Effects of flavonoids on membrane adaptation of food-associated bacteria.

The effects of naringenin and the biflavonoids amentoflavone and tetrahydroamentoflavone on select bacterial lipids (carotenoids, fatty acids, and menaquinones) and membrane fluidity based on Laurdan generalized polarization were investigated. For this purpose, the pigment-forming food-associated microorganisms Staphylococcus xylosus (DSM 20266T and J70), Staphylococcus carnosus DSM 20501T, and Micrococcus luteus (ATCC 9341 and J3) were studied. The results suggest an envelope stress response by microorganisms due to flavonoids and an employment of adaptive mechanisms using carotenoids, fatty acids, and menaquinones. The flavonoid monomer naringenin impacted carotenoids, fatty acids, menaquinones, and membrane fluidity. Naringenin significantly influenced the carotenoid profile, particularly by an increase in the relative proportion of 4,4'-diaponeurosporenoic acid in Staphylococcus xylosus. Amentoflavone caused changes mainly in the membrane of Micrococcus luteus and decreased the menaquinone content. Tetrahydroamentoflavone mainly affected the carotenoids in the investigated strains. The noticeably different CCS value of tetrahydroamentoflavone compared to naringenin and amentoflavone revealed further insights into the structure-dependent effects of flavonoids. This study provides valuable insights into the response of pigment-forming food-associated microorganisms to naringenin, amentoflavone, and tetrahydroamentoflavone, which is important for the targeted and safe application of the latter as natural preservatives and useful for further research on the mechanisms of action.

Engineered CRISPR/Cas9 System for Transcriptional Gene Silencing in Arthrobacter Species Indicates Bacterioruberin is Indispensable for Growth at Low Temperatures.

Pink-pigmented Arthrobacter species produce the rare C50 carotenoid bacterioruberin, which is suspected to be part of the cold adaptation mechanism. In silico analysis of the repertoire of genes encoded by the Arthrobacter agilis and Arthrobacter bussei genome revealed the biosynthetic pathway of bacterioruberin. Although genetic analysis is an essential tool for studying the physiology of Arthrobacter species, genetic manipulation of Arthrobacter is always time and labor intensive due to the lack of genetic engineering tools. Here we report the construction and application of a CRISPR/deadCas9 system (pCasiART) for gene silencing in Arthrobacter species. The engineered system pCasiART is suitable for the Golden Gate assembly of spacers, enabling rapid and accurate construction of adapted systems. In addition, pCasiART has been developed to provide an efficient transcription inhibition system for genome-wide gene silencing. The gene silencing of the phytoene synthase (CrtB), the first enzyme in bacterioruberin biosynthesis, suppressed bacterioruberin biosynthesis in Arthrobacter agilis and Arthrobacter bussei, resulting in a lack of pink pigmentation, reduction of biomass production, and growth rates at low temperatures.

Exogenous fatty acids affect membrane properties and cold adaptation of Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen that can grow at very low temperatures close to the freezing point of food and other matrices. Maintaining cytoplasmic membrane fluidity by changing its lipid composition is indispensable for growth at low temperatures. Its dominant adaptation is to shorten the fatty acid chain length and, in some strains, increase in addition the menaquinone content. To date, incorporation of exogenous fatty acid was not reported for Listeria monocytogenes. In this study, the membrane fluidity grown under low-temperature conditions was affected by exogenous fatty acids incorporated into the membrane phospholipids of the bacterium. Listeria monocytogenes incorporated exogenous fatty acids due to their availability irrespective of their melting points. Incorporation was demonstrated by supplementation of the growth medium with polysorbate 60, polysorbate 80, and food lipid extracts, resulting in a corresponding modification of the membrane fatty acid profile. Incorporated exogenous fatty acids had a clear impact on the fitness of the Listeria monocytogenes strains, which was demonstrated by analyses of the membrane fluidity, resistance to freeze-thaw stress, and growth rates. The fatty acid content of the growth medium or the food matrix affects the membrane fluidity and thus proliferation and persistence of Listeria monocytogenes in food under low-temperature conditions.

The C50 carotenoid bacterioruberin regulates membrane fluidity in pink-pigmented Arthrobacter species.

Carotenoids have several crucial biological functions and are part of the cold adaptation mechanism of some bacteria. Some pink-pigmented Arthrobacter species produce the rare C50 carotenoid bacterioruberin, whose function in these bacteria is unclear and is found mainly in halophilic archaea. Strains Arthrobacter agilis DSM 20550T and Arthrobacter bussei DSM 109896T show an increased bacterioruberin content if growth temperature is reduced from 30 down to 10 °C. In vivo anisotropy measurements with trimethylammonium-diphenylhexatriene showed increased membrane fluidity and a broadening phase transition with increased bacterioruberin content in the membrane at low-temperature growth. Suppression of bacterioruberin synthesis at 10 °C using sodium chloride confirmed the function of bacterioruberin in modulating membrane fluidity. Increased bacterioruberin content also correlated with increased cell resistance to freeze-thaw stress. These findings confirmed the adaptive function of bacterioruberin for growth at low temperatures for pink-pigmented Arthrobacter species.

Menaquinone-mediated regulation of membrane fluidity is relevant for fitness of Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen with the ability to grow at low temperatures down to - 0.4 °C. Maintaining cytoplasmic membrane fluidity by changing the lipid membrane composition is important during growth at low temperatures. In Listeria monocytogenes, the dominant adaptation effect is the fluidization of the membrane by shortening of fatty acid chain length. In some strains, however, an additional response is the increase in menaquinone content during growth at low temperatures. The increase of this neutral lipid leads to fluidization of the membrane and thus represents a mechanism that is complementary to the fatty acid-mediated modification of membrane fluidity. This study demonstrated that the reduction of menaquinone content for Listeria monocytogenes strains resulted in significantly lower resistance to temperature stress and lower growth rates compared to unaffected control cultures after growth at 6 °C. Menaquinone content was reduced by supplementation with aromatic amino acids, which led to a feedback inhibition of the menaquinone synthesis. Menaquinone-reduced Listeria monocytogenes strains showed reduced bacterial cell fitness. This confirmed the adaptive function of menaquinones for growth at low temperatures of this pathogen.

Arthrobacter bussei sp. nov., a pink-coloured organism isolated from cheese made of cow's milk.

A pink-coloured bacterium (strain KR32T) was isolated from cheese and assigned to the 'Arthrobacter agilis group'. Members of the 'pink Arthrobacter agilis group' form a stable clade (100 % bootstrap value) and contain the species Arthrobacter agilis, Arthrobacter ruber and Arthrobacter echini, which share ≥99.0 % 16S rRNA gene sequence similarity. Isolate KR32T showed highest 16S rRNA gene sequence similarity (99.9 %) to A. agilis DSM 20550T. Additional multilocus sequence comparison confirmed the assignment of strain KR32T to the clade 'pink A. agilis group'. Average nucleotide identity and digital DNA-DNA hybridization values between isolate KR32T and A. agilis DSM 20550T were 82.85 and 26.30 %, respectively. The G+C content of the genomic DNA of isolate KR32T was 69.14 mol%. Chemotaxonomic analysis determined anteiso-C15 : 0 as the predominant fatty acid and MK-9(H2) as the predominant menaquinone. Polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and monoacyldimannosyl-monoacylglycerol. The peptidoglycan type of the isolate was A3α. The carotenoid bacterioruberin was detected as the major pigment. At 10 °C, strain KR32T grew with increased concentrations of bacterioruberin and production of unsaturated fatty acids. Strain KR32T was a Gram-stain-positive, catalase-positive, oxidase-positive and coccus-shaped bacterium with optimal growth at 27-30 °C and pH 8. The results of phylogenetic and phenotypic analyses enabled the differentiation of the isolate from other closely related species of the 'pink A. agilis group'. Therefore, strain KR32T represents a novel species for which the name Arthrobacter bussei sp. nov. is proposed. The type strain is KR32T (=DSM 109896T=LMG 31480T=NCCB 100733T).

Increased Isoprenoid Quinone Concentration Modulates Membrane Fluidity in Listeria monocytogenes at Low Growth Temperatures.

Listeria monocytogenes is a food pathogen capable of growing at a broad temperature range from 50°C to refrigerator temperatures. A key requirement for bacterial activity and growth at low temperatures is the ability to adjust the membrane lipid composition to maintain cytoplasmic membrane fluidity. In this study, we confirmed earlier findings that the extents of fatty acid profile adaptation differed between L. monocytogenes strains. We were able to demonstrate for isolates from food that growth rates at low temperatures and resistance to freeze-thaw stress were not impaired by a lower adaptive response of the fatty acid composition. This indicated the presence of a second adaptation mechanism besides temperature-regulated fatty acid synthesis. For strains that showed weaker adaptive responses in their fatty acid profiles to low growth temperature, we could demonstrate a significantly higher concentration of isoprenoid quinones. Three strains even showed a higher quinone concentration after growth at 6°C than at 37°C, which is contradictory to the reduced respiratory activity at lower growth temperatures. Analyses of the membrane fluidity in vivo by measuring generalized polarization and anisotropy revealed modulation of the transition phase. Strains with increased quinone concentrations showed an expanded membrane transition phase in contrast to strains with pronounced adaptations of fatty acid profiles. The correlation between quinone concentration and membrane transition phase expansion was confirmed by suppression of quinone synthesis. A reduced quinone concentration resulted in a narrower transition phase. Expansion of the phase transition zone by increasing the concentration of non-fatty acid membrane lipids is discussed as an additional mechanism improving adaptation to temperature shifts for L. monocytogenes strains.IMPORTANCEListeria monocytogenes is a foodborne pathogen with an outstanding temperature range for growth. The ability for growth at temperatures close to the freezing point constitutes a serious contamination potential for cold stored food. The only known mechanism of the species for adaptation of membrane fluidity is modification of the membrane fatty acid composition. We were able to demonstrate that, at least for some strains, this adaptation mechanism is supported by regulation of the menaquinone concentration. The increase of this neutral membrane lipid is correlated with fluidization of the membrane under low-temperature conditions and therefore represents a fatty acid-independent mechanism for adaptation to low temperatures.

Electron Accepting Units of the Diheme Cytochrome c TsdA, a Bifunctional Thiosulfate Dehydrogenase/Tetrathionate Reductase.

The enzymes of the thiosulfate dehydrogenase (TsdA) family are wide-spread diheme c-type cytochromes. Here, redox carriers were studied mediating the flow of electrons arising from thiosulfate oxidation into respiratory or photosynthetic electron chains. In a number of organisms, including Thiomonas intermedia and Sideroxydans lithotrophicus, the tsdA gene is immediately preceded by tsdB encoding for another diheme cytochrome. Spectrophotometric experiments in combination with enzymatic assays in solution showed that TsdB acts as an effective electron acceptor of TsdA in vitro when TsdA and TsdB originate from the same source organism. Although TsdA covers a range from -300 to +150 mV, TsdB is redox active between -100 and +300 mV, thus enabling electron transfer between these hemoproteins. The three-dimensional structure of the TsdB-TsdA fusion protein from the purple sulfur bacterium Marichromatium purpuratum was solved by X-ray crystallography to 2.75 Å resolution providing insights into internal electron transfer. In the oxidized state, this tetraheme cytochrome c contains three hemes with axial His/Met ligation, whereas heme 3 exhibits the His/Cys coordination typical for TsdA active sites. Interestingly, thiosulfate is covalently bound to Cys330 on heme 3. In several bacteria, including Allochromatium vinosum, TsdB is not present, precluding a general and essential role for electron flow. Both AvTsdA and the MpTsdBA fusion react efficiently in vitro with high potential iron-sulfur protein from A. vinosum (Em +350 mV). High potential iron-sulfur protein not only acts as direct electron donor to the reaction center in anoxygenic phototrophs but can also be involved in aerobic respiratory chains.