Comparative Proteomics Reveals the Anaerobic Lifestyle of Meat-Spoiling Pseudomonas Species

Abstract

The ability of certain Pseudomonas (P.) species to grow or persist in anoxic habitats by either denitrification, acetate fermentation, or arginine fermentation has been described in several studies as a special property. Previously, we had isolated strains belonging to the species P. lundensis, P. weihenstephanensis, and P. fragi from anoxic modified atmosphere packaged (MAP) minced beef and further proved their anaerobic growth in vitro on agar plates. This follow-up study investigated the anaerobic growth of two strains per respective species in situ on inoculated chicken breast filet under 100% N2 modified atmosphere. We were able to prove anaerobic growth of all six strains on chicken breast filet with cell division rates of 0.2–0.8/day. Furthermore, we characterized the anaerobic metabolic lifestyle of these Pseudomonas strains by comparative proteomics, upon their cultivation in meat simulation media, which were constantly gassed with either air or 100% N2 atmospheres. From these proteomic predictions, and respective complementation by physiological experiments, we conclude that the Pseudomonas strains P. fragi, P. weihenstephanensis, P. lundensis exhibit a similar anaerobic lifestyle and employ arginine fermentation via the arginine deiminase (ADI) pathway to grow anaerobically also on MAP meats. Furthermore, glucose fermentation to ethanol via the ED-pathway is predicted to enable long term survival but no true growth, while respiratory growth with nitrate as alternative electron acceptor or glucose fermentation to acetate could be excluded due to absence of essential genes. The citric acid cycle is partially bypassed by the glyoxylate shunt, functioning as the gluconeogenetic route without production of NADH2 under carbon limiting conditions as e.g., in packaged meats. Triggered by an altered redox balance, we also detected upregulation of enzymes involved in protein folding as well as disulfide bonds isomerization under anoxic conditions as a counteracting mechanism to reduce protein misfolding. Hence, this study reveals the mechanisms enabling anaerobic grow and persistence of common meat-spoiling Pseudomonas species, and further complements the hitherto limited knowledge of the anaerobic lifestyle of Pseudomonas species in general.

A Survey of the Microbial Communities of Commercial Presliced, Packaged Deli-Style Ham Throughout Storage

“…It is clear that pseudomonads are more complex and adaptable than they are given credit; generalizations of behavior at the genus level have led to a misunderstanding regarding the environments in which they can flourish. This large genus does contain obligate aerobes but also contains obligate respirators or anoxic persisters that can grow without the presence of oxygen, utilizing additional fermentative pathways (Kolbeck et al, 2021). It has even been observed that Pseudomonas fragi strains in vacuum-packaged beef utilize more protolytic behavior and less aerobic respiration behaviors than counterparts stored aerobically (De Filippis et al, 2018).…”

Nitrogen transformation processes catalyzed by manure microbiomes in earthen pit and concrete storages on commercial dairy farms

“…For example, Proteiniclasticum, Luteimonas, and Proteiniphilum are known to degrade and live on proteins using an inventory of proteases, peptidases and amino acid deaminases [44][45][46] (Additional file 2: Table S7-8). Similarly, Pseudomonas, Hydrogenophaga, Flavobacterium, and those from the Rhodobacteraceae family could obtain ammonia nitrogen from urea [47][48][49][50] (Additional file 2: Tables S6 and S7). As the pH for both storages ranged from 6.92 to 7.85 (Additional file 2: Table S2) and the pKa of ammonia is 9.2, not more than 4% of this compound will occur in the deprotonated or NH 3 form which could be released to the atmosphere (Additional file 2: Table S2).…”

Spoilage Pseudomonas survive common thermal processing schedules and grow in emulsified meat during extended vacuum storage

“…isolated from vacuum packaged meat are able to grow under strictly anaerobic conditions and that arginine fermentation is the primary pathway anaerobic spoilage Pseudomonas spp. use for growth (Hilgarth et al., 2019; Kolbeck et al., 2021). Although the metabolic reasons for this phenomenon require further investigation, advances in microbial sampling techniques and proteomic analysis have generated data that rebut the traditional understanding of the role of Pseudomonas spp.…”