Adaptation to life on land at high O2 via transition from ferredoxin-to NADH-dependent redox balance.

Pyruvate : ferredoxin oxidoreductase (PFO) and iron only hydrogenase ([Fe]-HYD) are common enzymes among eukaryotic microbes that inhabit anaerobic niches. Their function is to maintain redox balance by donating electrons from food oxidation via ferredoxin (Fd) to protons, generating H2 as a waste product. Operating in series, they constitute a soluble electron transport chain of one-electron transfers between FeS clusters. They fulfil the same function-redox balance-served by two electron-transfers in the NADH- and O2-dependent respiratory chains of mitochondria. Although they possess O2-sensitive FeS clusters, PFO, Fd and [Fe]-HYD are also present among numerous algae that produce O2. The evolutionary persistence of these enzymes among eukaryotic aerobes is traditionally explained as adaptation to facultative anaerobic growth. Here, we show that algae express enzymes of anaerobic energy metabolism at ambient O2 levels (21% v/v), Chlamydomonas reinhardtii expresses them with diurnal regulation. High O2 environments arose on Earth only approximately 450 million years ago. Gene presence/absence and gene expression data indicate that during the transition to high O2 environments and terrestrialization, diverse algal lineages retained enzymes of Fd-dependent one-electron-based redox balance, while the land plant and land animal lineages underwent irreversible specialization to redox balance involving the O2-insensitive two-electron carrier NADH.

Origin and phylogenetic relationships of [4Fe-4S]-containing O2 sensors of bacteria.

The advent of environmental O2 about 2.5 billion years ago forced microbes to metabolically adapt and to develop mechanisms for O2 sensing. Sensing of O2 by [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion represents an ancient mechanism that is used by FNREc (Escherichia coli), FNRBs (Bacillus subtilis), NreBSa (Staphylococcus aureus) and WhiB3Mt (Mycobacterium tuberculosis). The phylogenetic relationship of these sensors was investigated. FNREc homologues are restricted to the proteobacteria and a few representatives from other phyla. Homologues of FNRBs and NreBSa are located within the bacilli, of WhiB3 within the actinobacteria. Archaea contain no homologues. The data reveal no similarity between the FNREc , FNRBs , NreBSa and WhiB3 sensor families on the sequence and structural levels. These O2 sensor families arose independently in phyla that were already present at the time O2 appeared, their members were subsequently distributed by lateral gene transfer. The chemistry of [4Fe-4S] and [2Fe-2S] cluster formation and interconversion appears to be shared by the sensor protein families. The type of signal output is, however, family specific. The homologues of FNREc and NreBSa vary with regard to the number of Cys residues that coordinate the cluster. It is suggested that the variants derive from lateral gene transfer and gained other functions.

The effect of a simple 3-step pressure relieving strategy for preventing pressure ulcers: an explorative longitudinal study from 2002-2011.

OBJECTIVE: Pressure ulcers (PUs) still form an important and distressing problem in Dutch nursing homes. Pressure ulcer prevention protocols are generally based on current guidelines. The authors developed an alternative 3-step protocol to help prevent pressure ulcers. The effects of this new 3-step protocol on the prevalence of pressure ulcers in patients at risk of developing PUs in the Avoord nursing homes in Etten-Leur/Zundert (Netherlands) were calculated. In addition, the protocol's general cost effects were explored. METHOD: Data on the prevalence of PUs and the use of preventive measures were derived from the annual independent International Prevalence Measurement of Care Problems of Maastricht University (Landelijke prevalentiemeting Zorgproblemen [LPZ]). This annual measurement was implemented in 1998 and measures care problems such as the prevalence of PUs and related preventive measures. Data on patients at risk of developing PUs at the Avoord nursing homes in Etten- Leur/Zundert (Netherlands) were analyzed and compared with national data between 2002 and 2011. RESULTS: The introduction of the 3-step protocol resulted in a significant reduction of the nosocomial prevalence of category 2-4 PUs. The prevalence was reduced from 8.7% to 0.5% during the first year and remained stable at about 2% throughout the rest of the study period. The prevalence at the national level also decreased during this period, but not as much, and was still significantly higher in 2011. The use of alternated systems decreased to almost 1%. Use of static air mattresses showed an almost linear rise in the Avoord nursing homes from the start of the implementation of the protocol, while the trend for both types of mattresses remained stable on a national level. Introducing the static air mattress instead of the more expensive alternating mattresses helped to reduce the mean daily costs of mattresses at the Avoord nursing homes by more than 70% compared to national figures. The workload of the nursing staff decreased as well due to the reduction of repositioning. CONCLUSION: The introduction of the 3-step protocol showed to be effective. The prevalence of PUs and the mean daily costs were reduced to, and have been sustained at, a significantly lower level. .

Molecular data from the chloroplast rpoC1 gene suggest a deep and distinct dichotomy of contemporary spermatophytes into two monophyla: gymnosperms (including Gnetales) and angiosperms.

Partial sequences of the rpoC1 gene from two species of angiosperms and three species of gymnosperms (8330 base pairs) were determined and compared. The data obtained support the hypothesis that angiosperms and gymnosperms are monophyletic and none of the recent groups of the latter is sister to angiosperms.

Chloroplast class I and class II aldolases are bifunctional for fructose-1,6-biphosphate and sedoheptulose-1,7-biphosphate cleavage in the Calvin cycle.

Class I and class II aldolases are products of two evolutionary non-related gene families. The cytosol and chloroplast enzymes of higher plants are of the class I type, the latter being bifunctional for fructose-1,6- and sedoheptulose-1,7-P2 in the Calvin cycle. Recently, class II aldolases were detected for the cytosol and chloroplasts of the lower alga Cyanophora paradoxa. The respective chloroplast enzyme has been shown here to be also bifunctional for fructose-1,6- and sedoheptulose-1,7-P2. Kinetics, also including fructose-1-P, were determined for all these enzymes. Apparently, aldolases are multifunctional enzymes, irrespective of their class I or class II type.

Multiple recruitment of class-I aldolase to chloroplasts and eubacterial origin of eukaryotic class-II aldolases revealed by cDNAs from Euglena gracilis.

The photosynthetic protist Euglena gracilis is one of few organisms known to possess both class-I and class-II fructose-1,6-bisphosphate aldolases (FBA). We have isolated cDNA clones encoding the precursor of chloroplast class-I FBA and cytosolic class-II FBA from Euglena. Chloroplast class-I FBA is encoded as a single subunit rather than as a polyprotein, its deduced transit peptide of 139 amino acids possesses structural motifs neccessary for precursor import across Euglena's three outer chloroplast membranes. Evolutionary analyses reveal that the class-I FBA of Euglena was recruited to the chloroplast independently from the chloroplast class-I FBA of chlorophytes and may derive from the cytosolic homologue of the secondary chlorophytic endosymbiont. Two distinct subfamilies of class-II FBA genes are shown to exist in eubacteria, which can be traced to an ancient gene duplication which occurred in the common ancestor of contemporary gram-positive and proteobacterial lineages. Subsequent duplications involving eubacterial class-II FBA genes resulted in functional specialization of the encoded products for substrates other than fructose-1,6-bisphosphate. Class-II FBA genes of Euglena and ascomycetes are shown to be of eubacterial origin, having been acquired via endosymbiotic gene transfer, probably from the antecedants of mitochondria. The data provide evidence for the chimaeric nature of eukaryotic genomes.

Island colonization and evolution of the insular woody habit in Echium L. (Boraginaceae).

Numerous island-inhabiting species of predominantly herbaceous angiosperm genera are woody shrubs or trees. Such "insular woodiness" is strongly manifested in the genus Echium, in which the continental species of circummediterranean distribution are herbaceous, whereas endemic species of islands along the Atlantic coast of north Africa are woody perennial shrubs. The history of 37 Echium species was traced with 70 kb of noncoding DNA determined from both chloroplast and nuclear genomes. In all, 239 polymorphic positions with 137 informative sites, in addition to 27 informative indels, were found. Island-dwelling Echium species are shown to descend from herbaceous continental ancestors via a single island colonization event that occurred < 20 million years ago. Founding colonization appears to have taken place on the Canary Islands, from which the Madeira and Cape Verde archipelagos were invaded. Colonization of island habitats correlates with a recent origin of perennial woodiness from herbaceous habit and was furthermore accompanied by intense speciation, which brought forth remarkable diversity of forms among contemporary island endemics. We argue that the origin of insular woodiness involved response to counter-selection of inbreeding depression in founding island colonies.

cDNA cloning of a Sec61 homologue from the cryptomonad alga Pyrenomonas salina.

Sec61 is an endoplasmic reticulum transmembrane protein involved in the process of translocation of proteins across this membrane. To-date, the only cloned genes for Sec61 are derived from mammals and yeast. In this paper, we present the first full-length cDNA from a sec61 gene of a plant cell. Comparison of the predicted protein sequence with all known Sec61 proteins, as well as with the bacterial/plastome-encoded homologue SecY, demonstrates a high degree of similarity among the SecY/Sec61 family.

The smallest known eukaryotic genomes encode a protein gene: towards an understanding of nucleomorph functions.

Cryptomonads are unicellular algae with plastids surrounded by four membranes. Between the two pairs of membranes lies a periplastidal compartment that harbours a DNA-containing organelle, termed the nucleomorph. The nucleomorph is the vestigial nucleus of a phototrophic, eukaryotic endosymbiont. Subcloning of parts of one nucleomorph chromosome revealed a gene coding for an Hsp70 protein. We demonstrate the expression of this nucleomorph protein-coding gene and present a model for protein transport from the host to the endosymbiont compartment.

Non-coding chloroplast DNA for plant molecular systematics at the infrageneric level.

With primers constructed against highly conserved regions of tRNA genes (trnTUGU, trnLUAA and trnFGAA) in chloroplast DNA, we have amplified two different non-coding spacers and one intron from four species within the genus Echium L. (Boraginaceae) and from two confamilial outgroups. The trnTUGU-trnLUAA intergenic spacer contains a greater number of polymorphic sites than the trnLUAA intron or the trnLUAA-trnFGAA intergenic spacer. We analyzed a total of 11 kb of sequence data from this non-coding DNA. Total nucleotide divergence between Echium species is on the order of 1% for these regions, all of which possess infrageneric length polymorphisms. The latter two regions contain indels which occur only in the 14 Macaronesian Island endemic species of Echium studied and suggest that these may form a monophyletic group.

Mosses do express conventional, distantly B-type-related phytochromes. Phytochrome of Physcomitrella patens (Hedw.).

We have screened a cDNA library of the moss Physcomitrella patens (Hedw.) for phytochrome sequences. The isolated sequences turned out to encode a phytochrome dissimilar to the phytochrome type postulated for the moss Ceratodon [(1992) Plant Mol. Biol. 20, 1003-1017] Physcomitrella phytochrome was completely alignable to fern phytochrome (Selaginella) and phytochromes of higher plants. The frequency of clones encoding this phytochrome indicated that a Ceratodon-like type should only be expressed, if at all, with lower frequencies than the sequenced phytochrome cDNA. Sequence differences between lower plant phytochromes are small as compared to phytochrome types of higher plants.

Secondary structure and phylogeny of the chloroplast 23S rRNA gene from the brown alga Pylaiella littoralis.

The entire nucleotide sequence of a 23S rRNA gene from the brown alga Pylaiella littoralis (L.) Kjellm has been determined. The predicted length of the 23S rRNA is 2948 nucleotides, including the 4.5S rRNA-like region at the 3' end of the molecule. The putative transcript has been folded into a secondary structure by comparison to existing structure models, and the predicted helical regions were inspected by identifying compensatory downstream base changes. The 23S rRNA secondary structure presented here has features that are unique to P. littoralis (no other chromophyte or red algal 23S rRNA sequences are yet available), but has none of the features specific to the chloroplast rRNAs of green plants and green algae. The Pylaiella sequence was aligned with analogous plastidial and eubacterial gene sequences, and the alignment was used to construct a phylogenetic tree. The plastidial sequences formed a coherent cluster closely associated with the 23S rRNA of the cyanobacterium Anacystis nidulans. Within the plastid group, the P. littoralis sequence was most closely related to that of Euglena gracilis confirming earlier analyses based upon 16S rRNA sequences.

Molecular analysis of the Ubiquitous (Uq) transposable element system of Zea mays.

The Uq transposable element of maize is the most widely dispersed among different maize populations and genetic testerstrains. Despite intensive genetic characterization, little is known about its molecular structure. In order to obtain information relevant to this topic, we have cloned and sequenced three ruq receptors. Surprisingly, they are all Ds1-like receptor types of the Ac-Ds transposon family. Based on our molecular data, we present a model to explain the functional differences associated with the differential expression of the Uq and Ac transposon systems.

Evolution of the Rubisco operon from prokaryotes to algae: structure and analysis of the rbcS gene of the brown alga Pylaiella littoralis.

The rbcS gene coding for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the brown alga Pylaiella littoralis is located within the plastid genome and is transcribed as a single polycistronic mRNA with the gene for the large subunit of Rubisco, rbcL. The structure of the Rubisco operon from P. littoralis was determined. Molecular phylogenies for rbcS and rbcL with a wide range of prokaryotes and eukaryotes were constructed which are congruent with recent evidence for polyphyletic plastid origins. Both rbcL and rbcS of the beta-purple bacterium Alcaligenes eutrophus clearly cluster with the rhodophyte and chromophyte proteins. The data suggest that the Rubisco operons of red algal and chromophytic plastids derive from beta-purple eubacterial antecedents, rather than the cyanobacterial lineage of eubacteria from which other of their genes derive. This implies a lateral transfer of Rubisco genes from beta-purple eubacterial ancestors to the cyanobacterial ancestor of rhodophyte and chromophyte plastids.

Strong functional GC pressure in a light-regulated maize gene encoding subunit GAPA of chloroplast glyceraldehyde-3-phosphate dehydrogenase: implications for the evolution of GAPA pseudogenes.

The light-regulated nuclear gene encoding subunit A of chloroplast glyceraldehyde-3-phosphate dehydrogenase (subunit GAPA, gene Gpa1) from maize is extremely G + C rich (67% in codons). The genomic surroundings of this gene have been characterized together with the sequences of two strongly conserved Gpa pseudogenes isolated from a genomic maize library by differential cDNA hybridization. The comparisons show that the high G + C content of the maize gene is maintained independently of the surrounding noncoding sequences, which are G + C poor (42%), and only as long as the gene encodes a functional protein. After nonfunctionalization, Gpa pseudogenes rapidly loose G + C mainly due to enhanced turnover of CpG and CpXpG methylation sites. These results suggest that the maize Gpa1 gene is under strong functional GC pressure, due to constraints (CpG island) probably exerted at the transcriptional level. They also indicate that Gpa pseudogenes are methylated and that methylation was either the cause or the immediate consequence of their nonfunctionalization. It can be concluded further that the progenitor of pseudogenes 1 and 2 was a second functional Gpa gene (Gpa'), which, after duplication, accelerated in evolutionary rate due to relaxation of selective constraints. This is in agreement with the neutral theory of evolution. Comparison of Gpa intron sequences reveals a gradient of divergence: the more 3' the position of an intron the more its sequence has diverged between the three Gpa genes. A speculative model is presented explaining these observations in terms of a homologous recombination of genes with their reverse-transcribed pre-mRNAs.

Intron conservation across the prokaryote-eukaryote boundary: structure of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize.

The nuclear gene encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from maize has been cloned and sequenced. The gene is G + C rich in its coding sequences and, in addition, contains a CpG-rich region surrounding the promoter. Further upstream several enhancer-like repetitions have been identified that may control the light- and phytochrome-mediated expression of this gene. The gene is interrupted by three introns. Introns 1 and 2 are located within the sequence encoding the transit peptide, dividing it into three parts, each containing one of the three major homology blocks typical for transit peptides of nucleus-encoded chloroplast proteins. Intron 3 is located at codon 166 (glycine) at the same nucleotide position as intron 1 in the GAPDH gene from the nematode Caenorhabditis elegans, suggesting that this intron was present in the parental GAPDH gene from which these two modern descendants originated. Intron 3 divides the GAPDH protein into its two constituent domains, the NAD-binding and the catalytic domain, immediately after helix alpha 1 at a position homologous to that of intron 9 in the gene for maize alcohol dehydrogenase, thereby confirming the prediction of Brändén et al. on the basis of gene-protein structure correlations in maize alcohol dehydrogenase for the placement of introns in the GAPDH gene [Brändén, C.I., Eklund, H., Cambillau, C. & Pryor, A.J. (1984) EMBO J. 3, 1307-1310]. These results suggest that intron 3 is an archetypical relic of early GAPDH and alcohol dehydrogenase evolution, whereas introns 1 and 2 were implicated in the evolution of chloroplast transit peptides.

An overview of occupational safety and health guidelines for Superfund sites.

An overview of the NIOSH occupational safety and health guidelines for Superfund activities is presented. Critical elements are outlined for site safety plans, site surveys and monitoring plans. The gathering of accurate and adequate information to prepare a plan of action to clean up abandoned hazardous waste sites is the first step and continues throughout any Superfund activity. Major emphasis is placed on selecting personal protective equipment with consideration for preventing heat stress. Decontamination is stressed to prevent exposure of other site workers as well as off-site personnel and the public. Worker training and medical monitoring are key components in a comprehensive occupational safety and health program for hazardous waste workers.