Mechanism for nitrogen isotope fractionation during ammonium assimilation by Escherichia coli K12.

Organisms that use ammonium as the sole nitrogen source discriminate between [(15)N] and [(14)N] ammonium. This selectivity leaves an isotopic signature in their biomass that depends on the external concentration of ammonium. To dissect how differences in discrimination arise molecularly, we examined a wild-type (WT) strain of Escherichia coli K12 and mutant strains with lesions affecting ammonium-assimilatory proteins.

Using genomic sequencing for classical genetics in E. coli K12.

We here develop computational methods to facilitate use of 454 whole genome shotgun sequencing to identify mutations in Escherichia coli K12. We had Roche sequence eight related strains derived as spontaneous mutants in a background without a whole genome sequence. They provided difference tables based on assembling each genome to reference strain E.

The Rut pathway for pyrimidine degradation: novel chemistry and toxicity problems.

The Rut pathway is composed of seven proteins, all of which are required by Escherichia coli K-12 to grow on uracil as the sole nitrogen source. The RutA and RutB proteins are central: no spontaneous suppressors arise in strains lacking them. RutA works in conjunction with a flavin reductase (RutF or a substitute) to catalyze a novel reaction.

Genetic evidence for an essential oscillation of transmembrane-spanning segment 5 in the Escherichia coli ammonium channel AmtB.

Ammonium channels, called Amt or Mep, concentrate NH(4)(+) against a gradient. Each monomer of the trimer has a pore through which substrate passes and a C-terminal cytoplasmic extension. The importance of the C-terminal extension to AmtB activity remains unclear.

An Rh1-GFP fusion protein is in the cytoplasmic membrane of a white mutant strain of Chlamydomonas reinhardtii.

The major Rhesus (Rh) protein of the green alga Chlamydomonas reinhardtii, Rh1, is homologous to Rh proteins of humans. It is an integral membrane protein involved in transport of carbon dioxide. To localize a fusion of intact Rh1 to the green fluorescent protein (GFP), we used as host a white (lts1) mutant strain of C.

The ultrastructure of a Chlamydomonas reinhardtii mutant strain lacking phytoene synthase resembles that of a colorless alga.

Chlamydomonas reinhardtii strains lacking phytoene synthase, the first enzyme of carotenoid biosynthesis, are white. They lack carotenoid pigments, have very low levels of chlorophyll, and can grow only heterotrophically in the dark. Our electron and fluorescence microscopic studies showed that such a mutant strain (lts1-204) had a proliferated plastid envelope membrane but no stacks of thylakoid membranes within the plastid.

Epistatic effects of the protease/chaperone HflB on some damaged forms of the Escherichia coli ammonium channel AmtB.

The Escherichia coli ammonium channel AmtB is a trimer in which each monomer carries a pore for substrate conduction and a cytoplasmic C-terminal extension of approximately 25 residues. Deletion of the entire extension leaves the protein with intermediate activity, but some smaller lesions in this region completely inactivate AmtB, as do some lesions in its cytoplasmic loops. We here provide genetic evidence that inactivation depends on the essential protease HflB, which appears to cause inactivation not as a protease but as a chaperone.

Transcription factors bind thousands of active and inactive regions in the Drosophila blastoderm.

Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. We used whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior-posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos.

The W148L substitution in the Escherichia coli ammonium channel AmtB increases flux and indicates that the substrate is an ion.

The Amt/Mep ammonium channels are trimers in which each monomer contains a long, narrow, hydrophobic pore. Whether the substrate conducted by these pores is NH(3) or NH(4)(+) remains controversial. Substitution of leucine for the highly conserved tryptophan 148 residue at the external opening to Escherichia coli AmtB pores allowed us to address this issue.

The Chlamydomonas genome reveals the evolution of key animal and plant functions.

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella.

A previously undescribed pathway for pyrimidine catabolism.

The b1012 operon of Escherichia coli K-12, which is composed of seven unidentified ORFs, is one of the most highly expressed operons under control of nitrogen regulatory protein C. Examination of strains with lesions in this operon on Biolog Phenotype MicroArray (PM3) plates and subsequent growth tests indicated that they failed to use uridine or uracil as the sole nitrogen source and that the parental strain could use them at room temperature but not at 37 degrees C. A strain carrying an ntrB(Con) mutation, which elevates transcription of genes under nitrogen regulatory protein C control, could also grow on thymidine as the sole nitrogen source, whereas strains with lesions in the b1012 operon could not.

Spontaneous mutations in the ammonium transport gene AMT4 of Chlamydomonas reinhardtii.

Evidence in several microorganisms indicates that Amt proteins are gas channels for NH(3) and CH(3)NH(2), and this has been confirmed structurally. Chlamydomonas reinhardtii has at least four AMT genes, the most reported for a microorganism. Under nitrogen-limiting conditions all AMT genes are transcribed and Chlamydomonas is sensitive to methylammonium toxicity.

The chromosome of Shigella flexneri bacteriophage Sf6: complete nucleotide sequence, genetic mosaicism, and DNA packaging.

Shigella flexneri temperate bacteriophage Sf6 is of interest in part because its prophage expresses the oac gene that alters the antigenic properties of the surface O-antigen polysaccharide of its host bacterium. We have determined the complete sequence of its 39,044 bp genome. The sequence shows that Sf6 is a member of the canonical lambdoid phage group, and like other phages of this type has a highly mosaic genome.

Lack of the Rhesus protein Rh1 impairs growth of the green alga Chlamydomonas reinhardtii at high CO2.

Although Rhesus (Rh) proteins are best known as antigens on human red blood cells, they are not restricted to red cells or to mammals, and hence their primary biochemical functions can be studied in more tractable organisms. We previously established that the Rh1 protein of the green alga Chlamydomonas reinhardtii is highly expressed in cultures bubbled with air containing high CO(2) (3%), conditions under which Chlamydomonas grows rapidly. By RNA interference, we have now obtained Chlamydomonas rh mutants (epigenetic), which are among the first in nonhuman cells.