Dataset Information

Impact of modified atmospheres on growth and metabolism of meat-spoilage relevant Photobacterium spp. as predicted by comparative proteomics

ABSTRACT: Modified atmosphere packaging (MAP) is a common strategy to selectively prevent the growth of certain species of meat spoiling bacteria. While studies on the effectiveness of MAP are still scarce on a putative control over the population of photobacteria detected as meat spoilers, they could develop means to enhance safety and quality of raw meat. This study aims to determine the impact on photobacteria of two modified atmospheres: with high oxygen concentration (red and white meats), and free oxygen MAP (white meats and seafood). We have conducted growth experiments of the two main species found on meat, Photobacterium carnosum (P.) and P. phosphoreum, on a meat simulation media under different gas mixtures of nitrogen, oxygen and carbon dioxide representing air-, high oxygen- and vacuum-like conditions with and without carbon dioxide present. Growth was monitored based on optical density, and samples were taken during exponential growth for a comparative proteomic analysis that allowed the determination of the effects of the different gases and their synergy. Growth under air atmosphere appears optimal particularly for P. carnosum, with enhancement of energy metabolism, respiration, oxygen consuming reactions, and a predicted preference for lipids as carbon source. However, all the other atmospheres show some degree of growth reduction. An increase in oxygen concentration leads to an increase in enzymes counteracting oxidative stress for both species, and enhancement of heme utilization and iron-sulfur cluster assembly proteins for P. phosphoreum. Absence of oxygen appears to switch the metabolism towards fermentative pathways, where either ribose (P. phosphoreum), or glycogen (P. carnosum) appear to be the preferred substrates. Additionally, it promotes the use of alternative electron donors/acceptors, mainly formate and nitrate/nitrite. Stress response is manifested as enhanced expression of enzymes able to produce ammonia (e.g. carbonic anhydrase, hydroxylamine reductase) and regulate osmotic stress. Our results suggest that photobacteria do not sense the environmental levels of carbon dioxide but rather adapt to their own anaerobic metabolism. The regulation in presence of carbon dioxide is limited and strain-specific under anaerobic conditions. However, when oxygen at air-like concentration is present together with carbon dioxide the oxidative stress appears enhanced compared to air conditions (very low carbon dioxide), explained if both gases have a synergistic effect. This is further supported by the increase in oxygen concentration in presence of carbon dioxide. The atmosphere is able to fully inhibit P. carnosum, heavily reduce P. phosphoreum growth in vitro and trigger diversification of energy production with higher energetic cost, highlighting the importance of concomitant bacteria for their growth on raw meat under said atmosphere.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Photobacterium Phosphoreum Photobacterium Carnosum

SUBMITTER: Christina Ludwig

LAB HEAD: Christina Ludwig

PROVIDER: PXD031343 | Pride | 2022-05-31

REPOSITORIES: Pride

ACCESS DATA

Similar Datasets

Project description:Oxygen and carbon dioxide are common protective gases used in modified atmosphere packaging (MAP) of meat. Within the package, they selectively suppress members of the spoilage microbiome, reshaping it to adapted species concomitantly growing upon MAP. Thus, this species must exhibit adaptation mechanisms to withstand the inhibitory effect of carbon dioxide and oxygen, and cope with selective nutrition on MAP meat. In order to uncover these mechanisms, the typical representative meat-spoiling bacteria Brochothrix (B.) thermosphacta TMW2.2101 and four lactic acid bacteria (LAB) Carnobacterium (C.) divergens TMW2.1577, C. maltaromaticum TMW2.1581, Leuconostoc (L.) gelidum subsp. gelidum TMW2.1618 and L. gelidum subsp. gasicomitatum TMW2.1619 were grown in a meat simulation medium under a controlled, sterile environment, aerated constantly with either air, 100%_N2, 30%_CO2/70%_O2 or 30%_CO2/70%_N2. Growth dynamics were monitored and a label-free quantitative mass spectrometric approach was employed to determine changes within the bacterial proteomes in response to the different gas atmospheres. Revealed bacterial tolerance to modified atmospheres (MA) comprise two possible scenarios: Either bacteria were intrinsically adapted to MA, exhibiting no proteomic regulation of enzymes (L. gelidum subsp. gelidum and gasicomitatum) or, tolerance was provided by varying specific metabolic adaptation (B. thermosphacta, C. divergens, C. maltaromaticum). In detail, metabolic adaptation mechanisms to oxygen comprised an enhanced oxidative stress reduction response, adjustment of the pyruvate metabolism and catabolic oxygen consuming reactions. Adaptation to carbon dioxide was characterized by an upregulation of proteins involved in intracellular pH homeostasis, maintenance of osmotic balance and alteration of the fatty acid composition of the cell membrane. We furthermore predict species-specific strategies for different and preferential carbon source utilization enabling a non-competitive coexistence on meat and resulting in a synergistic spoilage. We conclude that a gas atmosphere containing 30%_CO2/70%_O2 has no inhibitory effect on the analyzed prominent meat-spoiling bacteria whereas 30%_CO2/70%_N2 predictively inhibits C. divergens TMW21577 and B. thermosphacta TMW2.2101 but not the other three species. This gives a mechanistically explanation of their acknowledged status as typical spoilage organisms on packaged meats.

Project description: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 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 fillet under 100% N2 modified atmosphere. We were able to prove anaerobic growth of all six strains on chicken breast fillet 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 disulphide 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.

Project description:The success of turkey breeding for rapid growth rate and larger breast size has coincided with an increasing incidence of a meat quality defect described as pale, soft and exudative (PSE). We hypothesized that this defect, which is associated with an abnormally rapid rate of postmortem metabolism, derives from altered expression of genes involved in metabolic regulation. Our objective was to use deep transcriptome RNA sequence analysis (RNAseq) to identify differentially expressed genes between normal and PSE turkey breasts. Following harvest of turkey breasts (n = 43), the pH at 15 min post-slaughter and percent marinade uptake at 24h post-slaughter were determined. Breast samples were classified as normal or PSE based on marinade uptake (high = normal; low = PSE). Total RNA from samples with the highest (n=4) and lowest (n=4) marinade uptake were isolated and sequenced using the Illumina GAIIX platform. Of 21,340 gene loci discovered by RNAseq, 8480 loci completely matched the turkey reference genome, and 480 genes were differentially expressed (false discovery rate, FDR<0.05) between normal and PSE samples. Two highlights were the genes nephroblastoma overexpressed (NOV), upregulated about 38-fold and pyruvate dehydrogenase kinase isoform 4 (PDK4), downregulated 14-fold in PSE samples. Pathway analysis suggested that several biological functions, including carbohydrate metabolism and energy production, were affected by meat quality. Because PDK4 regulates conversion of pyruvate to acetyl CoA, differences in regulation of oxidative metabolism may exist among turkeys. Accelerated early postmortem metabolism would result in faster pH decline in PSE meat. This hypothesis was supported by the fact that decreased expression of PDK4 was associated with lower pH in PSE samples (pH[PSE] = 5.59M-BM-10.09, pH[normal] = 5.77M-BM-10.17). The RNAseq results provided a greater molecular mechanistic understanding of development of PSE turkey, which will be a foundation for new intervention strategies to prevent development of this defect. The mRNA profiles of normal and PSE turkey breast muscle were generated by deep sequencing using Illumina GAIIx platform. Multiplexing was performed (2 samples/lane). Afterwards, difference in gene expression between normal and PSE samples were tested.

Dataset Information

Impact of modified atmospheres on growth and metabolism of meat-spoilage relevant Photobacterium spp. as predicted by comparative proteomics

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure