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.

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.

Carbon Source-Dependent Reprogramming of Anaerobic Metabolism in Staphylococcus aureus.

To be a successful pathogen, Staphylococcus aureus has to adapt its metabolism to the typically oxygen- and glucose-limited environment of the host. Under fermenting conditions and in the presence of glucose, S. aureus uses glycolysis to generate ATP via substrate-level phosphorylation and mainly lactic acid fermentation to maintain the redox balance by reoxidation of NADH equivalents. However, it is less clear how S. aureus proceeds under anoxic conditions and glucose limitation, likely representing the bona fide situation in the host. Using a combination of proteomic, transcriptional, and metabolomic analyses, we show that in the absence of an abundant glycolysis substrate, the available carbon source pyruvate is converted to acetyl coenzyme A (AcCoA) in a pyruvate formate-lyase (PflB)-dependent reaction to produce ATP and acetate. This process critically depends on derepression of the catabolite control protein A (CcpA), leading to upregulation of pflB transcription. Under these conditions, ethanol production is repressed to prevent wasteful consumption of AcCoA. In addition, our global and quantitative characterization of the metabolic switch prioritizing acetate over lactate fermentation when glucose is absent illustrates examples of carbon source-dependent control of colonization and pathogenicity factors.IMPORTANCE Under infection conditions, S. aureus needs to ensure survival when energy production via oxidative phosphorylation is not possible, e.g., either due to the lack of terminal electron acceptors or by the inactivation of components of the respiratory chain. Under these conditions, S. aureus can switch to mixed-acid fermentation to sustain ATP production by substrate level phosphorylation. The drop in the cellular NAD+/NADH ratio is sensed by the repressor Rex, resulting in derepression of fermentation genes. Here, we show that expression of fermentation pathways is further controlled by CcpA in response to the availability of glucose to ensure optimal resource utilization under growth-limiting conditions. We provide evidence for carbon source-dependent control of colonization and virulence factors. These findings add another level to the regulatory network controlling mixed-acid fermentation in S. aureus and provide additional evidence for the lifestyle-modulating effect of carbon sources available to S. aureus.

A Core Genome Multilocus Sequence Typing Scheme for Enterococcus faecalis.

Among enterococci, Enterococcus faecalis occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of E. faecalis complicates both molecular investigations and phylogenetic analyses. Whole-genome sequencing (WGS) enables unraveling of epidemiological linkages and putative transmission events between humans, animals, and food. Core genome multilocus sequence typing (cgMLST) aims to combine the discriminatory power of classical multilocus sequence typing (MLST) with the extensive genetic data obtained by WGS. By sequencing a representative collection of 146 E. faecalis strains isolated from hospital outbreaks, food, animals, and colonization of healthy human individuals, we established a novel cgMLST scheme with 1,972 gene targets within the Ridom SeqSphere+ software. To test the E. faecalis cgMLST scheme and assess the typing performance, different collections comprising environmental and bacteremia isolates, as well as all publicly available genome sequences from the NCBI and SRA databases, were analyzed. In more than 98.6% of the tested genomes, >95% good cgMLST target genes were detected (mean, 99.2% target genes). Our genotyping results not only corroborate the known epidemiological background of the isolates but exceed previous typing resolution. In conclusion, we have created a powerful typing scheme, hence providing an international standardized nomenclature that is suitable for surveillance approaches in various sectors, linking public health, veterinary public health, and food safety in a true One Health fashion.

Comparative proteome analysis in an Escherichia coli CyDisCo strain identifies stress responses related to protein production, oxidative stress and accumulation of misfolded protein.

BACKGROUND: The Twin-arginine translocation (Tat) pathway of Escherichia coli has great potential for the export of biopharmaceuticals to the periplasm due to its ability to transport folded proteins, and its proofreading mechanism that allows correctly folded proteins to translocate. Coupling the Tat-dependent protein secretion with the formation of disulfide bonds in the cytoplasm of E. coli CyDisCo provides a powerful platform for the production of industrially challenging proteins. In this study, we investigated the effects on the E. coli cells of exporting a folded substrate (scFv) to the periplasm using a Tat signal peptide, and the effects of expressing an export-incompetent misfolded variant. RESULTS: Cell growth is decreased when either the correctly folded or misfolded scFv is expressed with a Tat signal peptide. However, only the production of misfolded scFv leads to cell aggregation and formation of inclusion bodies. The comprehensive proteomic analysis revealed that both conditions, recombinant protein overexpression and misfolded protein accumulation, lead to downregulation of membrane transporters responsible for protein folding and insertion into the membrane while upregulating the production of chaperones and proteases involved in removing aggregates. These conditions also differentially affect the production of transcription factors and proteins involved in DNA replication. The most distinct stress response observed was the cell aggregation caused by elevated levels of antigen 43. Finally, Tat-dependent secretion causes an increase in tatA expression only after induction of protein expression, while the subsequent post-induction analysis revealed lower tatA and tatB expression levels, which correlate with lowered TatA and TatB protein abundance. CONCLUSIONS: The study identified characteristic changes occurring as a result of the production of both a folded and a misfolded protein, but also highlights an exclusive unfolded stress response. Countering and compensating for these changes may result in higher yields of pharmaceutically relevant proteins exported to the periplasm.

Proteomic analysis of the food spoiler Pseudomonas fluorescens ITEM 17298 reveals the antibiofilm activity of the pepsin-digested bovine lactoferrin.

Pseudomonas fluorescens is implicated in food spoilage especially under cold storage. Due to its ability to form biofilm P. fluorescens resists to common disinfection strategies increasing its persistance especially across fresh food chain. Biofilm formation is promoted by several environmental stimuli, but gene expression and protein changes involved in this lifestyle are poorly investigated in this species. In this work a comparative proteomic analysis was performed to investigate metabolic pathways of underlying biofilm formation of the blue cheese pigmenting P. fluorescens ITEM 17298 after incubation at 15 and 30 °C; the same methodology was also applied to reveal the effects of the bovine lactoferrin hydrolysate (HLF) used as antibiofilm agent. At 15 °C biofilm biomass and motility increased, putatively sustained by the induction of regulators (PleD, AlgB, CsrA/RsmA) involved in these phenotypic traits. In addition, for the first time, TycC and GbrS, correlated to indigoidine synthesis (blue pigment), were detected and identified. An increase of virulence factors amounts (leukotoxin and PROKKA_04561) were instead found at 30 °C. HLF caused a significant reduction in biofilm biomass; indeed, at 15 °C HLF repressed PleD, TycC and GbrS and induced the negative regulators of alginate biosynthesis; at both temperatures induced the cyclic-di-GMP-binding biofilm dispersal mediator (PROKKA_02061). In conclusion, in this work protein determinats of biofilm formation were revelead in ITEM 17298 under the low temperature; the synthesis of these latter were inhibited by HLF confirming its possible exploitation as antibiofilm agent for biotechnological applications in cold stored foods.

Stability of Proteins Out of Service: the GapB Case of Bacillus subtilis.

Bacillus subtilis possesses two glyceraldehyde-3-phosphate dehydrogenases with opposite roles, the glycolytic NAD-dependent GapA and the NADP-dependent GapB enzyme, which is exclusively required during gluconeogenesis but not active under conditions promoting glycolysis. We propose that proteins that are no longer needed will be recognized and proteolyzed by Clp proteases and thereby recycled. To test this postulation, we analyzed the stability of the glycolytic enzyme GapA and the gluconeogenetic enzyme GapB in the presence and absence of glucose. It turned out that GapA remained rather stable under both glycolytic and gluconeogenetic conditions. In contrast, the gluconeogenetic enzyme GapB was degraded after a shift from malate to glucose (i.e., from gluconeogenesis to glycolysis), displaying an estimated half-life of approximately 3 h. Comparative in vivo pulse-chase labeling and immunoprecipitation experiments of the wild-type strain and isogenic mutants identified the ATP-dependent ClpCP protease as the enzyme responsible for the degradation of GapB. However, arginine protein phosphorylation, which was recently described as a general tagging mechanism for protein degradation, did not seem to play a role in GapB proteolysis, because GapB was also degraded in a mcsB mutant, lacking arginine kinase, in the same manner as in the wild type.IMPORTANCE GapB, the NADP-dependent glyceraldehyde-3-phosphosphate dehydrogenase, is essential for B. subtilis under gluconeogenetic conditions. However, after a shift to glycolytic conditions, GapB loses its physiological function within the cell and becomes susceptible to degradation, in contrast to GapA, the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, which remains stable under glycolytic and gluconeogenetic conditions. Subsequently, GapB is proteolyzed in a ClpCP-dependent manner. According to our data, the arginine kinase McsB is not involved as adaptor protein in this process. ClpCP appears to be in charge in the removal of inoperable enzymes in B. subtilis, which is a strictly regulated process in which the precise recognition mechanism(s) remains to be identified.

Bacillus pumilus KatX2 confers enhanced hydrogen peroxide resistance to a Bacillus subtilis PkatA::katX2 mutant strain.

BACKGROUND: Bacillus pumilus cells exhibit a significantly higher resistance to hydrogen peroxide compared to closely related Bacilli like Bacillus subtilis. RESULTS: In this study we analyzed features of the catalase KatX2 of B. pumilus as one of the most important parts of the cellular response to hydrogen peroxide. KatX2, the vegetative catalase expressed in B. pumilus, was compared to the vegetative catalase KatA of B. subtilis. Data of our study demonstrate that B. pumilus can degrade toxic concentrations of hydrogen peroxide faster than B. subtilis. By replacing B. subtilis katA gene by katX2 we could significantly enhance its resistance to H2O2 and its potential to eliminate this toxic compound. Mutant cells showed a 1.5- to 2-fold higher survival to toxic concentrations of hydrogen peroxide compared to wild type cells. Furthermore, we found reversible but also irreversible oxidations of the KatX2 protein which, in contrast to KatA, contains several cysteine residues. CONCLUSIONS: Our study indicates that the catalase KatX2 plays a major role in the increased resistance of B. pumilus to oxidative stress caused by hydrogen peroxide. Resistance to hydrogen peroxide of other Bacilli can be enhanced by exchanging the native catalase in the cells with katX2.

Biotransformation and reduction of estrogenicity of bisphenol A by the biphenyl-degrading Cupriavidus basilensis.

The biphenyl-degrading Gram-negative bacterium Cupriavidus basilensis (formerly Ralstonia sp.) SBUG 290 uses various aromatic compounds as carbon and energy sources and has a high capacity to transform bisphenol A (BPA), which is a hormonally active substance structurally related to biphenyl. Biphenyl-grown cells initially hydroxylated BPA and converted it to four additional products by using three different transformation pathways: (a) formation of multiple hydroxylated BPA, (b) ring fission, and (c) transamination followed by acetylation or dimerization. Products of the ring fission pathway were non-toxic and all five products exhibited a significantly reduced estrogenic activity compared to BPA. Cell cultivation with phenol and especially in nutrient broth (NB) resulted in a reduced biotransformation rate and lower product quantities, and NB-grown cells did not produce all five products in detectable amounts. Thus, the question arose whether enzymes of the biphenyl degradation pathway are involved in the transformation of BPA and was addressed by proteomic analyses.

Proteome and carbon flux analysis of Pseudomonas aeruginosa clinical isolates from different infection sites.

Pseudomonas aeruginosa is known as opportunistic pathogen frequently isolated from different infection sites. To investigate the expression rates of P. aeruginosa proteins commonly expressed by different clinical isolates, absolute protein quantities were determined employing a gel-free and data-independent LC-IMS(E) approach. Moreover, the metabolic diversity of these isolates was investigated by (13) C-metabolic flux analyses. 812 proteins were reproducibly identified and absolutely quantified for the reference strain P. aeruginosa PAO1, 363 of which were also identified and relatively quantified in all isolates. Whilst the majority of these proteins were expressed in constant amounts, expression rates of 42 proteins were highly variable between the isolates. Notably, the outer membrane protein OprH and the response regulator PhoP were strongly expressed in burned wounds isolates compared to lung/urinary tract isolates. Moreover, proteins involved in iron/amino acids uptake were found to be highly abundant in urinary tract isolates. The fluxome data revealed a conserved glycolysis, and a niche-specific divergence in fluxes through the glyoxylate shunt and the TCA cycle among the isolates. The integrated proteome/fluxome analysis did not indicate straightforward correlation between the protein amount and flux, but rather points to additional layers of regulation that mediate metabolic adaption of P. aeruginosa to different host environments. All MS data have been deposited in the ProteomeXchange with identifier PXD002373 (http://proteomecentral.proteomexchange.org/dataset/PXD002373).

Coping with anoxia: a comprehensive proteomic and transcriptomic survey of denitrification.

Ralstonia eutropha H16 is a denitrifying microorganism able to use nitrate and nitrite as terminal electron acceptors under oxygen deprivation. To identify proteins showing an altered expression pattern in response to oxygen supply, R. eutropha cells grown aerobically and anaerobically were compared in a comprehensive proteome and transcriptome approach. Nearly 700 proteins involved in several processes including respiration, formation of cell appendages, and DNA and cofactor biosynthesis were found to be differentially expressed. A combination of 1D gel-LC and conventional 2D gel analysis of six consecutive sample points covering the entire denitrification sequence revealed a detailed view on the shifting abundance of the key proteins of denitrification. Denitrification- or anaerobiosis-induced alterations of the respiratory chain included a distinct expression pattern for multiple terminal oxidases. Alterations in the central carbon metabolism were restricted to a few key functions including the isoenzymes for aconitase and isocitrate dehydrogenase. Although R. eutropha is a strictly respiratory bacterium, the abundance of certain fermentation enzymes was increased. This work represents a comprehensive survey of denitrification on the proteomic and transcriptomic levels and provides unique insight into how R. eutropha adapts its metabolism to low oxygen conditions.

CtsR, the Gram-positive master regulator of protein quality control, feels the heat.

Protein quality networks are required for the maintenance of proper protein homeostasis and essential for viability and growth of all living organisms. Hence, regulation and coordination of these networks are critical for survival during stress as well as for virulence of pathogenic species. In low GC, Gram-positive bacteria central protein quality networks are under the control of the global repressor CtsR. Here, we provide evidence that CtsR activity during heat stress is mediated by intrinsic heat sensing through a glycine-rich loop, probably in all Gram-positive species. Moreover, a function for the recently identified arginine kinase McsB is confirmed, however, not for initial inactivation and dissociation of CtsR from the DNA, but for heat-dependent auto-activation of McsB as an adaptor for ClpCP-mediated degradation of CtsR.

The phosphoproteome and its physiological dynamics in Staphylococcus aureus.

Phosphorylation events on proteins during growth and stress/starvation can represent crucial regulation processes inside the bacterial cell. Therefore, serine, threonine and tyrosine phosphorylation patterns were analyzed by two powerful complementary proteomic methods for the human pathogen Staphylococcus aureus. Using 2D-gel analysis with a phosphosensitive stain (Pro-Q Diamond) and gel-free titanium dioxide based phosphopeptide enrichment, 103 putative phosphorylated proteins with successfully mapped 68 different phosphorylation sites were found in the soluble proteome of S. aureus. Additionally, in a proof of concept study, 8 proteins phosphorylated on arginine residues have been identified. Most important for functional analyses of S. aureus, proteins related to pathogenicity and virulence were found to be phosphorylated: the virulence regulator SarA, the potential antimicrobial target FbaA and the elastin-binding protein EbpS. Besides newly identified phosphorylation sites we compared our dataset with existing data from literature and subsequent experiments revealed additional phosphorylation events on highly conserved localizations in FbaA. Differential analysis of phosphorylation signals on the 2D-gels showed significant changes in phosphorylation under different physiological conditions for 10 proteins. Among these, we were able to detect newly appearing signals for phosphorylated isoforms of FdaB and HchA under nitrosative stress conditions.

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.

Genes for laminarin degradation are dispersed in the genomes of particle-associated Maribacter species.

Laminarin is a cytosolic storage polysaccharide of phytoplankton and macroalgae and accounts for over 10% of the world's annually fixed carbon dioxide. Algal disruption, for example, by viral lysis releases laminarin. The soluble sugar is rapidly utilized by free-living planktonic bacteria, in which sugar transporters and the degrading enzymes are frequently encoded in polysaccharide utilization loci. The annotation of flavobacterial genomes failed to identify canonical laminarin utilization loci in several particle-associated bacteria, in particular in strains of Maribacter. In this study, we report in vivo utilization of laminarin by Maribacter forsetii accompanied by additional cell growth and proliferation. Laminarin utilization coincided with the induction of an extracellular endo-laminarinase, SusC/D outer membrane oligosaccharide transporters, and a periplasmic glycosyl hydrolase family 3 protein. An ABC transport system and sugar kinases were expressed. Endo-laminarinase activity was also observed in Maribacter sp. MAR_2009_72, Maribacter sp. Hel_I_7, and Maribacter dokdonensis MAR_2009_60. Maribacter dokdonensis MAR_2009_71 lacked the large endo-laminarinase gene in the genome and had no endo-laminarinase activity. In all genomes, genes of induced proteins were scattered across the genome rather than clustered in a laminarin utilization locus. These observations revealed that the Maribacter strains investigated in this study participate in laminarin utilization, but in contrast to many free-living bacteria, there is no co-localization of genes encoding the enzymatic machinery for laminarin utilization.

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 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.

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.

Complete Genome Sequence of Citrobacter braakii GW-Imi-1b1, Isolated from Hospital Wastewater in Greifswald, Germany.

The imipenem-resistant Citrobacter braakii strain GW-Imi-1b1 was isolated from a hospital wastewater sample in Greifswald, Germany. The genome comprises one chromosome (5.09 Mb), one prophage (41.9 kb), and 13 plasmids (2 to 140.9 kb). The genome harbors 5,322 coding sequences, shows a high potential for genomic mobility, and includes genes encoding proteins for multiple drug resistances.

The natural product chlorotonil A preserves colonization resistance and prevents relapsing Clostridioides difficile infection.

Clostridioides difficile infections (CDIs) remain a healthcare problem due to high rates of relapsing/recurrent CDIs (rCDIs). Breakdown of colonization resistance promoted by broad-spectrum antibiotics and the persistence of spores contribute to rCDI. Here, we demonstrate antimicrobial activity of the natural product class of chlorotonils against C. difficile. In contrast to vancomycin, chlorotonil A (ChA) efficiently inhibits disease and prevents rCDI in mice. Notably, ChA affects the murine and porcine microbiota to a lesser extent than vancomycin, largely preserving microbiota composition and minimally impacting the intestinal metabolome. Correspondingly, ChA treatment does not break colonization resistance against C. difficile and is linked to faster recovery of the microbiota after CDI. Additionally, ChA accumulates in the spore and inhibits outgrowth of C. difficile spores, thus potentially contributing to lower rates of rCDI. We conclude that chlorotonils have unique antimicrobial properties targeting critical steps in the infection cycle of C. difficile.