and p38 mitogen-activated protein kinase (11).Accumulating evidence has revealed tyrosine phosphorylation as an important mechanism for modulation of HCN channel properties (12–16). An acute increase in tyrosine phosphorylation of I_f or HCN channels increases the channel activity, including an increase in the current amplitude, a positive shift of the voltage-dependent activation, an acceleration of activation kinetics, and an increase in whole cell conductance (12–15). Recently, we discovered that the cell surface expression of HCN2 channels can be remarkably inhibited by tyrosine dephosphorylation mediated by receptor-like protein tyrosine phosphatase α (RPTPα) and increased by tyrosine phosphorylation via Src kinase after long term treatment (17).D553N, a missense HCN4 mutant, was recently identified in a patient with cardiac arrhythmia associated with depressed HCN gating properties (18). Functional and structural assays revealed that D553N expresses little ionic currents, which is possibly due to the defective channel trafficking so that the channels cannot reach the plasma membrane for normal functions (18).The Src kinase family has nine members (19). They are closely related and share the same regulatory function. Three of them, Src, Fyn, and Yes, are ubiquitously expressed in a variety of tissues including neurons and myocytes (19, 20). Without stimulation, they are inactive. However, mutation of key tyrosine residue results in the constitutively active form of the kinase, SrcY529F, FynY531F, and YesY537F, respectively (15, 21, 22). Using these Src kinases, we show in this report a novel approach that can restore the surface expression of D553N for normal current expression via tyrosine phosphorylation.     

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

Protein–protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems.Protein–protein interactions (PPIs)¹ play a key role in defining protein functions in biological systems. Aberrant PPIs can have drastic effects on biochemical activities essential to cell homeostasis, growth, and proliferation, and thereby lead to various human diseases (1). Consequently, PPI interfaces have been recognized as a new paradigm for drug development. Therefore, mapping PPIs and their interaction interfaces in living cells is critical not only for a comprehensive understanding of protein function and regulation, but also for describing the molecular mechanisms underlying human pathologies and identifying potential targets for better therapeutics.Several strategies exist for identifying and mapping PPIs, including yeast two-hybrid, protein microarray, and affinity purification mass spectrometry (AP-MS) (2–5). Thanks to new developments in sample preparation strategies, mass spectrometry technologies, and bioinformatics tools, AP-MS has become a powerful and preferred method for studying PPIs at the systems level (6–9). Unlike other approaches, AP-MS experiments allow the capture of protein interactions directly from their natural cellular environment, thus better retaining native protein structures and biologically relevant interactions. In addition, a broader scope of PPI networks can be obtained with greater sensitivity, accuracy, versatility, and speed. Despite the success of this very promising technique, AP-MS experiments can lead to the loss of weak/transient interactions and/or the reorganization of protein interactions during biochemical manipulation under native purification conditions. To circumvent these problems, in vivo chemical cross-linking has been successfully employed to stabilize protein interactions in native cells or tissues prior to cell lysis (10–16). The resulting covalent bonds formed between interacting partners allow affinity purification under stringent and fully denaturing conditions, consequently reducing nonspecific background while preserving stable and weak/transient interactions (12–16). Subsequent mass spectrometric analysis can reveal not only the identities of interacting proteins, but also cross-linked amino acid residues. The latter provides direct molecular evidence describing the physical contacts between and within proteins (17). This information can be used for computational modeling to establish structural topologies of proteins and protein complexes (17–22), as well as for generating experimentally derived protein interaction network topology maps (23, 24). Thus, cross-linking mass spectrometry (XL-MS) strategies represent a powerful and emergent technology that possesses unparalleled capabilities for studying PPIs.Despite their great potential, current XL-MS studies that have aimed to identify cross-linked peptides have been mostly limited to in vitro cross-linking experiments, with few successfully identifying protein interaction interfaces in living cells (24, 25). This is largely because XL-MS studies remain challenging due to the inherent difficulty in the effective MS detection and accurate identification of cross-linked peptides, as well as in unambiguous assignment of cross-linked residues. In general, cross-linked products are heterogeneous and low in abundance relative to non-cross-linked products. In addition, their MS fragmentation is too complex to be interpreted using conventional database searching tools (17, 26). It is noted that almost all of the current in vivo PPI studies utilize formaldehyde cross-linking because of its membrane permeability and fast kinetics (10–16). However, in comparison to the most commonly used amine reactive NHS ester cross-linkers, identification of formaldehyde cross-linked peptides is even more challenging because of its promiscuous nonspecific reactivity and extremely short spacer length (27). Therefore, further developments in reagents and methods are urgently needed to enable simple MS detection and effective identification of in vivo cross-linked products, and thus allow the mapping of authentic protein contact sites as established in cells, especially for protein complexes.Various efforts have been made to address the limitations of XL-MS studies, resulting in new developments in bioinformatics tools for improved data interpretation (28–32) and new designs of cross-linking reagents for enhanced MS analysis of cross-linked peptides (24, 33–39). Among these approaches, the development of new cross-linking reagents holds great promise for mapping PPIs on the systems level. One class of cross-linking reagents containing an enrichment handle have been shown to allow selective isolation of cross-linked products from complex mixtures, boosting their detectability by MS (33–35, 40–42). A second class of cross-linkers containing MS-cleavable bonds have proven to be effective in facilitating the unambiguous identification of cross-linked peptides (36–39, 43, 44), as the resulting cross-linked products can be identified based on their characteristic and simplified fragmentation behavior during MS analysis. Therefore, an ideal cross-linking reagent would possess the combined features of both classes of cross-linkers. To advance the study of in vivo PPIs, we have developed a new XL-MS platform based on a novel membrane-permeable, enrichable, and MS-cleavable cross-linker, Azide-A-DSBSO (azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide), and multistage tandem mass spectrometry (MSⁿ). This new XL-MS strategy has been successfully employed to map in vivo PPIs from mammalian cells at both the proteome scale and the targeted protein complex level.     

Biochemical Characterization of Chloromethane Emission from the Wood-Rotting Fungus Phellinus pomaceus

Many wood-rotting fungi, including Phellinus pomaceus, produce chloromethane (CH₃Cl). P. pomaceus can be cultured in undisturbed glucose mycological peptone liquid medium to produce high amounts of CH₃Cl. The biosynthesis of CH₃Cl is catalyzed by a methyl chloride transferase (MCT), which appears to be membrane bound. The enzyme is labile upon removal from its natural location and upon storage at low temperature in its bound state. Various detergents failed to solubilize the enzyme in active form, and hence it was characterized by using a membrane fraction. The enzyme had a sharp pH optimum between 7 and 7.2. Its apparent K_m for Cl⁻ (ca. 300 mM) was much higher than that for I⁻ (250 μM) or Br⁻ (11 mM). A comparison of these K_m values to the relative in vivo methylation rates for different halides suggests that the real K_m for Cl⁻ may be much lower, but the calculated value is high because the CH₃Cl produced is used immediately in a coupled reaction. Among various methyl donors tested, S-adenosyl-l-methionine (SAM) was the only one that supported significant methylation by MCT. The reaction was inhibited by S-adenosyl-l-homocysteine, an inhibitor of SAM-dependent methylation, suggesting that SAM is the natural methyl donor. These findings advance our comprehension of a poorly understood metabolic sector at the origin of biogenic emissions of halomethanes, which play an important role in atmospheric chemistry.Halogenated organic compounds are ubiquitous in nature (29). They participate in the depletion of stratospheric ozone and have a profound impact on atmospheric chemistry (4, 18, 24). Although the dominant sources of these compounds are biogenic emissions (12, 25, 26, 28), their significance to the emitter organisms is rather poorly understood, with only a few indications of the roles they might play. In fungi, halomethanes serve as methyl group donors for the biosynthesis of esters, anisoles, and veratryl alcohol (9, 11). In algae, halomethanes are by-products of reactions in which scavenging of H₂O₂ releases HOBr, which is presumed to be a defense molecule against bacteria, fungi, and herbivores (23, 27). A recent report (28) that a marine alga, Endocladia muricata, and a salt-tolerant plant, Mesembryanthemum crystallinum, could methylate Cl⁻ ions to chloromethane (CH₃Cl) triggered speculation that this may be a mechanism for Cl⁻ detoxification and salt tolerance. The S-adenosyl-l-methionine (SAM)-dependent methyl chloride transferase (MCT) that catalyzes this reaction was partially purified from E. muricata (28). The enzyme can also use I⁻ and Br⁻ as substrates.These results suggest possibilities for engineering a Cl⁻ detoxification capability into crop plants, many of which are sensitive to Cl⁻ (6, 17). Wood-rotting fungi of the family Hymenochaetaceae are the most efficient producers of CH₃Cl (5, 7, 13). Phellinus pomaceus converts Cl⁻ to CH₃Cl with over 90% efficiency, even at extremely low concentrations of the ion (7). A low MCT activity was detected in cell extracts of this fungus (28).Halomethanes are the primary carriers of halogens between the biosphere and the atmosphere (4, 18) and therefore play pivotal roles in the effect of halogens on atmospheric chemistry and the integrity of the ozone layer (24). Since biogenic sources are major contributors of atmospheric halomethanes (7, 12, 18, 25, 28), attempts to understand atmospheric composition must include an understanding of the metabolic processes underlying the generation of these gases. In addition, engineering a Cl⁻ detoxification capability into plants depends on the identification of novel metabolic pathways and an understanding of their regulation. Within this dual context, our objective was to determine the biochemical nature of the CH₃Cl-evolving system of P. pomaceus.     

Sequence Diversity of the oprI Gene,Coding for Major Outer Membrane Lipoprotein I,among rRNA Group I Pseudomonads

The sequence of oprI, the gene coding for the major outer membrane lipoprotein I, was determined by PCR sequencing for representatives of 17 species of rRNA group I pseudomonads, with a special emphasis on Pseudomonas aeruginosa and Pseudomonas fluorescens. Within the P. aeruginosa species, oprI sequences for 25 independent isolates were found to be identical, except for one silent substitution at position 96. The oprI sequences diverged more for the other rRNA group I pseudomonads (85 to 91% similarity with P. aeruginosa oprI). An accumulation of silent and also (but to a much lesser extent) nonsilent substitutions in the different sequences was found. A clustering according to the respective presence and/or positions of the HaeIII, PvuII, and SphI sites could also be obtained. A sequence cluster analysis showed a rather widespread distribution of P. fluorescens isolates. All other rRNA group I pseudomonads clustered in a manner that was in agreement with other studies, showing that the oprI gene can be useful as a complementary phylogenetic marker for classification of rRNA group I pseudomonads.Pseudomonads are increasingly being recognized as important microorganisms in our biosphere, and Pseudomonas aeruginosa and Pseudomonas fluorescens are two important representatives of this genus. As a typical opportunist, P. aeruginosa is more and more involved in a variety of often fatal nosocomial infections, in which it accounts for more than 11% of all isolates recovered (29). In cystic fibrosis, one of the most common autosomal recessive genetic diseases, it is a characteristic pathogen responsible for most of the cases of morbidity and mortality (16, 38). In general, fluorescent pseudomonads, including P. aeruginosa, Pseudomonas putida, P. fluorescens, and other species, are frequently found as rhizosphere microorganisms, in some cases promoting plant growth (11, 19, 20).P. fluorescens and P. aeruginosa are also found as inherent flora of mineral water (14, 39). Identification of fluorescent pseudomonads is often tedious and not reliable. Indeed, the present taxonomy of this group is far from clear at the finer taxonomic level, as polyphasic investigations have demonstrated (4, 13, 18, 26). Ribosomal RNAs have been applied as molecular markers with great success to unravel the rough phylogenetic structure which, at the finer level, is not always in complete agreement with the genotypic and phenotypic similarities deduced from other parts of the genome. Horizontal gene transfer, chromosomal mutation hot spots, and internal genomic rearrangements are probably the bases of these discrepancies at the species and subspecies levels. These arguments, together with the importance of discriminating phenotypic tests in routine identifications, support a polyphasic approach in bacterial taxonomy (2, 8–10, 13, 37, 40). Additional phylogenetic information requires the identification of molecules, like the recA or the gyrB genes, that are widely distributed, large enough to contain a substantial amount of information, and conserved to an appropriate degree (24, 46). In the phylogenetic tree published by Woese (43), species with the same generic name were allocated in phylogenetically distant groups. This was the case for the “genus” Pseudomonas, which is known to be a dump of assemblages of distantly related species (3, 8–10, 17). Taxonomic rearrangements of the genus Pseudomonas sensu stricto resulted in the splitting of the genus and as a logical consequence, the present genus Pseudomonas is restricted to the rRNA group I organisms, with P. aeruginosa as the type species in this group (27, 28, 37, 42, 44, 45).The oprI gene, coding for the outer membrane lipoprotein I of P. aeruginosa (5), was found to be conserved among the fluorescent pseudomonads and was considered to be a possible phylogenetic marker (6, 31). In this study, we tested whether the oprI gene could be a useful detection and identification target molecule as well as a complementary phylogenetic marker for rRNA group I pseudomonads. Also, we examined to what extent the sequence variation of the oprI gene reflects the species diversity in P. aeruginosa and P. fluorescens.