Molecular analysis of two genes of the Escherichia coli gab cluster: nucleotide sequence of the glutamate:succinic semialdehyde transaminase gene (gabT) and characterization of the succinic semialdehyde dehydrogenase gene (gabD).

We have characterized two genes of the Escherichia coli K-12 gab cluster, which encodes the enzymes of the 4-aminobutyrate degradation pathway. The nucleotide sequence of gabT, coding for glutamate:succinic semialdehyde transaminase (EC 2.6.1.19), alternatively known as 4-aminobutyrate transaminase, was determined. The structural gene consists of 1,281 nucleotides specifying a protein of 426 amino acids with a molecular mass of 45.76 kDa. The protein shows significant homologies to the ornithine transaminases from Saccharomyces cerevisiae and from rat and human mitochondria. Three functionally and structurally important amino acid residues of the transaminase were identified by sequence comparison studies, and evolutionary relationships of the aminotransferases are discussed. The gabD gene, encoding succinic semialdehyde dehydrogenase (EC 1.2.1.16), was cloned and shown to be located adjacent to the 5' end of gabT. Expression studies with subfragments of the initially cloned DNA region revealed a maximal size of 1.7 kb for gabD. Both genes are cotranscribed from a promoter located upstream of gabD.     

Complete amino acid sequence of the cytochrome subunit and amino-terminal sequence of the flavin subunit of flavocytochrome c (sulfide dehydrogenase) from Chlorobium thiosulfatophilum

The complete amino acid sequence of the 86-residue heme subunit of flavocytochrome c (sulfide dehydrogenase) from the green phototrophic bacterium Chlorobium thiosulfatophilum strain Tassajara has been determined as follows: APEQSKSIPRGEILSLSCAGCHGTDGKSESIIPTIYGRSAEYIESALLDFKSGA- RPSTVMGRHAKGYSDEEIHQIAEYFGSLSTMNN. The subunit has a single heme-binding site near the N terminus, consisting of a pair of cysteine residues at positions 18 and 21. The out-of-plane ligands are apparently contributed by histidine 22 and methionine 60. The molecular weight including heme is 10,014. The heme subunit is apparently homologous to small cytochromes c by virtue of the location of the heme-binding site and its extraplanar ligands. However, the amino acid sequence is closer to Paracoccus sp. cytochrome c554(548) (37%) than it is to the heme subunit from Pseudomonas putida p-cresol methylhydroxylase flavocytochrome c (20%). The flavocytochrome c heme subunit is only 14% similar to the small cytochrome c555 also found in Chlorobium. Secondary structure predictions suggest N- and C-terminal helices as expected, but the midsection of the protein probably folds somewhat differently from the small cytochromes of known three-dimensional structure such as Pseudomonas cytochrome c551. Analyses of the residues near the exposed heme edges of the cytochrome subunits of P. putida and C. thiosulfatophilum flavocytochromes c (assuming homology to proteins of known structure) indicate that charged residues are not conserved, suggesting that electrostatic interactions are not involved in the association of the heme and flavin subunits. The N-terminal sequence of the flavoprotein subunit of flavocytochrome has also been determined. It shows no similarity to the comparable region of the p-cresol methylhydroxylase flavoprotein subunit from P. putida. The flavin-binding hexapeptide, isolated and sequenced earlier (Kenney, W. C., McIntire, W., and Yamanaka, T. (1977) Biochim. Biophys. Acta 483, 467-474), is situated at positions 40-46.     

Proton NMR study of the comparative electronic/magnetic properties and dynamics of the acid in equilibrium with alkaline transition in a series of ferricytochromes c'

The proton NMR spectra of ferricytochrome c' from Rhodopseudomonas palustris, Rhodospirillum molischianum, Rhodospirillum rubrum, and Chromatium vinosum have been investigated for the purpose of further elucidating the common spectral and/or structural properties for this subclass of cytochromes in the acidic and alkaline forms, and to characterize in detail the dynamics and structural basis for this acid in equilibrium with alkaline transition. The identification of strongly upfield-shifted meso-H peaks in all but C. vinosum ferricytochrome c' at weakly acidic to neutral pH is consistent with, but not proof for, S = 3/2 character for the spin state of C. vinosum, but argues for primarily S = 5/2 character for the other three proteins. Hence, we conclude that the quantum mechanically mixed S = 3/2, S = 5/2 spin ground state of neutral pH C. vinosum ferricytochrome c' is an anomaly rather than a characteristic of this class of proteins. The 1H NMR spectra of ferricytochromes c' at alkaline pH again exhibit strong similarities among all members except that for C. vinosum. Two pK values are observed for ferricytochrome c' for R. molischianum and C. vinosum, of which the higher value pK is accompanied by significant line broadening, as found earlier for the proteins from both R. rubrum and R. palustris. Detailed analysis of the exchange line broadening for all four proteins reveals that hydrolysis is the rate-limiting step, with base catalysis occurring at about the same rate in the diffusion control limit for all four proteins. The variable first order dissociation rates of the alkaline species reveal differential stabilities of that species in the order R. palustris greater than R. molischianum greater than R. rubrum much greater than C. vinosum. The rates of exchange of the axial His imidazole labile proton was determined by linewidth and saturation transfer analysis and shown to occur via base catalysis at the same diffusion control rate as found for the acid----alkaline transition for the oxidized protein, and support the proposal that the acid----alkaline transition involves simply the abstraction of a proton from the neutral His imidazole to yield an imidazolate.     

The association of bacterial mutagenicity of hydrocarbon-derived 'bay-region' dihydrodiols with the Iball indices for carcinogenicity and with the extents of DNA-binding on mouse skin of the parent hydrocarbons.

The mutagenic activities of benz[alpha]anthracene, 7-methylbenz[alpha]anthracene, 7,12-dimethylbenz[alpha]anthracene, 3-methylcholanthrene and benzo[alpha]pyrene, together with those of the trans-dihydrodiols derived from these hydrocarbons that would be expected to yield 'bay-region' vicinal diolepoxides on further metabolism have been examined in assays with S. typhimurium TA100 using post-mitochondrial supernatant fractions prepared from the livers of 3-methylcholanthrene-treated rats. Mutagenic activities obtained have been compared with: (a) the extents of reaction with DNA that occur in mouse skin following treatment with these hydrocarbons; (b) the carcinogenicities of the hydrocarbons expressed as Iball indices; (c) their activities as tumour-initiating agents on mouse skin. Close positive associations were found between the microsome-mediated mutagenicities of the dihydrodiols that could yield "bay-region" diol-epoxides and: (a) the extents of reaction with DNA in hydrocarbon-treated mouse skin; (b) the carcinogenic potencies of the parent hydrocarbons; although these correlations are not perfect, the mutagenic activities of the hydrocarbons themselves in microsome-mediated assays with S. typhimurium show no correlation with their extents of DNA binding on mouse skin and a poor correlation with their activities as initiating agents. These comparisons also indicated a statistically-significant positive correlation between carcinogenicity and the in vivo DNA binding on mouse skin treated with the hydrocarbons. Differences in the metabolic pathways by which polycyclic hydrocarbons are activated in vivo and in vitro are discussed in relation to the improved correlations found with the dihydrodiols.     

Liver-microsome-mediated formation of alkylating agents from vinyl bromide and vinyl chloride.

When a mixture of vinyl chloride/oxygen or vinyl bromide/air was passed through a mouse-liver microsomal system, volatile alkylating metabolites were trapped by reaction with excess 4-(4-nitrobenzyl)pyridine. The absorption spectra of the adducts, either from vinyl bromide or vinyl chloride, were identical with that obtained by reaction of chloroethylene oxide with 4-(4-nitrobenzyl) pyridine. Chloroethylene oxide decomposes in aqueous solution with a half-life of 1.6 minutes. After reaction of chloroethylene oxide and 2-chloroacetaldehyde with adenosine and Sephadex chromatography the binding products were compared with those formed in the presence of vinyl chloride, mouse-liver microsomes and adenosine. A common product of these reactions was tentatively characterized as 3-β-ribofuranosyl-imidazo-[2,1-i]purine.     

Heat stress responses and population genetics of the kelp Laminaria digitata (Phaeophyceae) across latitudes reveal differentiation among North Atlantic populations

To understand the thermal plasticity of a coastal foundation species across its latitudinal distribution, we assess physiological responses to high temperature stress in the kelp Laminaria digitata in combination with population genetic characteristics and relate heat resilience to genetic features and phylogeography. We hypothesize that populations from Arctic and cold‐temperate locations are less heat resilient than populations from warm distributional edges. Using meristems of natural L. digitata populations from six locations ranging between Kongsfjorden, Spitsbergen (79°N), and Quiberon, France (47°N), we performed a common‐garden heat stress experiment applying 15°C to 23°C over eight days. We assessed growth, photosynthetic quantum yield, carbon and nitrogen storage, and xanthophyll pigment contents as response traits. Population connectivity and genetic diversity were analyzed with microsatellite markers. Results from the heat stress experiment suggest that the upper temperature limit of L. digitata is nearly identical across its distribution range, but subtle differences in growth and stress responses were revealed for three populations from the species’ ecological range margins. Two populations at the species’ warm distribution limit showed higher temperature tolerance compared to other populations in growth at 19°C and recovery from 21°C (Quiberon, France), and photosynthetic quantum yield and xanthophyll pigment responses at 23°C (Helgoland, Germany). In L. digitata from the northernmost population (Spitsbergen, Norway), quantum yield indicated the highest heat sensitivity. Microsatellite genotyping revealed all sampled populations to be genetically distinct, with a strong hierarchical structure between southern and northern clades. Genetic diversity was lowest in the isolated population of the North Sea island of Helgoland and highest in Roscoff in the English Channel. All together, these results support the hypothesis of moderate local differentiation across L. digitata's European distribution, whereas effects are likely too weak to ameliorate the species’ capacity to withstand ocean warming and marine heatwaves at the southern range edge.     

Expression levels of the metalloproteinase ADAM8 critically regulate proliferation,migration and malignant signalling events in hepatoma cells

Hepatocellular carcinoma (HCC) is one of the most common metastatic tumours. Tumour growth and metastasis depend on the induction of cell proliferation and migration by various mediators. Here, we report that the A Disintegrin and Metalloproteinase (ADAM) 8 is highly expressed in murine HCC tissues as well as in murine and human hepatoma cell lines Hepa1-6 and HepG2, respectively. To establish a dose-dependent role of different ADAM8 expression levels for HCC progression, ADAM8 expression was either reduced via shRNA- or siRNA-mediated knockdown or increased by using a retroviral overexpression vector. These two complementary approaches revealed that ADAM8 expression levels correlated positively with proliferation, clonogenicity, migration and matrix invasion and negatively with apoptosis of hepatoma cells. Furthermore, the analysis of pro-migratory and proliferative signalling pathways revealed that ADAM8 expression level was positively associated with expression of β1 integrin as well as with the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), Src kinase and Rho A GTPase. Finally, up-regulation of promigatory signalling and cell migration was also seen with a proteolytically inactive ADAM8 mutant. These findings reveal that ADAM8 is critically up-regulated in hepatoma cells contributes to cell proliferation and survival and furthermore induces pro-migratory signalling pathways independently of its proteolytic activity. By this, ADAM8 can promote cell functions most relevant for HCC growth and metastasis.     

Modulation of physiological performance by temperature and salinity in the sugar kelp Saccharina latissima

The sugar kelp Saccharina latissima experiences a wide range of environmental conditions along its geographical and vertical distribution range. Temperature and salinity are two critical drivers influencing growth, photosynthesis and biochemical composition. Moreover, interactive effects might modify the results described for single effects. In shallow water coastal systems, exposure to rising temperatures and low salinity are expected as consequence of global warming, increased precipitation and coastal run‐off. To understand the acclimation mechanisms of S. latissima to changes in temperature and salinity and their interactions, we performed a mechanistic laboratory experiment in which juvenile sporophytes from Brittany, France were exposed to a combination of three temperatures (0, 8 and 15°C) and two salinity levels (20 and 30 psu (practical salinity units)). After a temperature acclimation of 7 days, sporophytes were exposed to low salinity (20 psu) for a period of 11 days. Growth, and maximal quantum yield of photosystem II (Fv/Fm), pigments, mannitol content and C:N ratio were measured over time. We report for the first time in S. latissima a fivefold increase in the osmolyte mannitol in response to low temperature (0°C) compared to 8 and 15°C that may have ecological and economic implications. Low temperatures significantly affected all parameters, mostly in a negative way. Chlorophyll a, the accessory pigment pool, growth and Fv/Fm were significantly lower at 0°C, while the de‐epoxidation state of the xanthophyll cycle was increased at both 0 and 8°C compared to 15°C. Mannitol content and growth decreased with decreased salinity; in contrast, pigment content and Fv/Fm were to a large extent irresponsive to salinity. In comparison to S. latissima originating from an Arctic population, despite some reported differences, this study reveals a remarkably similar impact of temperature and salinity variation, reflecting the large degree of adaptability in this species.     

Effects of ocean acidification on growth and physiology of Ulva lactuca (Chlorophyta) in a rockpool‐scenario

Rising atmospheric CO₂‐concentrations will have severe consequences for a variety of biological processes. We investigated the responses of the green alga Ulva lactuca (Linnaeus) to rising CO₂‐concentrations in a rockpool scenario. U. lactuca was cultured under aeration with air containing either preindustrial pCO₂ (280 μatm) or the pCO₂ predicted by the end of the 21st century (700 μatm) for 31 days. We addressed the following question: Will elevated CO₂‐concentrations affect photosynthesis (net photosynthesis, maximum relative electron transport rate (rETR(max)), maximum quantum yield (Fv/Fm), pigment composition) and growth of U. lactuca in rockpools with limited water exchange? Two phases of the experiment were distinguished: In the initial phase (day 1–4) the Seawater Carbonate System (SWCS) of the culture medium could be adjusted to the selected atmospheric pCO₂ condition by continuous aeration with target pCO₂ values. In the second phase (day 4–31) the SWCS was largely determined by the metabolism of the growing U. lactuca biomass. In the initial phase, Fv/Fm and rETR(max) were only slightly elevated at high CO₂‐concentrations, whereas growth was significantly enhanced. After 31 days the Chl a content of the thalli was significantly lower under future conditions and the photosynthesis of thalli grown under preindustrial conditions was not dependent on external carbonic anhydrase. Biomass increased significantly at high CO₂‐concentrations. At low CO₂‐concentrations most adult thalli disintegrated between day 14 and 21, whereas at high CO₂‐concentrations most thalli remained integer until day 31. Thallus disintegration at low CO₂‐concentrations was mirrored by a drastic decline in seawater dissolved inorganic carbon and HCO₃^?. Accordingly, the SWCS differed significantly between the treatments. Our results indicated a slight enhancement of photosynthetic performance and significantly elevated growth of U. lactuca at future CO₂‐concentrations. The accelerated thallus disintegration at high CO₂‐concentrations under conditions of limited water exchange indicates additional CO₂ effects on the life cycle of U. lactuca when living in rockpools.     

The Bacillus cereus Hbl and Nhe Tripartite Enterotoxin Components Assemble Sequentially on the Surface of Target Cells and Are Not Interchangeable

Bacillus cereus is a spore-forming, Gram-positive bacterium commonly associated with outbreaks of food poisoning. It is also known as an opportunistic pathogen causing clinical infections such as bacteremia, meningitis, pneumonia, and gas gangrene-like cutaneous infections, mostly in immunocompromised patients. B. cereus secretes a plethora of toxins of which four are associated with the symptoms of food poisoning. Two of these, the non-hemolytic enterotoxin Nhe and the hemolysin BL (Hbl) toxin, are predicted to be structurally similar and are unique in that they require the combined action of three toxin proteins to induce cell lysis. Despite their dominant role in disease, the molecular mechanism of their toxic function is still poorly understood. We report here that B. cereus strain ATCC 10876 harbors not only genes encoding Nhe, but also two copies of the hbl genes. We identified Hbl as the major secreted toxin responsible for inducing rapid cell lysis both in cultured cells and in an intraperitoneal mouse toxicity model. Antibody neutralization and deletion of Hbl-encoding genes resulted in significant reductions of cytotoxic activity. Microscopy studies with Chinese Hamster Ovary cells furthermore showed that pore formation by both Hbl and Nhe occurs through a stepwise, sequential binding of toxin components to the cell surface and to each other. This begins with binding of Hbl-B or NheC to the eukaryotic membrane, and is followed by the recruitment of Hbl-L₁ or NheB, respectively, followed by the corresponding third protein. Lastly, toxin component complementation studies indicate that although Hbl and Nhe can be expressed simultaneously and are predicted to be structurally similar, they are incompatible and cannot complement each other.