Deletion of the archaeal histone in Methanosarcina mazei Gö1 results in reduced growth and genomic transcription.
ABSTRACT HMm is the only archaeal histone in Methanosarcina mazei Göl and recombinant HMm, synthesized by expression of MM1825 in Escherichia coli, has been purified and confirmed to have the DNA binding and compaction properties characteristic of an archaeal histone. Insertion of a puromycin resistance conferring cassette (pac) into MM1825 was not lethal but resulted in mutants (M. mazei MM1825::pac) that have impaired ability to grow on methanol and trimethylamine. Loss of HMm also resulted in increased sensitivity to UV light and decreased transcript levels for approximately 25% of all M. mazei genes. For most genes, the transcript decrease was 3- to 10-fold, but transcripts of MM483 (small heat-shock protein), MM1688 (trimethylamine:corrinoid methyl transferase) and MM3195 (transcription regulator), were reduced 100-, 100- and 25-fold, respectively, in M. mazei MM1825::pac cells. Transcripts of only five adjacent genes that appear to constitute an aromatic amino acid biosynthetic operon were elevated in M. mazei MM1825::pac cells. Complementary synthesis of HMm from a plasmid transformed into M. mazei MM1825::pac restored wild-type growth and transcript levels.
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ABSTRACT: Methansarcina mazei Gö1 DNA arrays were constructed and used to evaluate the genomic expression patterns of cells grown on either of two alternative methanogenic substrates, acetate or methanol, as sole carbon and energy source. Analysis of differential transcription across the genome revealed two functionally grouped sets of genes that parallel the central biochemical pathways in, and reflect many known features of, acetate and methanol metabolism. These include the acetate-induced genes encoding acetate activating enzymes, acetyl-CoA synthase/CO dehydrogenase, and carbonic anhydrase. Interestingly, additional genes expressed at significantly higher levels during growth on acetate included two energy-conserving complexes (the Ech hydrogenase, and the A1A0-type ATP synthase). Many previously unknown features included the induction by acetate of genes coding for ferredoxins and flavoproteins, an aldehyde:ferredoxin oxidoreductase, enzymes for the synthesis of aromatic amino acids, and components of iron, cobalt and oligopeptide uptake systems. In contrast, methanol-grown cells exhibited elevated expression of genes assigned to the methylotrophic pathway of methanogenesis. Expression of genes for components of the translation apparatus was also elevated in cells grown in the methanol medium relative to acetate, and was correlated with the faster growth rate observed on the former substrate. These experiments provide the first comprehensive insight into substrate-dependent gene expression in a methanogenic archaeon. This genome-wide approach, coupled with the complementary molecular and biochemical tools, should greatly accelerate the exploration of Methanosarcina cell physiology, given the present modest level of our knowledge of these large archaeal genomes.Molecular and General Genetics 06/2005; 273(3):225-39. · 2.88 Impact Factor
Article: Bacterial chromatin.[show abstract] [hide abstract]
ABSTRACT: Recent studies have revealed that the bacterial nucleoid is a dynamic entity that alters its overall structure in response to changes in both growth rate and growth phase. These structural changes are correlated with, and might be driven by, changes in the distribution and utilization of DNA supercoiling. In turn, these parameters in addition to the delimitation of topological domains are dependent both on the relative proportions of the abundant nucleoid-associated proteins and on transcriptional activity. The domain structure itself is dynamic.Current Opinion in Genetics & Development 11/2005; 15(5):507-14. · 7.47 Impact Factor
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ABSTRACT: Histone preparations from Methanothermus fervidus (HMf) contain two small polypeptides, HMfA and HMfB, which in solution are dimers and compact DNA to form nucleosome-like structures. These archaeal nucleosome-like structures constrain positive DNA supercoils, in contrast to the negatively supercoiled DNA in eukaryal nucleosomes. HMfA has been found to make up as much as 80% of HMf preparations synthesized by M. fervidus cells during the exponential growth phase of batch cultures but to decrease to approximately 50% as cultures enter the stationary phase. By using a nondenaturing polyacrylamide gel system at pH 6.1, we have demonstrated that HMf preparations contain HMfA homodimers, HMfB homodimers, and HMfA-HMfB heterodimers and that heating a mixture of recombinant HMfA and HMfB homodimers at 95 degrees C for 5 min generates HMfA-HMfB heterodimers. Circular dichroism spectroscopy indicates that HMfA and HMfB have very similar secondary structures, but based on agarose gel electrophoretic mobility shifts, DNA topology assays, and electron microscopy, they have different DNA binding properties. HMfA binding to DNA could be detected at lower protein/DNA ratios than HMfB, but HMfB binding resulted in more extensive DNA compaction. The increased HMfB synthesized in cells approaching the stationary phase and the highly compacted state of HMfB-bound DNA are consistent with preparations for the impending period of limited genome activity.Proceedings of the National Academy of Sciences 01/1995; 91(26):12624-8. · 9.74 Impact Factor