Cutinase-like biodegradable plastic-degrading enzymes from phylloplane yeasts have cutinase activity

Phylloplane yeast genera Pseudozyma and Cryptococcus secrete biodegradable plastic (BP)-degrading enzymes, termed cutinase-like enzymes (CLEs). Although, CLEs contain highly conserved catalytic sites, the whole protein exhibits ≤ 30% amino acid sequence homology with cutinase. In this study, we analyzed whether CLEs exhibit cutinase activity. Seventeen Cryptococcus magnus strains, which degrade BP at 15 °C, were isolated from leaves, and identified the DNA sequence of the CLE in one of the strains. Cutin was prepared from tomato leaves and treated with CLEs from three Cryptococcus species (C. magnus, Cryptococcus flavus, and Cryptococcus laurentii) and Pseudozyma antarctia (PaE). A typical cutin monomer, 10,16-dihydroxyhexadecanoic acid, was detected in extracts of the reaction solution via gas chromatography-mass spectrometry, showing that cutin was indeed degraded by CLEs. In addition to the aforementioned monomer, separation analysis via thin-layer chromatography detected high-molecular-weight products resulting from the breakdown of cutin by PaE, indicating that PaE acts as an endo-type enzyme.

Identification of the PA1113 Gene Product as an ABC Transporter Involved in the Uptake of Carbenicillin in Pseudomonas aeruginosa PAO1

The resistance of Pseudomonas aeruginosa to antibiotics is multi factorial and complex. Whereas efflux pumps such as MexAB-OprM have been thought to predominate, here we show that a novel ATP Binding Cassette (ABC) transporter that mediates influx of carbenicillin from the periplasm to the cytoplasm and away from its cell wall target plays an important role in the resistance of P. aeruginosa to this antibiotic. Treatment of P. aeruginosa with verapamil, an inhibitor of ABC transporters in eukaryotic cells, increases its sensitivity to carbenicillin. Using amino acid sequence homology with known verapamil protein targets as a probe, we determined that the PA1113 gene product, an ABC transporter, mediates carbenicillin uptake into the bacterial cytoplasm. Docking and pharmacological analyses showed that verapamil and carbenicillin compete for the same site on the PA1113 gene protein, explaining the inhibitory effect of verapamil on carbenicillin uptake, and furthermore suggest that the PA1113 ABC transporter accounts for about 30% of P. aeruginosa carbenicillin resistance. Our findings demonstrate that the PA1113 gene product helps mediate carbenicillin resistance by transporting it away from its cell wall target and represents a promising new therapeutic target.

Identification of a Cytopathogenic Toxin from Sneathia amnii

ABSTRACT Sneathia amnii is a poorly characterized emerging pathogen that has been implicated in amnionitis and urethritis. We found that S. amnii damages fetal membranes, and we identified and purified a cytotoxic exotoxin that lyses human red blood cells and damages cells from fetal membranes. The gene appears to be cotranscribed with a second gene that encodes a protein with identity to two-partner system transporters, suggesting that it is the “A,” or secreted component of a type Vb system. The toxin is 1,881 amino acids with a molecular weight of approximately 200 kDa. It binds to red blood cell membranes and forms pores with a diameter of 2.0 to 3.0 nm, resulting in osmolysis. Because it appears to be the “A” or passenger component of a two-partner system, we propose to name this novel cytotoxin/hemolysin CptA for cytopathogenic toxin component A. IMPORTANCE Sneathia amnii is a very poorly characterized emerging pathogen that can affect pregnancy outcome and cause urethritis and other infections. To date, nothing is known about its virulence factors or pathogenesis. We have identified and isolated a cytotoxin, named CptA for cytopathogenic toxin, component A, that is produced by S. amnii. CptA is capable of permeabilizing chorionic trophoblasts and lysing human red blood cells and, thus, may play a role in virulence. Except for small domains conserved among two-partner secretion system passenger proteins, the cytotoxin exhibits little amino acid sequence homology to known toxins. In this study, we demonstrate the pore-forming activity of this novel toxin.

The LisH Domain-Containing N-Terminal Fragment is Important for the Localization, Dimerization, and Stability of Katnal2 in Tetrahymena

Katanin-like 2 protein (Katnal2) orthologs have a tripartite domain organization. Two highly conserved regions, an N-terminal LisH (Lis-homology) domain and a C-terminal AAA catalytic domain, are separated by a less conserved linker. The AAA domain of Katnal2 shares the highest amino acid sequence homology with the AAA domain of the canonical katanin p60. Katnal2 orthologs are present in a wide range of eukaryotes, from protists to humans. In the ciliate Tetrahymena thermophila, a Katnal2 ortholog, Kat2, co-localizes with the microtubular structures, including basal bodies and ciliary outer doublets, and this co-localization is sensitive to levels of microtubule glutamylation. The functional analysis of Kat2 domains suggests that an N-terminal fragment containing a LisH domain plays a role in the subcellular localization, dimerization, and stability of Kat2.

GhYGL1d, a pentatricopeptide repeat protein, is required for chloroplast development in cotton

Background: The pentatricopeptide repeat (PPR) gene family, which contains multiple 35-amino acid repeats, constitutes one of the largest gene families in plants. PPR proteins function in organelles to target specific transcripts and are involved in plant development and growth. However, the function of PPR proteins in cotton is still unknown. Results: In this study, we characterized a PPR gene YELLOW-GREEN LEAF (GhYGL1d) that is required for cotton plastid development. The GhYGL1d gene has a DYW domain in C-terminal and is highly express in leaves, localized to the chloroplast fractions. GhYGL1d share high amino acid-sequence homology with AtECB2. In atecb2 mutant, overexpression of GhYGL1d rescued the seedling lethal phenotype and restored the editing of accD and ndhF transcripts. Silencing of GhYGL1d led to the reduction of chlorophyll and phenotypically yellow-green leaves in cotton. Compared with wild type, GhYGL1d-silenced cotton showed significant deformations of thylakoid structures. Furthermore, the transcription levels of plastid-encoded polymerase (PEP) and nuclear-encoded polymerase (NEP) dependent genes were decreased in GhYGL1d-silenced cotton. Conclusions: Our data indicate that GhYGL1d not only contributes to the editing of accD and ndhF genes, but also affects the expression of NEP- and PEP-dependent genes to regulate the development of thylakoids, and therefore regulates leaf variegation in cotton.

GhYGL1d, a pentatricopeptide repeat protein, is required for chloroplast development in cotton

Background: The pentatricopeptide repeat (PPR) gene family, which contains multiple 35-amino acid repeats, constitutes one of the largest gene families in plants. PPR proteins function in organelles to target specific transcripts and are involved in plant development and growth. However, the function of PPR proteins in cotton is still unknown. Results: In this study, we characterized a PPR gene YELLOW-GREEN LEAF (GhYGL1d) that is required for cotton plastid development. The GhYGL1d gene has a DYW domain in C-terminal and is highly express in leaves, localized to the chloroplast fractions. GhYGL1d share high amino acid-sequence homology with AtECB2. In atecb2 mutant, overexpression of GhYGL1d rescued the seedling lethal phenotype and restored the editing of accD and ndhF transcripts. Silencing of GhYGL1d led to the reduction of chlorophyll and phenotypically yellow-green leaves in cotton. Compared with wild type, GhYGL1d-silenced cotton showed significant deformations of thylakoid structures. Furthermore, the transcription levels of plastid-encoded polymerase (PEP) and nuclear-encoded polymerase (NEP) dependent genes were decreased in GhYGL1d-silenced cotton. Conclusions: Our data indicate that GhYGL1d not only contributes to the editing of accD and ndhF genes, but also affects the expression of NEP- and PEP-dependent genes to regulate the development of thylakoids, and therefore regulates leaf variegation in cotton.

GhYGL1d, a pentatricopeptide repeat protein, is required for chloroplast development in cotton

Background: The pentatricopeptide repeat (PPR) gene family, which contains multiple 35-amino acid repeats, constitutes one of the largest gene families in plants. PPR proteins function in organelles to target specific transcripts and are involved in plant development and growth. However, the function of PPR proteins in cotton is still unknown. Results: In this study, we characterized a PPR gene YELLOW-GREEN LEAF (GhYGL1d) that is required for cotton plastid development. The GhYGL1d gene has a DYW domain in C-terminal and is highly express in leaves, localized to the chloroplast fractions. GhYGL1d share high amino acid-sequence homology with AtECB2. In atecb2 mutant, overexpression of GhYGL1d rescued the seedling lethal phenotype and restored the editing of accD and ndhF transcripts. Silencing of GhYGL1d led to the reduction of chlorophyll and phenotypically yellow-green leaves in cotton. Compared with wild type, GhYGL1d-silenced cotton showed significant deformations of thylakoid structures. Furthermore, the transcription levels of plastid-encoded polymerase (PEP) and nuclear-encoded polymerase (NEP) dependent genes were decreased in GhYGL1d-silenced cotton. Conclusions: Our data indicate that GhYGL1d not only contributes to the editing of accD and ndhF genes, but also affects the expression of NEP- and PEP-dependent genes to regulate the development of thylakoids, and therefore regulates leaf variegation in cotton.

GhYGL1, a pentatricopeptide repeat protein, is required for chloroplast development in cotton

Background The pentatricopeptide repeat (PPR) gene family, which contains multiple 35-amino acid repeats, constitutes one of the largest gene families in plants. Pentatricopeptide repeat genes function in organelles to target specific transcripts and are involved in plant development and growth. However, the function of PPR genes in cotton is still unknown. Results In this study, we characterized a PPR gene (GhYGL1), producing a yellow green leaf phenotype, that is required for cotton plastid development. The GhYGL1 gene has a DYW domain in C-terminal and is highly express in leaves, localized to the chloroplast fractions. GhYGL1 share high amino acid-sequence homology with AtECB2. In ecb2-mutated Arabidopsis, overexpression of GhYGL1 rescued the seedling lethal phenotype and restored the editing of accD and ndhF transcripts. Silencing of GhYGL1 led to the reduction of chlorophyll and phenotypically yellow-green leaves in cotton. Compared with wild type, GhYGL1-silenced cotton showed significant deformations of thylakoid structures. Furthermore, the expression levels of plastid-encoded polymerase- (PEP) and nuclear-encoded polymerase- (NEP) dependent genes were significantly decreased in GhYGL1-silenced cotton. Conclusions Our data indicate that GhYGL1 not only controls the editing of accD and ndhF genes, but also controls the expression of NEP- and PEP-dependent genes to regulate the development of thylakoids, and therefore regulates leaf variegation in cotton.

Fast and sensitive protein sequence homology searches using hierarchical cluster BLAST

The throughput of DNA sequencing continues to increase, allowing researchers to analyze genomes of interest at greater depths. An unintended consequence of this data deluge is the increased cost of analyzing these datasets. As a result, genome and metagenome annotation pipelines are left with a few options: (i) search against smaller reference databases, (ii) use faster, but less sensitive, algorithms to assess sequence similarities, or (iii) invest in computing hardware specifically designed to improve BLAST searches such as GPGPU systems and/or large CPU-rich clusters.We present a pipeline that improves the speed of amino acid sequence homology searches with a minimal decrease in sensitivity and specificity by searching against hierarchical clusters. Briefly, the pipeline requires two homology searches: the first search is against a clustered version of the database and the second is against sequences belonging to clusters with a hit from the first search. We tested this method using two assembled viral metagenomes and three databases (Swiss-Prot, Metagenomes Online, and UniRef100). Hierarchical cluster homology searching proved to be 12-times faster than BLASTp and produced alignments that were nearly identical to BLASTp (precision=0.99; recall=0.97). This approach is ideal when searching large collections of sequences against large databases.