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Co-ordinating metabolism and growth is a key challenge for all organisms. Despite fluctuating environments, cells must produce the same metabolic outputs to thrive. The mechanisms underlying this 'growth homeostasis' are known in bacteria and eukaryotes, but remain unexplored in archaea. In the model archaeon Halobacterium salinarum, the transcription factor TrmB regulates enzyme-coding genes in diverse metabolic pathways in response to glucose. However, H. salinarum is thought not to catabolize glucose. To resolve this discrepancy, we demonstrate that TrmB regulates the gluconeogenic production of sugars incorporated into the cell surface S-layer glycoprotein. Additionally, we show that TrmB-DNA binding correlates with instantaneous growth rate, likely because S-layer glycosylation is proportional to growth. This suggests that TrmB transduces a growth rate signal to co-regulated metabolic pathways including amino acid, purine, and cobalamin biosynthesis. Remarkably, the topology and function of this growth homeostatic network appear conserved across domains despite extensive alterations in protein components., (© 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

The bacterio-opsin gene (bop) of Halobacterium halobium is located within a cluster with three other genes. Growth conditions of high light intensity and low oxygen tension induce bop gene cluster expression. To identify putative regulatory factor binding sites upstream of the bop gene, we have compared sequences upstream of the bop gene with the corresponding sequences from two other genes in the bop gene cluster. Conserved sequence motifs were observed which may mediate the effect of high light intensity and/or low oxygen tension on bop gene expression. Based on these motifs, a set of mutants was constructed which contained deletions upstream of the bop gene. These constructs were tested in a host strain where bop gene expression is independent of oxygen regulation and in another strain where it is regulated by oxygen and light. The minimal upstream sequence required for both light- and oxygen-regulated bop gene expression was determined to be 54 bp.

The retinal protein halorhodopsin (HR), a light-driven chloride pump from Halobacterium halobium, was homologously overexpressed in this archaebacterium. Two DNA expression systems differing in their promoter region were investigated. The halopsin, hop, promoter coupled to the hop gene gave an increased level of HR synthesis. However, the extent of expression was driven by the copy number of the shuttle vector and did not reach the magnitude of the bacterio-opsin, bop, promoter system. Employing a gene fusion approach, the promoter for the bop gene was used to drive expression of the hop gene. A shuttle vector containing a bop-hop-cartridge was transformed into a HR-deficient strain and blueish-coloured transformants were obtained. The bop promoter expressed HR to an extent where a specific membrane fraction resembled the crystalline purple membrane of BR in terms of the lipid to protein ratio. HR could, therefore, be easily isolated in a natural membrane-bound state. This allows for direct use in biophysical studies without the application of detergents. This was the first successful overexpression of a 7-helical transmembrane protein and may be extended to other proteins of this family.