A fundamental paradigm in microbiology was created by Christian Gram's pioneering 1884 staining technique that characterised the vast majority of the bacterial world as either 'Gram-positive' or 'Gram-negative'. Subsequently, it was recognised that the hallmark of typical Gram-positive bacteria is a single cell membrane (monoderm), whereas Gram-negative bacteria possess a second, outer membrane (OM, diderm). Examination of the distribution of these cell envelope archetypes at higher taxonomic levels has led to the significant insights that [1] the majority of the bacterial world is diderm, [2] most bacteria belong to two large groups ('superphyla'), the Gracilicutes (diderm) and Terrabacteria (diderm or monoderm) and [3] that the likely root of the bacterial evolutionary tree between these superphyla suggests that the 'last bacterial common ancestor' was diderm. However, the earlier 'last universal common ancestor' (primordial protocell) was most likely monoderm. This makes the question 'how did bacterial OM evolve?' central to understanding bacterial evolution. Notably, no well accepted hypothesis for the mechanism of OM evolution has yet been proposed. Slightly counterintuitively, it appears that the defining features of an OM are not characteristic lipids but certain proteins present. Prominent among the definitive OM proteins are bacterial lipoproteins which are modified with a lipid anchor unique to bacteria. OM lipoproteins play crucial roles in OM assembly and stabilisation, particularly those anchored at the inner face of the OM (a 'protein head down, lipid tail up' orientation). In most Gracilicutes, lipoproteins are delivered from the cell membrane to the OM by the Lol ABC exporter system, which has been extensively characterised in the model diderm Escherichia coli. The central role of lipoproteins in OM biology suggests a novel hypothesis for OM evolution. It has been long established that monoderm bacteria can release lipoproteins to their extracellular environment ('shedding'). Thus, we hypothesise that released lipoprotein(s) may have reassociated with the cell wall peptidoglycan and/or surface proteins of monoderm bacteria. Such interactions would position the lipoprotein lipid group towards the bacterial surface i.e., matching the 'protein head down, lipid tail up' orientation of OM lipoproteins. Over evolutionary timescales, such interactions may have become robust enough to form a rudimentary lipid barrier (proto-OM), which was then strengthened by intercalation with released membrane lipids. We propose to test this hypothesis by engineering a lipoprotein hyper-secretion phenotype in a monoderm host, Geobacillus stearothermophilus. To achieve this, we will exploit the E. coli Lol system for OM lipoprotein localisation, inserting the lolCDE lipoprotein exporter genes into the chromosome of the monoderm Geobacillus host. We predict that this system will extract lipoproteins from the Geobacillus cell membrane and, in the absence of an OM, thus create a hyper-secretion phenotype.