Archaeal grpE: transcription in two different morphologic stages of Methanosarcina mazei and comparison with dnaK and dnaJ

Abstract

Transcription of the heat shock gene grpE was studied in two different morphologic stages of the archaeon Methanosarcina mazei S-6 that differ in resistance to physical and chemical traumas: single cells and packets. While single cells are directly exposed to environmental changes, such as temperature elevations, cells in packets are surrounded by intercellular and peripheral material that keeps them together in a globular structure which can reach several millimeters in diameter. grpE transcript levels determined by Northern (RNA) blotting peaked after a 15-min heat shock in single cells. In contrast, the highest transcript levels in packets were observed after the longest heat shock tested, 60 min. The same response profiles were demonstrated by primer extension experiments and S1 nuclease analysis. A comparison of the grpE response to heat shock with those of dnaK and dnaJ showed that the grpE transcript level was the most increased, closely followed by that of the dnaK transcript, with that of the dnaJ gene being the least augmented. Transcription of grpE started at the same site under normal and heat shock temperatures, and the transcript was consistently ϳ700 bases long. Codon usage patterns revealed that the three archaeal genes use most codons and have the same codon preference for 61% of the amino acids.A gene encoding a GrpE homolog has recently been cloned from the genome of the archaeon Methanosarcina mazei S-6 and sequenced (6). It is the first example of a grpE heat shock gene found within the domain Archaea. The Archaea are organisms phylogenetically distinct from Bacteria (eubacteria) and Eucarya (eucaryotes) (41). However, Archaea resemble eucaryotes in several molecular biological features more than they resemble bacteria and are closer to eucaryotes than bacteria (15,28,30,31,41). A case in point is gene regulation, since there are data indicating that Archaea use a transcription regulation machinery similar to that of eucaryotes (10,24,28,33). Whether this is the case also for archaeal heat shock genes has not been established. To address this question, it is necessary to first determine the expression pattern of the known archaeal heat shock genes and then proceed to analyze the regulation mechanisms at the molecular level.Among the Archaea, M. mazei is particularly interesting because it is one of a small group of organisms, the methanosarcinae (22,23), that undergo a growth cycle with morphologic conversions (3,25,32,42). The cycle includes a unicellular form or stage, named single cells, and two multicellular forms, called lamina and packets. The latter are globular structures with zonal heterogeneity and intercellular connective material that extends the outer surface of the packets and wraps the cells together. This connective material is composed chiefly of a heteropolysaccharide (14, 16) which confers to the packets resistance to traumas of various types: mechanical, physical (e.g., heat), and chemical (unpublished observations). This resistance is in contrast to the relative fragilit...

Deep sequencing analysis of the Methanosarcina mazei Gö1 transcriptome in response to nitrogen availability

“…Of the cDNAs, 26.4% (NF) and 31.6% (NS) mapped to intergenic regions (IGRs) of the genome in the enriched (ϩ) libraries, as compared with 21% and 28%, respectively, in the cognate nonenriched (Ϫ) libraries, indicating depletion of processed rRNAs and tRNAs in favor of IGR transcripts. Furthermore, a strong enrichment of cDNAs clustering toward the TSS of several mRNA genes analyzed in previous studies was observed [e.g., of grpE and glnA 1 (24,25)]; the 5Ј ends of the grpE and glnA 1 cDNA clusters exactly match the previously identified TSS (Table S1). Fig.…”

Evolution of a Protein-Folding Machine: Genomic and Evolutionary Analyses Reveal Three Lineages of the Archaeal hsp70(dnaK) Gene

“…In previous work, it was demonstrated that the genes in the triad 5¢-grpE-dnaK-dnaJ-3¢ in M. mazei S-6 are transcribed individually from a different promoter for each gene, rather than as an operon from a single promoter as in many bacteria Conway de Macario et al 1995). The structure and organization of the triad in M. mazei S-6 are very similar to those in the other Methanosarcina species studied in this work, all of which display Locus I Type I.…”

“…However, we noticed that the structural features common to Firmicutes and Methanosarcinales were also common to many other bacterial lineages, suggesting the possibility that modern-age Firmicutes and Methanosarcinales may have independently inherited the structural organization of their loci from a primitive prokaryotic ancestor. Furthermore, although in M mazei S-6 and probably in other Methanosarcinales, each gene of Locus I is controlled by a separate promoter Conway de Macario et al 1995), the dnaK locus of Firmicutes is polycistronic, as has been observed in B. subtilis (Homuth et al 1997), Listeria monocytogenes (Hanawa et al 2000), and Clostridium acetobutylicum (Narberhaus et al 1992) or predicted in Staphylococcus aureus (Wang et al 2004). This difference in functionality of the intergenic regions of the locus in Firmicutes vs. Mathanosarcinales may suggest that their similarity in length could have evolved independently in the two lineages.…”

Integration of Foreign DNA in an Intergenic Region of the ArchaeonMethanosarcina mazeiWithout Effect on Transcription of Adjacent Genes

“…This organism, therefore, affords a unique archaeal model suitable to study development of complex multicellular structures in response to environmental changes that might have played a role in evolution, and in response to stressors occurring today in nature and in the laboratory. Also, M. mazei is the only archaeon whose stress (heat-shock) genes dnaK, dnaJ, and grpE have been cloned and sequenced, and their constitutive and induced expression patterns determined Conway de Macario et al, 1995). Consequently, it is now possible to study the response of M. mazei to a variety of stressors not only at the microscopic (morphologic) but also at the molecular genetic level.…”

“…Considerable advances have been made in the understanding of the physiology and metabolism of methanosarcinas and their role in anaerobic bioconversions (Maestrojuán & Boone, 1991;Zinder, 1993), and in the analysis of the structure, organization, and expression of the dnaK-locus genes in M. mazei Conway de Macario et al, 1995). However, the lack of a transformation system stands in the way of progress in the molecular genetic analysis of the in vivo stress-gene expression, and of the effects of manufactured gene modifications, whether in the protein-coding or in the regulatory regions.…”