Identification of protein stability determinants in chloroplasts

Although chloroplast protein stability has long been recognised as a major level of post‐translational regulation in photosynthesis and gene expression, the factors determining protein stability in plastids are largely unknown. Here, we have identified stability determinants in vivo by producing plants with transgenic chloroplasts that express a reporter protein whose N‐ and C‐termini were systematically modified. We found that major stability determinants are located in the N‐terminus. Moreover, testing of all 20 amino acids in the position after the initiator methionine revealed strong differences in protein stability and indicated an important role of the penultimate N‐terminal amino acid residue in determining the protein half life. We propose that the stability of plastid proteins is largely determined by three factors: (i) the action of methionine aminopeptidase (the enzyme that removes the initiator methionine and exposes the penultimate N‐terminal amino acid residue), (ii) an N‐end rule‐like protein degradation pathway, and (iii) additional sequence determinants in the N‐terminal region.     

Immobilized pH gradient-driven paper-based IEF: a new method for fractionating complex peptide mixtures before MS analysis

Introduction  

The vast difference in the abundance of different proteins in biological samples limits the determination of the complete proteome of a cell type, requiring fractionation of proteins and peptides before MS analysis.     

Selenite reduction by a denitrifying culture: batch- and packed-bed reactor studies

Selenite reduction by a bacterial consortium enriched from an oil refinery waste sludge was studied under denitrifying conditions using acetate as the electron donor. Fed-batch studies with nitrate as the primary electron acceptor showed that accumulation of nitrite led to a decrease in the extent of selenite reduction. Also, when nitrite was added as the primary electron acceptor, rapid selenite reduction was observed only after nitrite was significantly depleted from the medium. These results indicate that selenite reduction was inhibited at high nitrite concentrations. In addition to batch experiments, continuous-flow selenite reduction experiments were performed in packed-bed columns using immobilized enrichment cultures. These experiments were carried out in three phases: in phase I, a continuous nitrate feed with different inlet selenite concentration was applied; in phase II, nitrate was fed in a pulsed fashion; and in phase III, nitrate was fed in a continuous mode but at much lower concentrations than the other two phases. During the phase I experiments, little selenite was removed from the influent. However, when the column was operated in the pulse feed strategy (phase II) or in the continuous mode with low nitrate levels (phase III), significant quantities of selenium were removed from solution and retained in the immobilization matrix in the column. Thus, immobilized denitrifying cultures can be effective in removing selenium from waste streams, but nitrate-limited operating conditions might be required.     

FdhTU-modulated formate dehydrogenase expression and electron donor availability enhance recovery of Campylobacter jejuni following host cell infection

Campylobacter jejuni is a food-borne bacterial pathogen that colonizes the intestinal tract and causes severe gastroenteritis. Interaction with host epithelial cells is thought to enhance severity of disease, and the ability of C. jejuni to modulate its metabolism in different in vivo and environmental niches contributes to its success as a pathogen. A C. jejuni operon comprising two genes that we designated fdhT (CJJ81176_1492) and fdhU (CJJ81176_1493) is conserved in many bacterial species. Deletion of fdhT or fdhU in C. jejuni resulted in apparent defects in adherence and/or invasion of Caco-2 epithelial cells when assessed by CFU enumeration on standard Mueller-Hinton agar. However, fluorescence microscopy indicated that each mutant invaded cells at wild-type levels, instead suggesting roles for FdhTU in either intracellular survival or postinvasion recovery. The loss of fdhU caused reduced mRNA levels of formate dehydrogenase (FDH) genes and a severe defect in FDH activity. Cell infection phenotypes of a mutant deleted for the FdhA subunit of FDH and an ΔfdhU ΔfdhA double mutant were similar to those of a ΔfdhU mutant, which likewise suggested that FdhU and FdhA function in the same pathway. Cell infection assays followed by CFU enumeration on plates supplemented with sodium sulfite abolished the ΔfdhU and ΔfdhA mutant defects and resulted in significantly enhanced recovery of all strains, including wild type, at the invasion and intracellular survival time points. Collectively, our data indicate that FdhTU and FDH are required for optimal recovery following cell infection and suggest that C. jejuni alters its metabolic potential in the intracellular environment.     

Synthetic nanopores as a test case for ion channel theories: the anomalous mole fraction effect without single filing

The predictions of a theory for the anomalous mole fraction effect (AMFE) are tested experimentally with synthetic nanopores in plastic. The negatively charged synthetic nanopores under consideration are highly cation selective and 50 Å in diameter at their smallest point. These pores exhibit an AMFE in mixtures of Ca²⁺ and monovalent cations. An AMFE occurs when the conductance through a pore is lower in a mixture of salts than in the pure salts at the same concentration. For ion channels, the textbook interpretation of the AMFE is that multiple ions move through the pore in coordinated, single-file motion. However, because the synthetic nanopores are so wide, their AMFE shows that single filing is not necessary for the AMFE. It is shown that the AMFE in the synthetic nanopores is explained by a theory of preferential ion selectivity. The unique properties of the synthetic nanopores allow us to experimentally confirm several predictions of this theory. These same properties make synthetic nanopores an interesting new platform to test theories of ion channel permeation and selectivity in general.     

POR C of Arabidopsis thaliana: a third light- and NADPH-dependent protochlorophyllide oxidoreductase that is differentially regulated by light

During the sequencing of the genome of Arabidopsis thaliana a gene has been identified that encodes a novel NADPH-protochlorophyllide oxidoreductase (POR)-like protein (accession number AC 002560). This protein has been named POR C. We have expressed the POR C protein in Escherichia coli and have determined its in vitro activity. POR C shows the characteristics of a light-dependent and NADPH-requiring POR similar to POR A and POR B. The expression of the POR C gene differs markedly from that of the POR A and POR B genes. In contrast to the POR A and POR B mRNAs, the POR C mRNA has been shown previously to accumulate only after the beginning of illumination. In light-adapted mature plants only POR B and POR C mRNAs were detectable. The amounts of both mRNAs show pronounced diurnal rhythmic fluctuations. While the oscillations of POR B mRNA are under the control of the circadian clock, those of POR C mRNA are not. Another difference between POR B and POR C was found in seedlings that were grown under continuous white light. The concentration of POR C mRNA rapidly declined and soon dropped beyond the limit of detection, after these seedlings were transferred to the dark. On the other hand, POR B mRNA was unaffected by this light/dark shift. When seedlings were exposed to different light intensities, the amounts of POR B mRNA remained the same, while POR A and POR C mRNAs were modulated in an inverse way by these light intensity changes. POR A mRNA was still detectable in seedlings grown under low light intensities but disappeared at higher light intensities, while the mRNA concentration of POR C rose with increasing light intensities. These different responses to light suggest that the functions of the three PORs of Arabidopsis are not completely redundant, but may allow the plant to adapt its needs for chlorophyll biosynthesis more selectively by using preferentially one of the three enzymes under a given light regime.     

Klaus Apel (1942–2017): a pioneer of photosynthesis research

We present here a Tribute to Klaus Apel (1942–2017), a photosynthesis pioneer—an authority on plant molecular genetics—in five parts. The first section is a prologue. The second section deals with a chronological discussion of Apel’s research life, prepared by the editor Govindjee; it is based on a website article at the Boyce Thompson Institute (BTI) by Patricia Waldron (https://btiscience.org/explore-bti/news/post/bti-says-goodbye-klaus-apel/), as approved for use here by Keith C. Hannon and David Stern of BTI. The third section, which focuses on Apel’s pioneering work on singlet oxygen-mediated EXECUTER-dependent signaling in plants, is written by two of us (J-DR and CK). The fourth section includes three selected reminiscences, one from BTI and two from ETH (Eidgenössische Technische Hochschule). This tribute ends with section five, which is a very brief presentation of Klaus Apel’s personal life, by Wiebke Apel.