Lysozyme and Penicillin Inhibit the Growth of Anaerobic Ammonium-Oxidizing Planctomycetes

Abstract

bAnaerobic ammonium-oxidizing (anammox) planctomycetes oxidize ammonium in the absence of molecular oxygen with nitrite as the electron acceptor. Although planctomycetes are generally assumed to lack peptidoglycan in their cell walls, recent genome data imply that the anammox bacteria have the genes necessary to synthesize peptidoglycan-like cell wall structures. In this study, we investigated the effects of two antibacterial agents that target the integrity and synthesis of peptidoglycan (lysozyme and penicillin G) on the anammox bacterium Kuenenia stuttgartiensis. The effects of these compounds were determined in both short-term batch incubations and long-term (continuous-cultivation) growth experiments in membrane bioreactors. Lysozyme at 1 g/liter (20 mM EDTA) lysed anammox cells in less than 60 min, whereas penicillin G did not have any observable short-term effects on anammox activity. Penicillin G (0.5, 1, and 5 g/liter) reversibly inhibited the growth of anammox bacteria in continuous-culture experiments. Furthermore, transcriptome analyses of the penicillin G-treated reactor and the control reactor revealed that penicillin G treatment resulted in a 10-fold decrease in the ribosome levels of the cells. One of the cell division proteins (Kustd1438) was downregulated 25-fold. Our results suggested that anammox bacteria contain peptidoglycan-like components in their cell wall that can be targeted by lysozyme and penicillin G-sensitive proteins were involved in their synthesis. Finally, we showed that a continuous membrane reactor system with free-living planktonic cells was a very powerful tool to study the physiology of slow-growing microorganisms under physiological conditions.

Physiological characterization of anaerobic ammonium oxidizing bacterium ‘Candidatus Jettenia caeni’

“…Jettenia caeni’ cells reduced 15 NO 3 − to 15 NH 4 + via 15 NO 2 − . Notably, this experiment was performed in the presence of penicillin G, which is an inhibitor of most heterotrophs but does not affect anammox bacteria (Jetten et al ., ; Okabe et al ., ; Hu et al ., ); therefore, the reduction of 15 NO 3 − to 15 NH 4 + via 15 NO 2 − was likely mediated by ‘ Ca . Jettenia caeni’ cells.…”

Ecology and physiology of anaerobic ammonium oxidizing bacteria

“…Jettenia' is considered a freshwater genus. The recent physiological study Egli et al, 2001Güven et al, 2005Strous et al 2006Dapena-Mora et al, 2007van der Star et al 2008Rattray et al, 2008Kartal et al, 2012Hu et al, 2013bKartal et al, 2013Russ et al, 2014Zhao et al, 2014Oshiki et al, 2011, b Strous et al, 1998, 1999bJetten et al, 2005Carvajal-Arroyo et al, 2013Puyol et al, 2013Kartal et al, 2008Gori et al, 2011Carvajal-Arroyo et al, 2013Puyol et al, 2013Lotti et al, 2014Kartal et al, 2007a…”

“…Of note is that some interesting physiological aspects of anammox bacteria have been deduced from the genomic information and subsequently demonstrated by physiological and/or biochemical experiments: e.g. iron/manganese oxidation and reduction , reduction of NO 2 − to NO and subsequent hydrazine biosynthesis , involvement of anammox bacterial NirK in NO2 − reduction (Hira et al, 2012) and the presence of peptidoglycan structures (Hu et al, 2013b;van Teeseling et al, 2015) and of membrane S-layer (van Teeseling et al, 2014). This fact indicates that the current anammox bacterial genome data are excellent material to examine the physiological characteristics without cultivation.…”

Evolutionary Cell Biology of Division Mode in the Bacterial Planctomycetes-Verrucomicrobia- Chlamydiae Superphylum

“…Nevertheless, the growth of the anammox planctomycetes Cand. Kuenenia stuttgartiensis has been shown to be inhibited by lysozyme and penicillin, which both act on PG (Hu et al, 2013). PG had also been detected in some, but not all, verrucomicrobial species investigated (Yoon et al, 2007a,b, 2008a,b, 2010).…”

“…It is unlikely that the sacculi consisted exclusively of proteins, as proteins of mesophilic organisms cannot resist hours of boiling in SDS and would be denatured under those conditions. Attempts to determine the presence of PG in Planctomycetes never involved lysozyme treatment, until recently (Giovannoni et al, 1987; Hu et al, 2013; Jeske et al, 2015; van Teeseling et al, 2015). …”