Reply to “Concerns about Transmission Electron Micrographs in Recent Article by Chen and Williams”

T his Letter to the Editor concerns a recent article published in mBio (1). I am offering my comments on this article in the spirit of the correct interpretation of scientific data. My areas of expertise are in electron microscopy and in prokaryotic predators known as Bdellovibrio and Bdellovibrio-like organisms (BALOs). I have a recent article in the International Journal of Systematic and Evolutionary Microbiology (2), which will confirm this. I understand the concept behind Dr. William's experiments to coinfect a marine bacterium, Vibrio vulnificus, with both bacterio-phage and Bacteriovorax. However, the transmission electron mi-crographs in Fig. 1 of this article (1) do not support his conclusions. The cells in Fig. 1B to D that he refers to as bdelloplasts look to me like plasmolyzed V. vulnificus cells. The fixation method he used (3) was for encapsulated Escherichia coli strains and not for a marine bacterium. Most marine bacteria are fixed in the presence of salts at a concentration equivalent to that of seawater to maintain osmotic stability. This point aside, two things are usually seen inside a bdelloplast: the prey cell protoplast (with the nutrients) and the growing BALO (see the thin section in Fig. 4 in reference 3). In Fig. 1B to D of reference 1, only one structure is seen inside V. vulnificus. To me, this must be the protoplast of the prey cell, as there are bacteriophages there. Where is the BALO? In Fig. 1D, a growing aseptate filament of a bacterial BALO never looks like that (in good fixation conditions). There are no projections and attachments to the outer cell wall—which is why I think this looks like a plasmolyzed cell. Therefore, the legend to Fig. 1 is incorrect, in that the electron micrographs do not show a predator (Bacte-riovorax) residing inside the prey cell. I also have experience with scanning electron microscopy (2). The Bacteriovorax predators in Fig. 2 do not look well preserved. In Fig. 2A, it looks constricted in the middle. In Fig. 2B, it has an unusual shape for a vibrio— " ballooned out. " Also, how do we know where the bacteriophage CK2 is on the surface of the Vibrio cell? There are no arrows to point them out, and what I do see on the surface look like outer membrane blebs or vesicles. Another point that is not clear in this mBio article is where the agents of coinfection …