The microbiome of the lichen Lobaria pulmonaria varies according to climate on a subcontinental scale.

The Lobaria pulmonaria holobiont comprises algal, fungal, cyanobacterial and bacterial components. We investigated L. pulmonaria's bacterial microbiome in the adaptation of this ecologically sensitive lichen species to diverse climatic conditions. Our central hypothesis posited that microbiome composition and functionality aligns with subcontinental-scale (a stretch of ~1100 km) climatic parameters related to temperature and precipitation. We also tested the impact of short-term weather dynamics, sampling season and algal/fungal genotypes on microbiome variation. Metaproteomics provided insights into compositional and functional changes within the microbiome. Climatic variables explained 41.64% of microbiome variation, surpassing the combined influence of local weather and sampling season at 31.63%. Notably, annual mean temperature and temperature seasonality emerged as significant climatic drivers. Microbiome composition correlated with algal, not fungal genotype, suggesting similar environmental recruitment for the algal partner and microbiome. Differential abundance analyses revealed distinct protein compositions in Sub-Atlantic Lowland and Alpine regions, indicating differential microbiome responses to contrasting environmental/climatic conditions. Proteins involved in oxidative and cellular stress were notably different. Our findings highlight microbiome plasticity in adapting to stable climates, with limited responsiveness to short-term fluctuations, offering new insights into climate adaptation in lichen symbiosis.

The long-chain flavodoxin FldX1 improves the biodegradation of 4-hydroxyphenylacetate and 3-hydroxyphenylacetate and counteracts the oxidative stress associated to aromatic catabolism in Paraburkholderia xenovorans.

BACKGROUND: Bacterial aromatic degradation may cause oxidative stress. The long-chain flavodoxin FldX1 of Paraburkholderia xenovorans LB400 counteracts reactive oxygen species (ROS). The aim of this study was to evaluate the protective role of FldX1 in P. xenovorans LB400 during the degradation of 4-hydroxyphenylacetate (4-HPA) and 3-hydroxyphenylacetate (3-HPA). METHODS: The functionality of FldX1 was evaluated in P. xenovorans p2-fldX1 that overexpresses FldX1. The effects of FldX1 on P. xenovorans were studied measuring growth on hydroxyphenylacetates, degradation of 4-HPA and 3-HPA, and ROS formation. The effects of hydroxyphenylacetates (HPAs) on the proteome (LC-MS/MS) and gene expression (qRT-PCR) were quantified. Bioaugmentation with strain p2-fldX1 of 4-HPA-polluted soil was assessed, measuring aromatic degradation (HPLC), 4-HPA-degrading bacteria, and plasmid stability. RESULTS: The exposure of P. xenovorans to 4-HPA increased the formation of ROS compared to 3-HPA or glucose. P. xenovorans p2-fldX1 showed an increased growth on 4-HPA and 3-HPA compared to the control strain WT-p2. Strain p2-fldX1 degraded faster 4-HPA and 3-HPA than strain WT-p2. Both WT-p2 and p2-fldX1 cells grown on 4-HPA displayed more changes in the proteome than cells grown on 3-HPA in comparison to glucose-grown cells. Several enzymes involved in ROS detoxification, including AhpC2, AhpF, AhpD3, KatA, Bcp, CpoF1, Prx1 and Prx2, were upregulated by hydroxyphenylacetates. Downregulation of organic hydroperoxide resistance (Ohr) and DpsA proteins was observed. A downregulation of the genes encoding scavenging enzymes (katE and sodB), and gstA and trxB was observed in p2-fldX1 cells, suggesting that FldX1 prevents the antioxidant response. More than 20 membrane proteins, including porins and transporters, showed changes in expression during the growth of both strains on hydroxyphenylacetates. An increased 4-HPA degradation by recombinant strain p2-fldX1 in soil microcosms was observed. In soil, the strain overexpressing the flavodoxin FldX1 showed a lower plasmid loss, compared to WT-p2 strain, suggesting that FldX1 contributes to bacterial fitness. Overall, these results suggest that recombinant strain p2-fldX1 is an attractive bacterium for its application in bioremediation processes of aromatic compounds. CONCLUSIONS: The long-chain flavodoxin FldX1 improved the capability of P. xenovorans to degrade 4-HPA in liquid culture and soil microcosms by protecting cells against the degradation-associated oxidative stress.

Proteomic insight into arabinogalactan utilization by particle-associated Maribacter sp. MAR_2009_72.

Arabinose and galactose are major, rapidly metabolized components of marine particulate and dissolved organic matter. In this study, we observed for the first time large microbiomes for the degradation of arabinogalactan and report a detailed investigation of arabinogalactan utilization by the flavobacterium Maribacter sp. MAR_2009_72. Cellular extracts hydrolysed arabinogalactan in vitro. Comparative proteomic analyses of cells grown on arabinogalactan, arabinose, galactose, and glucose revealed the expression of specific proteins in the presence of arabinogalactan, mainly glycoside hydrolases (GH). Extracellular glycan hydrolysis involved five alpha-l-arabinofuranosidases affiliating with glycoside hydrolase families 43 and 51, four unsaturated rhamnogalacturonylhydrolases (GH105) and a protein with a glycoside hydrolase family-like domain. We detected expression of three induced TonB-dependent SusC/D transporter systems, one SusC, and nine glycoside hydrolases with a predicted periplasmatic location. These are affiliated with the families GH3, GH10, GH29, GH31, GH67, GH78, and GH115. The genes are located outside of and within canonical polysaccharide utilization loci classified as specific for arabinogalactan, for galactose-containing glycans, and for arabinose-containing glycans. The breadth of enzymatic functions expressed in Maribacter sp. MAR_2009_72 as response to arabinogalactan from the terrestrial plant larch suggests that Flavobacteriia are main catalysts of the rapid turnover of arabinogalactans in the marine environment.

Proteomic profiling of antibiotic-resistant Escherichia coli GW-AmxH19 isolated from hospital wastewater treated with physical plasma.

Microorganisms which are resistant to antibiotics are a global threat to the health of humans and animals. Wastewater treatment plants are known hotspots for the dissemination of antibiotic resistances. Therefore, novel methods for the inactivation of pathogens, and in particular antibiotic-resistant microorganisms (ARM), are of increasing interest. An especially promising method could be a water treatment by physical plasma which provides charged particles, electric fields, UV-radiation, and reactive species. The latter are foremost responsible for the antimicrobial properties of plasma. Thus, with plasma it might be possible to reduce the amount of ARM and to establish this technology as additional treatment stage for wastewater remediation. However, the impact of plasma on microorganisms beyond a mere inactivation was analyzed in more detail by a proteomic approach. Therefore, Escherichia coli GW-AmxH19, isolated from hospital wastewater in Germany, was used. The bacterial solution was treated by a plasma discharge ignited between each of four pins and the liquid surface. The growth of E. coli and the pH-value decreased during plasma treatment in comparison with the untreated control. Proteome and antibiotic resistance profile were analyzed. Concentrations of nitrite and nitrate were determined as long-lived indicative products of a transient chemistry associated with reactive nitrogen species (RNS). Conversely, hydrogen peroxide served as indicator for reactive oxygen species (ROS). Proteome analyses revealed an oxidative stress response as a result of plasma-generated RNS and ROS as well as a pH-balancing reaction as key responses to plasma treatment. Both, the generation of reactive species and a decreased pH-value is characteristic for plasma-treated solutions. The plasma-mediated changes of the proteome are discussed also in comparison with the Gram-positive bacterium Bacillus subtilis. Furthermore, no effect of the plasma treatment, on the antibiotic resistance of E. coli, was determined under the chosen conditions. The knowledge about the physiological changes of ARM in response to plasma is of fundamental interest to understand the molecular basis for the inactivation. This will be important for the further development and implementation of plasma in wastewater remediation.

Marine particle microbiomes during a spring diatom bloom contain active sulfate-reducing bacteria.

Phytoplankton blooms fuel marine food webs with labile dissolved carbon and also lead to the formation of particulate organic matter composed of living and dead algal cells. These particles contribute to carbon sequestration and are sites of intense algal-bacterial interactions, providing diverse niches for microbes to thrive. We analyzed 16S and 18S ribosomal RNA gene amplicon sequences obtained from 51 time points and metaproteomes from 3 time points during a spring phytoplankton bloom in a shallow location (6-10 m depth) in the North Sea. Particulate fractions larger than 10 µm diameter were collected at near daily intervals between early March and late May in 2018. Network analysis identified two major modules representing bacteria co-occurring with diatoms and with dinoflagellates, respectively. The diatom network module included known sulfate-reducing Desulfobacterota as well as potentially sulfur-oxidizing Ectothiorhodospiraceae. Metaproteome analyses confirmed presence of key enzymes involved in dissimilatory sulfate reduction, a process known to occur in sinking particles at greater depths and in sediments. Our results indicate the presence of sufficiently anoxic niches in the particle fraction of an active phytoplankton bloom to sustain sulfate reduction, and an important role of benthic-pelagic coupling for microbiomes in shallow environments. Our findings may have implications for the understanding of algal-bacterial interactions and carbon export during blooms in shallow-water coastal areas.

Characterizing the flavodoxin landscape in Clostridioides difficile.

Clostridioides difficile infections have become a major challenge in medical facilities. The bacterium is capable of spore formation allowing the survival of antibiotic treatment. Therefore, research on the physiology of C. difficile is important for the development of alternative treatment strategies. In this study, we investigated eight putative flavodoxins of C. difficile 630. Flavodoxins are small electron transfer proteins of specifically low potential. The unusually high number of flavodoxins in C. difficile suggests that they are expressed under different conditions. We determined high transcription levels for several flavodoxins during the exponential growth phase, especially for floX. Since flavodoxins are capable of replacing ferredoxins under iron deficiency conditions in other bacteria, we also examined their expression in C. difficile under low iron and no iron levels. In particular, the amount of fldX increased with decreasing iron concentration and thus could possibly replace ferredoxins. Moreover, we demonstrated that fldX is increasingly expressed under different oxidative stress conditions and thus may play an important role in the oxidative stress response. While increased fldX expression was detectable at both RNA and protein level, CD2825 showed increased expression only at mRNA level under H2O2 stress with sufficient iron availability and may indicate hydroxyl radical-dependent transcription. Although the exact function of the individual flavodoxins in C. difficile needs to be further investigated, the present study shows that flavodoxins could play an important role in several physiological processes and under infection-relevant conditions. IMPORTANCE: The gram-positive, anaerobic, and spore-forming bacterium Clostridioides difficile has become a vast problem in human health care facilities. The antibiotic-associated infection with this intestinal pathogen causes serious and recurrent inflammation of the intestinal epithelium, in many cases with a severe course. To come up with novel targeted therapies against C. difficile infections, a more detailed knowledge on the pathogen's physiology is mandatory. Eight putative flavodoxins, an extraordinarily high copy number of this type of small electron transfer proteins, are annotated for C. difficile. Flavodoxins are known to be essential electron carriers in other bacteria, for instance, during infection-relevant conditions such as iron limitation and oxidative stress. This work is a first and comprehensive overview on characteristics and expression profiles of the putative flavodoxins in the pathogen C. difficile.

The long-chain flavodoxin FldX1 improves the biodegradation of 4-hydroxyphenylacetate and 3-hydroxyphenylacetate and counteracts the oxidative stress associated to aromatic catabolism in Paraburkholderia xenovorans

Background Bacterial aromatic degradation may cause oxidative stress. The long-chain flavodoxin FldX1 of Paraburkholderia xenovorans LB400 counteracts reactive oxygen species (ROS). The aim of this study was to evaluate the protective role of FldX1 in P. xenovorans LB400 during the degradation of 4-hydroxyphenylacetate (4-HPA) and 3-hydroxy-phenylacetate (3-HPA). Methods The functionality of FldX1 was evaluated in P. xenovorans p2-fldX1 that overexpresses FldX1. The effects of FldX1 on P. xenovorans were studied measuring growth on hydroxyphenylacetates, degradation of 4-HPA and 3-HPA, and ROS formation. The effects of hydroxyphenylacetates (HPAs) on the proteome (LC-MS/MS) and gene expression (qRT-PCR) were quantified. Bioaugmentation with strain p2-fldX1 of 4-HPA-polluted soil was assessed, measuring aromatic degradation (HPLC), 4-HPA-degrading bacteria, and plasmid stability. Results The exposure of P. xenovorans to 4-HPA increased the formation of ROS compared to 3-HPA or glucose. P. xenovorans p2-fldX1 showed an increased growth on 4-HPA and 3-HPA compared to the control strain WT-p2. Strain p2-fldX1 degraded faster 4-HPA and 3-HPA than strain WT-p2. Both WT-p2 and p2-fldX1 cells grown on 4-HPA displayed more changes in the proteome than cells grown on 3-HPA in comparison to glucose-grown cells. Several enzymes involved in ROS detoxification, including AhpC2, AhpF, AhpD3, KatA, Bcp, CpoF1, Prx1 and Prx2, were upregulated by hydroxyphenylacetates. Downregulation of organic hydroperoxide resistance (Ohr) and DpsA proteins was observed. A downregulation of the genes encoding scavenging enzymes (katE and sodB), and gstA and trxB was observed in p2-fldX1 cells, suggesting that FldX1 prevents the antioxidant response. More than 20 membrane proteins, including porins and transporters, showed changes in expression during the growth of both strains on hydroxyphenylacetates. An increased 4-HPA degradation by recombinant strain p2-fldX1 in soil microcosms was observed. In soil, the strain overexpressing the flavodoxin FldX1 showed a lower plasmid loss, compared to WT-p2 strain, suggesting that FldX1 contributes to bacterial fitness. Overall, these results suggest that recombinant strain p2-fldX1 is an attractive bacterium for its application in bioremediation processes of aromatic compounds. Conclusions The long-chain flavodoxin FldX1 improved the capability of P. xenovorans to degrade 4-HPA in liquid culture and soil microcosms by protecting cells against the degradation-associated oxidative stress.