pH-dependent expression of periplasmic proteins and amino acid catabolism in Escherichia coli

Escherichia coli grows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors. Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and through lac gene fusions. Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1. 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent. At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, and YfiD), whereas acetate-repressed proteins were lowered (Pta, TnaA, DksA, AroK, and MalE). These responses could be mediated by the reuptake of acetate driven by changes in pH. The amplified proton gradient could also be responsible for the acid induction of the tricarboxylic acid (TCA) enzymes SucB and SucC. In addition to the autoinducer LuxS, low pH induced another potential autoinducer component, the LuxH homolog RibB. pH modulated the expression of several periplasmic and outer membrane proteins: acid induced YcdO and YdiY; base induced OmpA, MalE, and YceI; and either acid or base induced OmpX relative to pH 7. Two pH-dependent periplasmic proteins were redox modulators: Tpx (acid-induced) and DsbA (base-induced). The locus alx, induced in extreme base, was identified as ygjT, whose product is a putative membrane-bound redox modulator. The cytoplasmic superoxide stress protein SodB was induced by acid, possibly in response to increased iron solubility. High pH induced amino acid metabolic enzymes (TnaA and CysK) as well as lac fusions to the genes encoding AstD and GabT. These enzymes participate in arginine and glutamate catabolic pathways that channel carbon into acids instead of producing alkaline amines. Overall, these data are consistent with a model in which E. coli modulates multiple transporters and pathways of amino acid consumption so as to minimize the shift of its external pH toward either acidic or alkaline extreme.     

Sodium transport by endocytic vesicles in cultured Arabidopsis thaliana (L.) Heynh. cells

In Vitro Cellular & Developmental Biology - Plant - The involvement of endocytosis in Na+ internalization by suspension-cultured Arabidopsis thaliana (L.) Heynh. cells under salt stress was...     

Analysis of the biosynthetic gene cluster for the polyether antibiotic monensin in Streptomyces cinnamonensis and evidence for the role of monB and monC genes in oxidative cyclization

The analysis of a candidate biosynthetic gene cluster (97 kbp) for the polyether ionophore monensin from Streptomyces cinnamonensis has revealed a modular polyketide synthase composed of eight separate multienzyme subunits housing a total of 12 extension modules, and flanked by numerous other genes for which a plausible function in monensin biosynthesis can be ascribed. Deletion of essentially all these clustered genes specifically abolished monensin production, while overexpression in S. cinnamonensis of the putative pathway-specific regulatory gene monR led to a fivefold increase in monensin production. Experimental support is presented for a recently-proposed mechanism, for oxidative cyclization of a linear polyketide intermediate, involving four enzymes, the products of monBI, monBII, monCI and monCII. In frame deletion of either of the individual genes monCII (encoding a putative cyclase) or monBII (encoding a putative novel isomerase) specifically abolished monensin production. Also, heterologous expression of monCI, encoding a flavin-linked epoxidase, in S. coelicolor was shown to significantly increase the ability of S. coelicolor to epoxidize linalool, a model substrate for the presumed linear polyketide intermediate in monensin biosynthesis.     

High rabdosiin and rosmarinic acid production in Eritrichium sericeum callus cultures and the effect of the calli on masugi-nephritis in rats

During an investigation of plant cell cultures that might be useful in the treatment of renal disorders, we established a vigorously-growing E-4 callus culture of Eritrichium sericeum that produced large amounts of caffeic acid metabolites, (-)-rabdosiin (1.8% dry wt) and rosmarinic acid (4.6% dry wt). Elicitation of the calli by methyl jasmonate induced a 38% increase in total polyphenol production. The most efficient method of eliciting (-)-rabdosiin biosynthesis was through the treatment of E-4 calli with cuprum glycerate, which induced an increase in (-)-rabdosiin production of as much as 4.1% dry wt. Oral administration of E-4 callus biomass (100 mg/kg/d for 30 d) to rats with induced Masugi-nephritis caused an increase in diuresis and lowered creatinine excretion and proteinuria levels as compared with Masugi-nephritis untreated rats. While all of the Masugi-nephritis untreated rats began to suffer, near a quarter of the E-4 treated rats remained in good health. This result indicates that the E-4 culture has the potential to alleviate the symptoms associated with nephritis.     

Effective Synthesis of Fluorescently Labeled Morpholino Nucleoside Triphosphate Derivatives

Morpholino nucleoside triphosphates (A, U, G, C, T) bearing the active functional amino group tethered to morpholine residue and their fluorescently labeled derivatives were synthesized. All compounds were characterized by ¹H, ¹³C, and ³¹P NMR, and mass spectrometry. A possibility of using fluorescently labeled morpholino nucleoside triphosphates as chain terminators in DNA sequencing is discussed.     

Identification of Nosema bombi Fantham and Porter 1914 (Microsporidia) in Bombus impatiens and Bombus sandersoni from Great Smoky Mountains National Park (USA)

Ninety three bumble bees belonging to the genus Bombus, subgenus Pyrobombus (three Bombus vagans, seven Bombus bimaculatus, 17 B. sandersoni and 68 B. impatiens) from Great Smoky Mountains National Park were examined for microsporidia. Light microscopy of calcoflour and trichrome-stained smears, and PCR revealed infection with N. bombi in one specimen each of B. sandersoni and B. impatiens. Sizes and shapes of spores in both N. bombi isolates were similar to those described for European isolates of the microsporidium. A region of the rRNA gene from the B. impatiens isolate (1689 bp, accession GQ254295) aligned with homologous sequences from eight European isolates, with only three variable sites. Sequence variability of this region between novel isolates and the European ones was the same as among European isolates.     

Mass Spectrometry Defines the Stoichiometry of Ribosomal Stalk Complexes across the Phylogenetic Tree

The ribosomal stalk complex plays a crucial role in delivering translation factors to the catalytic site of the ribosome. It has a very similar architecture in all cells, although the protein components in bacteria are unrelated to those in archaea and eukaryotes. Here we used mass spectrometry to investigate ribosomal stalk complexes from bacteria, eukaryotes, and archaea in situ on the ribosome. Specifically we targeted ribosomes with different optimal growth temperatures. Our results showed that for the mesophilic bacterial ribosomes we investigated the stalk complexes are exclusively pentameric or entirely heptameric in the case of thermophilic bacteria, whereas we observed only pentameric stalk complexes in eukaryotic species. We also found the surprising result that for mesophilic archaea, Methanococcus vannielii, Methanococcus maripaludis, and Methanosarcina barkeri, both pentameric and heptameric stoichiometries are present simultaneously within a population of ribosomes. Moreover the ratio of pentameric to heptameric stalk complexes changed during the course of cell growth. We consider these differences in stoichiometry within ribosomal stalk complexes in the context of convergent evolution.Ribosomes universally translate the genetic code into proteins. They consist of two asymmetric subunits between which mRNA is decoded and amino acids are added to a growing peptide chain. On the large subunit there is a noticeable protrusion, observable by electron microscopy, known as the stalk complex (also denoted as L8) (1). This complex is involved in the binding and orientation of translation factors and exists with variable composition throughout all three domains of life. In bacteria we and others have shown previously that it is composed of either two or three dimers of the protein L12 (termed L7 when N-acetylated) attached to a single copy of the scaffolding protein L10 (2, 3). These assemblies of stalk proteins, either L10(L7/L12)₄ or L10(L7/L12)₆, are referred to as pentameric or heptameric stalk complexes hereafter. In eukaryotes there is an identical arrangement for the stalk complex but of unrelated proteins with no sequence homology to L10/L12. In this case P0 is the L10 equivalent scaffolding protein, and two different but related proteins (P1 and P2) take the place of L12 (nomenclature according to Ref. 4). In plants P3 occurs in addition to P1 and P2 (5). The P-proteins are named after their propensity for phosphorylation when attached to the ribosome. In yeast, but not in higher eukaryotes, P1 and P2 have both evolved into two α and β proteins (6). In archaea the stalk complex constituents, although named L10 and L12, share sequence homology with the P-proteins (7). L12 and its P1/P2 counterparts are the only ribosomal proteins that have acidic pI values, do not interact directly with rRNA, and are present in multiple copies on the ribosome.We have shown previously that by applying a combination of MS and tandem MS approaches to intact MDa particles such as ribosomes we can obtain precise information, especially regarding the overall stoichiometry and composition of different stalk complexes (2, 8–11). This is due to the fact that the stalk complex is readily observable in mass spectra of intact ribosomes (Fig. 1). This is in contrast to the situation in most crystallographic investigations where the dynamics and heterogeneity of the stalk prevent its high resolution structure determination. Because it dissociates readily in the mass spectrometer as an intact, oligomeric species we can exploit this property, and previously we have shown that for the mesophilic bacteria Bacillus subtilis and Escherichia coli the stalk complex is unequivocally a pentamer comprising L10 and four copies of L7/L12 (2). For ribosomes of the extreme thermophilic bacteria Thermus thermophilus and Thermatoga maritima we demonstrated that the stalk complex was in fact a heptamer, rather than the anticipated pentamer, comprising one L10 and six copies of L12 (2). This led us to speculate that heptameric stalk complexes were likely to be present on ribosomes from species growing at higher temperatures. Although classification of the species has changed since their discovery, the generally accepted consensus at present separates them into four distinct categories based on their optimal growth temperatures (OGTs)¹: thermophiles (>55 °C), moderate thermophiles (>65 °C), extreme thermophiles (>75 °C), and hyperthermophiles (>85 °C) (12).Open in a separate windowFig. 1.Mass spectrum of intact ribosomes from T. thermophilus. The mass spectrum of intact ribosomes from T. Thermophilus in 1 m ammonium acetate showing well resolved charge states for the 30 S subunit at 14,000–18,000 m/z and 70 S subunit at 25,000–28,000 m/z is shown. The 50 S subunit lacks charge state resolution probably because of the heterogeneity associated with the partial loss of the stalk complex, which appears as a series of well resolved charge states at 4000–6000 m/z. Individual ribosomal proteins and tRNA are observed at the lower m/z regions of the spectrum.Here we report on investigations of ribosome stalk compositions from a range of species from all three domains of life, including examples with different OGTs. For bacteria we extended our earlier findings by including in our analysis the thermophile Bacillus stearothermophilus (OGT 55 °C) and compared it with the extreme thermophile Thermus aquaticus (OGT of 70–75 °C). For eukaryotes we compared ribosomes from three animals (brine shrimp, silkworm, and rabbit) and a thermophilic red alga, Galdieria sulphuraria, the eukaryote with the highest known OGT of 56 °C. Within the archaea we targeted the mesophilic methanogens Methanococcus vannielii, Methanococcus maripaludis, and Methanosarcina barkeri with OGTs of 35, 35–40, and 37 °C, respectively. Methanogens, capable of producing methane, are the most common and widely dispersed of the archaea. M. maripaludis is a model species among the methanogenic archaea. M. barkeri, unlike most methanogens, which only use carbon dioxide, is able to ferment a variety of carbon sources (13). Surprisingly stalk complexes of different stoichiometries were present simultaneously on ribosomes from these mesophilic archaea. Our MS study of ribosomes isolated from M. vannielii and M. barkeri harvested at different stages of growth showed that the ratio of pentameric versus heptameric stalks changes during cell growth with pentameric species being predominant at the early stages and during the lag phase and the proportion of heptameric complexes increasing toward the latter stages of cell growth. Overall therefore in this study we widened our previous MS investigations into ribosomal stalk complexes and targeted species with different OGTs within bacterial, eukaryotic, and archaeal domains.     

TFAM knockdown-triggered mtDNA-nucleoid aggregation and a decrease in mtDNA copy number induce the reorganization of nucleoid populations and mitochondria-associated ER-membrane contacts

The correct organization of mitochondrial DNA (mtDNA) in nucleoids and the contacts of mitochondria with the ER play an important role in maintaining the mitochondrial genome distribution within the cell. Mitochondria-associated ER membranes (MAMs) consist of interacting proteins and lipids located in the outer mitochondrial membrane and ER membrane, forming a platform for the mitochondrial inner membrane-associated genome replication factory as well as connecting the nucleoids with the mitochondrial division machinery. We show here that knockdown of a core component of mitochondrial nucleoids, TFAM, causes changes in the mitochondrial nucleoid populations, which subsequently impact ER-mitochondria membrane contacts. Knockdown of TFAM causes a significant decrease in the copy number of mtDNA as well as aggregation of mtDNA nucleoids. At the same time, it causes significant upregulation of the replicative TWNK helicase in the membrane-associated nucleoid fraction. This is accompanied by a transient elevation of MAM proteins, indicating a rearrangement of the linkage between ER and mitochondria triggered by changes in mitochondrial nucleoids. Reciprocal knockdown of the mitochondrial replicative helicase TWNK causes a decrease in mtDNA copy number and modifies mtDNA membrane association, however, it does not cause nucleoid aggregation and considerable alterations of MAM proteins in the membrane-associated fraction. Our explanation is that the aggregation of mitochondrial nucleoids resulting from TFAM knockdown triggers a compensatory mechanism involving the reorganization of both mitochondrial nucleoids and MAM. These results could provide an important insight into pathological conditions associated with impaired nucleoid organization or defects of mtDNA distribution.