It is becoming clear that the regulation of gas vesicle biogenesis in Halobacterium salinarum NRC-1 is multifaceted and appears to integrate environmental and metabolic cues at both the transcriptional and posttranscriptional levels. The mechanistic details underlying this process, however, remain unclear. In this manuscript, we quantify the contribution of light scattering made by both intracellular and released gas vesicles isolated from Halobacterium salinarum NRC-1, demonstrating that each form can lead to distinct features in growth curves determined by optical density measured at 600？nm (OD600). In the course of the study, we also demonstrate the sensitivity of gas vesicle accumulation in Halobacterium salinarum NRC-1 on small differences in growth conditions and reevaluate published works in the context of our results to present a hypothesis regarding the roles of the general transcription factor tbpD and the TCA cycle enzyme aconitase on the regulation of gas vesicle biogenesis. 1. Introduction The halophilic archaeon Halobacterium salinarum NRC-1 regulates the production of gas vesicles in response to shifts in various environmental factors. While gas vesicles (protein complexes that sequester gasses likely through the hydrophobic exclusion of water) confer buoyancy, the functional relevance, long thought to be a means by which cells could escape anoxic subsurface environments for more oxygen-rich surface waters, has recently been brought into question [1]. In Halobacterium salinarum NRC-1, gas vesicle proteins are expressed from two gene clusters, gvp1 and gvp2. A copy of each gene cluster is found on the pNRC200 plasmid while pNRC100 encodes only the gvp1 cluster [2–4]. Both gvp gene clusters encode two divergent operons encoding gvpACNO and gvpDEFGHIJKLM, respectively [3]. Of the 14 genes in the gvp gene cluster, only 10 (gvpACODEFGJLM) have been characterized, and only a fraction is required for gas vesicle expression [4–6]. The number and relationships among environmental factors influencing gas vesicle biogenesis has revealed itself to be more complex than originally anticipated. Carbon sources and oxygen may both play a part in regulating the biosynthesis of gas vesicles at both the transcriptional and posttranscriptional levels. A number of studies have suggested that low dissolved oxygen content in the growth media can trigger gas vesicle biogenesis [7–10]. An extension of this hypothesis was used to explain the large increases in gas vesicle abundance observed in batch cultures entering the comparatively oxygen-depleted stationary
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