The Plant Mitochondrial Carrier Family: Functional and Evolutionary Aspects

Mitochondria play a key role in respiration and energy production and are involved in multiple eukaryotic but also in several plant specific metabolic pathways. Solute carriers in the inner mitochondrial membrane connect the internal metabolism with that of the surrounding cell. Because of their common basic structure, these transport proteins affiliate to the mitochondrial carrier family (MCF). Generally, MCF proteins consist of six membrane spanning helices, exhibit typical conserved domains and appear as homodimers in the native membrane. Although structurally related, MCF proteins catalyze the specific transport of various substrates, such as nucleotides, amino acids, dicarboxylates, cofactors, phosphate or H+. Recent investigations identified MCF proteins also in several other cellular compartments and therefore their localization and physiological function is not only restricted to mitochondria. MCF proteins are a characteristic feature of eukaryotes and bacterial genomes lack corresponding sequences. Therefore, the evolutionary origin of MCF proteins is most likely associated with the establishment of mitochondria. It is not clear whether the host cell, the symbiont, or the chimerical organism invented the ancient MCF sequence. Here, we try to explain the establishment of different MCF proteins and focus on the characteristics of members from plants, in particular from Arabidopsis thaliana

Brevibacterium from Austrian hard cheese harbor a putative histamine catabolism pathway and a plasmid for adaptation to the cheese environment

The genus Brevibacterium harbors many members important for cheese ripening. We performed real-time quantitative PCR (qPCR) to determine the abundance of Brevibacterium on rinds of Vorarlberger Bergkäse, an Austrian artisanal washed-rind hard cheese, over 160 days of ripening. Our results show that Brevibacterium are abundant on Vorarlberger Bergkäse rinds throughout the ripening time. To elucidate the impact of Brevibacterium on cheese production, we analysed the genomes of three cheese rind isolates, L261, S111, and S22. L261 belongs to Brevibacterium aurantiacum, whereas S111 and S22 represent novel species within the genus Brevibacterium based on 16S rRNA gene similarity and average nucleotide identity. Our comparative genomic analysis showed that important cheese ripening enzymes are conserved among the genus Brevibacterium. Strain S22 harbors a 22 kb circular plasmid which encodes putative iron and hydroxymethylpyrimidine/thiamine transporters. Histamine formation in fermented foods can cause histamine intoxication. We revealed the presence of a putative metabolic pathway for histamine degradation. Growth experiments showed that the three Brevibacterium strains can utilize histamine as the sole carbon source. The capability to utilize histamine, possibly encoded by the putative histamine degradation pathway, highlights the importance of Brevibacterium as key cheese ripening cultures beyond their contribution to cheese flavor production

Autochthonous facility-specific microbiota dominates washed-rind Austrian hard cheese surfaces and its production environment

Cheese ripening involves the succession of complex microbial communities that are responsible for the organoleptic properties of the final products. The food processing environment can act as a source of natural microbial inoculation, especially in traditionally manufactured products. Austrian Vorarlberger Bergkäse (VB) is an artisanal washed-rind hard cheese produced in the western part of Austria without the addition of external ripening cultures. Here, the composition of the bacterial communities present on VB rinds and on different processing surfaces from two ripening cellars was assessed by near full length 16S rRNA gene amplification, cloning and sequencing. Non-inoculated aerobic bacteria dominated all surfaces in this study. VB production conditions (long ripening time, high salt concentration and low temperatures) favor the growth of psychro- and halotolerant bacteria. Several bacterial groups, such as coryneforms, Staphylococcus equorum and Halomonas dominated VB and were also found on most environmental surfaces. Analysis of OTUs shared between different surfaces suggests that VB rind bacteria are inoculated naturally during the ripening from the processing environment and that cheese surfaces exert selective pressure on these communities, as only those bacteria better adapted flourished on VB rinds. This study analyzed VB processing environment microbiota and its relationship with VB rinds for the first time, elucidating that the processing environment and the cheese microbiota should be considered as microbiologically linked ecosystems with the goal of better defining the events that take place during cheese maturation

Virulence Pattern Analysis of Three Listeria monocytogenes Lineage I Epidemic Strains with Distinct Outbreak Histories

Strains of the food-borne pathogen Listeria (L.) monocytogenes have diverse virulence potential. This study focused on the virulence of three outbreak strains: the CC1 strain PF49 (serovar 4b) from a cheese-associated outbreak in Switzerland, the clinical CC2 strain F80594 (serovar 4b), and strain G6006 (CC3, serovar 1/2a), responsible for a large gastroenteritis outbreak in the USA due to chocolate milk. We analysed the genomes and characterized the virulence in vitro and in vivo. Whole-genome sequencing revealed a high conservation of the major virulence genes. Minor deviations of the gene contents were found in the autolysins Ami, Auto, and IspC. Moreover, different ActA variants were present. Strain PF49 and F80594 showed prolonged survival in the liver of infected mice. Invasion and intracellular proliferation were similar for all strains, but the CC1 and CC2 strains showed increased spreading in intestinal epithelial Caco2 cells compared to strain G6006. Overall, this study revealed long-term survival of serovar 4b strains F80594 and PF49 in the liver of mice. Future work will be needed to determine the genes and molecular mechanism behind the long-term survival of L. monocytogenes strains in organs

Development of New Measurements and Tools to Mitigate Fescue Toxicosis in Beef Cattle

The objective of this study was to identify new ways to determine the severity of fescue toxicosis and identify genetic differences in fescue impacted traits as a basis in understanding how cattle could be selected for tolerance to fescue toxicosis. We identified across breed and within breed differences in heat stress related traits and growth rate in pregnant cows exposed to toxic fescue. In addition, new biomarkers were identified to differentiate susceptible and tolerant cattle in the form of specific fecal and vaginal microbes. Finally, we identified differentially expressed (DE) genes in high versus low tolerant cattle on toxic fescue. These findings may allow more accurate diagnosis of fescue toxicosis and provide a glimpse into the genes and microorganisms that may impact tolerance or susceptibility to toxic fescue

Abundance and potential contribution of Gram-negative cheese rind bacteria from Austrian artisanal hard cheeses

Many different Gram-negative bacteria have been shown to be present on cheese rinds. Their contribution to cheese ripening is however, only partially understood until now. Here, cheese rind samples were taken from Vorarlberger Bergkäse (VB), an artisanal hard washed-rind cheese from Austria. Ripening cellars of two cheese production facilities in Austria were sampled at the day of production and after 14, 30, 90 and 160 days of ripening. To obtain insights into the possible contribution of Advenella, Psychrobacter, and Psychroflexus to cheese ripening, we sequenced and analyzed the genomes of one strain of each genus isolated from VB cheese rinds. Additionally, quantitative PCRs (qPCRs) were performed to follow the abundance of Advenella, Psychrobacter, and Psychroflexus on VB rinds during ripening in both facilities. qPCR results showed that Psychrobacter was most abundant on cheese rinds and the abundance of Advenella decreased throughout the first month of ripening and increased significantly after 30 days of ripening (p\u3c0.01). Psychrobacter and Psychroflexus increased significantly during the first 30 ripening days (p\u3c0.01), and decreased to their initial abundance during the rest of the ripening time (p\u3c0.05). Genome sequencing resulted in 17 to 27 contigs with assembly sizes of 2.7 Mbp for Psychroflexus, 3 Mbp for Psychrobacter, and 4.3 Mbp for Advenella. Our results reveal that each genome harbors enzymes shown to be important for cheese ripening in other bacteria such as: Cystathionine/Methionine beta or gamma-Lyases, many proteases and peptidases (including proline iminopeptidases), aminotransferases, and lipases. Thus, all three isolates have the potential to contribute positively to cheese ripening. In conclusion, the three species quantified were stable community members throughout the ripening process and their abundance on cheese rinds together with the results from genome sequencing suggest an important contribution of these bacteria to cheese ripening

Plasmids contribute to food processing environment–associated stress survival in three Listeria monocytogenes ST121, ST8, and ST5 strains

Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis and is commonly isolated from food and food production facilities. Many L. monocytogenes strains contain plasmids, though the contributions of plasmids to survival in food production environments is unknown. Three L. monocytogenes ST5, ST8, and ST121 strains containing plasmids, which harbor putative stress response genes, were cured of their plasmids. Wildtype (WT) and plasmid-cured strains were exposed to disinfectant, oxidative, heat, acid, or salt stress. After stress exposure, cells were plated for colony forming unit (CFU) counts to determine survivors. L. monocytogenes WT strains exposed to 0.01% (vol/vol) H2O2, 1% (vol/vol) lactic acid, and 15% (wt/vol) NaCl, pH 5 showed significantly higher counts of survivors compared to the plasmid-cured strains. The number of survivors for the ST5 WT strain exposed to 10 μg/mL benzalkonium chloride (BC) was significantly higher than in the plasmid-cured strain. The ST8 and ST5 strains were exposed to elevated temperature (50° and 55°C respectively); only the ST5 WT strain had significantly higher numbers of survivors than the plasmid-cured strains. Our data revealed that L. monocytogenes ST5, ST8, and ST121 plasmids contribute to tolerance against elevated temperature, salinity, acidic environments, oxidative stress and disinfectants

Comparison of intestinal permeability, morphology, and ileal microbial communities of commercial hens housed in conventional cages and cage-free housing systems

The gastrointestinal health of poultry can be impacted by a variety of factors including their environment. As egg production moves from conventional cage housing (CC) towards cage-free housing (CF), it is important to understand this impact on intestinal health. This study was conducted to determine if housing type impacted intestinal permeability, morphology, and microbial communities in commercial hens across housing systems. Hens were randomly selected from 2 rooms of CC (n = 25) and CF (n = 25) at a commercial facility. Birds were given fluorescein isothiocyanate dextran (FITC-D) by oral gavage to measure intestinal permeability. Jejunal and ileal samples were collected to evaluate villus height, crypt depth and their ratio. Ileal contents were collected for bacterial DNA isolation and 16S rRNA gene sequencing. Serum FITC-D was similar between housing type (P = 0.709). Hens housed in the CF had increased jejunal villus height and crypt depth compared to hens from the CC (P \u3c 0.002). Hens from the CC tended to have a greater villus height to crypt depth ratio in both the jejunum and ileum compared to the CF (P = 0.064; P = 0.091, respectively). Microbial community diversity measurements favored hens housed in the CC as ileal contents tended to have increased species richness (P = 0.059), had greater alpha diversity (P = 0.044), and had an increased number of over represented OTUs (46/64), including Romboutsia sp. (30.80%), Lactobacillus kitasatonis (17.16%), and Lactobacillus aviarius (11.15%). Correlations between microbial communities with intestinal traits identified significant association with the greatest number of correlations with FITC-D and ileal morphology. Many of these correlations identified microbial communities associated with expected traits; thus, providing limited functional data to microbial communities with limited information. The greater number of correlations of ileal morphology with ileal microbial communities suggesting local microbial communities contribute to the intestinal environment distant. In this limited study, several parameters favored hens from CC suggesting an advantage of this system for intestinal health. However, the lower intestinal health parameters observed in CF were not at levels to indicate detrimental effects

Beef cattle that respond differently to fescue toxicosis have distinct gastrointestinal tract microbiota

Tall fescue (Lolium arundinaceum) is a widely used forage grass which shares a symbiosis with the endophytic fungus Epichloë coenophiala. The endophyte produces an alkaloid toxin that provides herbivory, heat and drought resistance to the grass, but can cause fescue toxicosis in grazing livestock. Fescue toxicosis can lead to reduced weight gain and milk yields resulting in significant losses to the livestock industry. In this trial, 149 Angus cows across two farms were continuously exposed to toxic, endophyte-infected, fescue for a total of 13 weeks. Of those 149 cows, 40 were classified into either high (HT) or low (LT) tolerance groups according to their growth performance (weight gain). 20 HT and 20 LT cattle balanced by farm were selected for 16S rRNA gene and ITS1 region Illumina MiSeq amplicon sequencing to compare the fecal microbiota of the two tolerance groups. This study reveals significantly (P\u3c0.05) different bacterial and fungal microbiota between HT and LT cattle, and indicates that fungal phylotypes may be important for an animal’s response to fescue toxicosis: We found that fungal phylotypes affiliating to the Neocallimastigaceae, which are known to be important fiber-degrading fungi, were consistently more abundant in the HT cattle. Whereas fungal phylotypes related to the genus Thelebolus were more abundant in the LT cattle. This study also found more pronounced shifts in the microbiota in animals receiving higher amounts of the toxin. We identified fungal phylotypes which were consistently more abundant either in HT or LT cattle and may thus be associated with the respective animal’s response to fescue toxicosis. Our results thus suggest that some fungal phylotypes might be involved in mitigating fescue toxicosis. The results of our study provide a basis for investigating the functional role of the gastrointestinal microbiota in fescue toxicosis in more detail in the future