For decades bacteria have been classified into two main groups by whether or not they retain crystal violet, the so-called "Gram" stain. Gram-positive cells have a single membrane and a thick peptidoglycan (PG) cell wall, which retains the stain where as Gram-negative cells are enclosed by two membranes separated by a thin layer of PG, which does not retain the stain. While more recently the terms Gram "-positive" and "-negative" have fallen out of favor in the face of richer phylogenetic distinctions, the presence of either one or two enclosing membranes remains a fundamentally intriguing difference between bacterial species. Transport across the inner membrane (IM) of double-membraned bacteria and the single membrane of single-membraned bacteria is tightly regulated, as these membranes sustain proton gradients essential for metabolism. Outer membranes (OM)s of double-membraned bacteria are structurally and functionally quite different, containing large amounts of the immunologically important macromolecule lipopolysacharide (LPS, or "endotoxin") and numerous beta-barrel protein porins that allow passive diffusion of small molecules. Assuming the first cells were enclosed by a single membrane, it is unclear how and why second membranes evolved (Bos et al., 2007
; Lake, 2009
In the original bacterial classifications, Gram-positives were assigned to the phylum Firmicutes
. Many species of the bacterial phylum Firmicutes
respond to adverse growth conditions by forming endospores (Piggot and Hilbert, 2004
). Sporulation begins with DNA replication, chromosome segregation and packing, asymmetric positioning of the Z-ring, and septation (reviewed in (Margolin, 2002
)). This yields a mother cell and a daughter cell, or “prespore”, that are separated by a double-membraned septum. After septum formation the mother cell engulfs the prespore in a process morphologically similar to phagocytosis. Inside the mother cell the forespore matures, adding several layers of a protein coat and in some species an exosporium. Finally, when the mother cell lyses, the mature spore is released. These resting forms can remain viable for thousands of years without water or nutrients and can resist, among other environmental insults, UV irradiation, heat, pH extremes and oxidative damage (Setlow, 2007
). When favorable conditions return, the spores germinate and new progeny emerge via outgrowth.
For decades, the model organism for studying both sporulation and the "Gram-positive" cell type has been the bacterium Bacillus subtilis
. B. subtilis
was the first sporulating bacterium to have its genome sequenced and in many ways is an excellent model organism. Its natural competency has facilitated genetic and biochemical characterization and its large size has benefited traditional electron microscopy (EM) and light microscopy (LM) investigations. Largely because in EM images of sporulating Gram-positive cells, the septum was clearly thinner than the thick, vegetative cell wall (Bechtel and Bulla, 1976
), it has long been thought that any PG present in the septum is degraded before engulfment begins. Furthermore, little attention was paid to the fate of the OsM, since it was not part of the future germinating cell.
is part of a lesser-known family of the Firmicutes (the Veillonellaceae
), which form endospores, but which are surprisingly "Gram-negative": they stain Gram-negative, they are enveloped by two membranes and a thin cell wall, and their OMs contain LPS (Hofstad, 1978
; Hofstad and Kristoffersen, 1970
; Kane and Breznak, 1991
; Mergenhagen, 1965
; Rainey, 2009
). Like B. subtilis
, A. longum
forms endospores that are both pasteurization-resistant and calcium dipicolinate-containing (Kane and Breznak, 1991
). Germination results, however, in a double-membraned, Gram-negative cell, calling attention to the origin of the OM and the periplasmic PG.
Also unlike B. subtilis
, A. longum
cells are slender enough to image intact in a near-native state by electron cryo-tomography (ECT). Previous images of B. subtilis
and other sporulating cells were obtained by chemically fixing, dehydrating, plastic embedding, sectioning, and staining the samples. Such approaches sometimes fail to preserve important details or even introduce misleading artifacts (Pilhofer et al., 2010
). ECT involves neither plastic embedding nor staining, yielding "macromolecular" resolution, three-dimensional (3-D) images of biological samples in near-native, frozen-hydrated states (Ben-Harush et al.; Li and Jensen, 2009
). ECT has been used for example to identify the architectures of the bacterial flagellar motor and chemoreceptor arrays (Briegel et al., 2009
; Chen et al., 2011
; Liu et al., 2009
In this study, we imaged vegetative, sporulating and germinating A. longum cells and endospores with ECT. ECT analyses were supplemented with LM, immunofluorescent labeling, Western blotting, traditional EM, mass spectrometry, genome sequencing, and phylogenetic profiling. The images and analyses show that a thin PG layer persists in the septum throughout sporulation and germination, that a protein coat (likely SpoIVA) forms densely packed concentric rings on the mother side of the engulfing septum, and that a region of the IM of the mother cell is inverted and transformed to become the OM of the outgrowing cell. A. longum's peculiar characteristics as an endospore-forming Gram-negative cell suggest interesting new explanations for the evolution of all Firmicutes and the bacterial OM.