Requirement of a Membrane Potential for the Posttranslational Transfer of Proteins into Mitochondsria

Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocatio

Cryptic species in a well-known habitat: applying taxonomics to the amphipod genus Epimeria (Crustacea, Peracarida)

Taxonomy plays a central role in biological sciences. It provides a communication system for scientists as it aims to enable correct identification of the studied organisms. As a consequence, species descriptions should seek to include as much available information as possible at species level to follow an integrative concept of ‘taxonomics’. Here, we describe the cryptic species Epimeria frankei sp. nov. from the North Sea, and also redescribe its sister species, Epimeria cornigera. The morphological information obtained is substantiated by DNA barcodes and complete nuclear 18S rRNA gene sequences. In addition, we provide, for the first time, full mitochondrial genome data as part of a metazoan species description for a holotype, as well as the neotype. This study represents the first successful implementation of the recently proposed concept of taxonomics, using data from highthroughput technologies for integrative taxonomic studies, allowing the highest level of confidence for both biodiversity and ecological research

DNA-SEQUENCE DETERMINATION AND FUNCTIONAL-CHARACTERIZATION OF THE OCT-PLASMID-ENCODED ALKJKL GENES OF PSEUDOMONAS-OLEOVORANS

The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides. The alkJ gene encodes a protein of 59 kilodaltons. The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases. AlkJ is membrane-bound and converts aliphatic medium-chain-length alcohols into aldehydes. The properties of AlkJ suggest that it is linked to the electron transfer chain. AlkJ is necessary for growth on alkanes only in P. putida alcohol dehydrogenase (AlcA) mutants. AlkK is homologous to a range of proteins which act by an ATP-dependent covalent binding of AMP to their substrate. This list includes the acetate, coumarate and long-chain fatty acid CoA ligases. The alkK gene complements a fadD mutation in Escherichia coli, which shows that it indeed encodes an acyl-CoA synthetase. AlkK is a 60 kilodalton protein located in the cytoplasm. AlkL is homologous to OmpW, a Vibrio cholerae outer membrane protein of unknown function, and a hypothetical polypeptide encoded by ytt4 in E. coli. AlkL, OmpW and Ytt4 all have a signal peptide and end with a sequence characteristic of outer membrane proteins. The alkL gene product was found in the outer membrane of E. coli W3110 containing the alk-genes. The alkL gene can be deleted without a clear effect on growth rate. Its function remains unknown. The G+C content of the alkJKL genes is 45%, identical to that of the alkBFGH genes, and significantly lower than the G+C content of the OCT-plasmid and the P. putida chromosome

A HIGHLY THERMOSTABLE NEUTRAL PROTEASE FROM BACILLUS-CALDOLYTICUS - CLONING AND EXPRESSION OF THE GENE IN BACILLUS-SUBTILIS AND CHARACTERIZATION OF THE GENE-PRODUCT

By using a gene library of Bacillus caldolyticus constructed in phage lambda EMBL12 and selecting for proteolytically active phages on plates supplemented with 0.8% skim milk, chromosomal B. caldolyticus DNA fragments that specified proteolytic activity were obtained. Subcloning of one of these fragments in a protease-deficient Bacillus subtilis strain resulted in protease proficiency of the host. The nucleotide sequence of a 2-kb HinfI-MluI fragment contained an open reading frame (ORF) that specified a protein of 544 amino acids. This ORF was denoted as the B. caldolyticus npr gene, because the nucleotide and amino acid sequences of the ORF were highly similar to that of the Bacillus stearothermophilus npr gene. Additionally, the size, pH optimum, and sensitivity to the specific Npr inhibitor phosphoramidon of the secreted enzyme indicated that the B. caldolyticus enzyme was a neutral protease. The B. stearothermophilus and B. caldolyticus enzymes differed at only three amino acid positions. Nevertheless, the thermostability and optimum temperature of the B. caldolyticus enzyme were 7 to 8-degrees-C higher than those of the B. stearothermophilus enzyme. In a three-dimensional model of the B. stearothermophilus Npr the three substitutions (Ala-4 to Thr, Thr-59 to Ala, and Thr-66 to Phe) were present at solvent-exposed positions. The role of these residues in thermostability was analyzed by using site-directed mutagenesis. It was shown that all three amino acid substitutions contributed to the observed difference in thermostability between the neutral proteases from B. stearothermophilus and B. caldolyticus

Identification and organization of carbon dioxide fixation genes in Xanthobacter flavus H4-14

The genes encoding the large (cfxL) and small (cfxS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisC/O) from Xanthobacter flavus H4-14 were identified and characterized. The RuBisC/O genes are separated by 11 bp and cotranscribed in Escherichia coli from the lac promoter in the order cfxLS. Primer extension and R-loop experiments with RNA isolated from autotrophically grown X. flavus H4-14 showed that transcription of cfxL and cfxS initiated 22 bp upstream from cfxL and resulted in a mRNA of at least 2.3 kb. DNA sequence analysis identified the start of an open reading frame transcribed divergently from cfxL, and displaying significant similarities with genes belonging to the lysR family of transcriptional activators. Downstream from cfxS an additional open reading frame was identified with unknown function. Expression studies showed that the genes encoding fructosebisphosphatase (cfxF) and phosphoribulokinase (cfxP) are located downstream from cfxLS. The cfxF and cfxP genes are cotranscribed in the same direction as cfxLS in the order cfxFP.