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

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of National Academy of Sciences for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences of the United States of America 110 (2013): 8696-8701, doi:10.1073/pnas.1216683110.Organisms that use ammonium as the sole nitrogen source discriminate between [15N] and [14N] 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. We used isotope ratio mass spectrometry (MS) to assess the nitrogen isotopic composition of cell material when the strains were grown in batch culture at either high or low external concentrations of NH3 (achieved by controlling total NH4Cl and pH of the medium). At high NH3 (≥0.89 µM), discrimination against the heavy isotope by the WT strain (−19.2‰) can be accounted for by the equilibrium isotope effect for dissociation of NH4+ to NH3 + H+. NH3 equilibrates across the cytoplasmic membrane, and glutamine synthetase does not manifest an isotope effect in vivo. At low NH3 (≤0.18 µM), discrimination reflects an isotope effect for the NH4+ channel AmtB (−14.1‰). By making E. coli dependent on the low-affinity ammonium-assimilatory pathway, we determined that biosynthetic glutamate dehydrogenase has an inverse isotope effect in vivo (+8.8‰). Likewise, by making unmediated diffusion of NH3 across the cytoplasmic membrane rate-limiting for cell growth in a mutant strain lacking AmtB, we could deduce an in vivo isotope effect for transport of NH3 across the membrane (−10.9‰). The paper presents the raw data from which our conclusions were drawn and discusses the assumptions underlying them.This work was supported by NIH grant GM38361 to S.K. J.M.H. thanks WHOI for support as an emeritus scientist.2013-11-0

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. coli MG1655 (NC_000913). Due to the evolutionary distance to MG1655, these contained a large number of both false negatives and positives. By manual analysis of the dataset, we detected all the known mutations (24 at nine locations) and identified and genetically confirmed new mutations necessary and sufficient for the phenotypes we had selected in four strains. We then had Roche assemble contigs de novo, which we further assembled to full-length pseudomolecules based on synteny with MG1655. This hybrid method facilitated detection of insertion mutations and allowed annotation from MG1655. After removing one genome with less than the optimal 20- to 30-fold sequence coverage, we identified 544 putative polymorphisms that included all of the known and selected mutations apart from insertions. Finally, we detected seven new mutations in a total of only 41 candidates by comparing single genomes to composite data for the remaining six and using a ranking system to penalize homopolymer sequencing and misassembly errors. An additional benefit of the analysis is a table of differences between MG1655 and a physiologically robust E. coli wild-type strain NCM3722. Both projects were greatly facilitated by use of comparative genomics tools in the CoGe software package (http://genomevolution.org/)

Selective isolation of bacterial mRNA

Wendisch VF, Kustu S. Selective isolation of bacterial mRNA. 05.06.2001

Natural History of Transposition in the Green Alga Chlamydomonas reinhardtii: Use of the AMT4 Locus as an Experimental System

The AMT4 locus of the green alga Chlamydomonas reinhardtii, which we mapped to the long arm of chromosome 8, provides a good experimental system for the study of transposition. Most mutations that confer resistance to the toxic ammonium analog methylammonium are in AMT4 and a high proportion of spontaneous mutations are caused by transposon-related events. Among the 15 such events that we have characterized at the molecular level, 9 were associated with insertions of the retrotransposon TOC1, 2 with a small Gulliver-related transposon, and 1 with the Tcr1 transposon. We found that Tcr1 is apparently a foldback transposon with terminal inverted repeats that are much longer and more complex than previously realized. A duplication of Tcr1 yielded a configuration thought to be important for chromosomal evolution. Other mutations in AMT4 were caused by two mobile elements that have not been described before. The sequence of one, which we propose to call the Bill element, indicates that it probably transposes by way of a DNA intermediate and requires functions that it does not encode. The sequence of the other and bioinformatic analysis indicates that it derives from a miniature retrotransposon or TRIM, which we propose to call MRC1 (miniature retrotransposon of Chlamydomonas)

Aquaporin Z of Escherichia coli: Reassessment of Its Regulation and Physiological Role

Transcription of an aqpZ-lac fusion in a single copy on the Escherichia coli chromosome increased as cells entered the stationary growth phase. This was true in a variety of media, and increased transcription in enriched medium required the RpoS sigma factor. Expression of the aqpZ-lac fusion was not affected by up- or downshifts in osmolality. Disruption of aqpZ had no detectable adverse effects