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.

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.

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 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.

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.

Clostridioides difficile Modifies its Aromatic Compound Metabolism in Response to Amidochelocardin-Induced Membrane Stress.

Amidochelocardin is a broad-spectrum antibiotic with activity against many Gram-positive and Gram-negative bacteria. According to recent data, the antibiotic effect of this atypical tetracycline is directed against the cytoplasmic membrane, which is associated with the dissipation of the membrane potential. Here, we investigated the effect of amidochelocardin on the proteome of Clostridioides difficile to gain insight into the membrane stress physiology of this important anaerobic pathogen. For the first time, the membrane-directed action of amidochelocardin was confirmed in an anaerobic pathogen. More importantly, our results revealed that aromatic compounds potentially play an important role in C. difficile upon dissipation of its membrane potential. More precisely, a simultaneously increased production of enzymes required for the synthesis of chorismate and two putative phenazine biosynthesis proteins point to the production of a hitherto unknown compound in response to membrane depolarization. Finally, increased levels of the ClnAB efflux system and its transcriptional regulator ClnR were found, which were previously found in response to cationic antimicrobial peptides like LL-37. Therefore, our data provide a starting point for a more detailed understanding of C. difficile's way to counteract membrane-active compounds. IMPORTANCE C. difficile is an important anaerobe pathogen causing mild to severe infections of the gastrointestinal tract. To avoid relapse of the infection following antibiotic therapy, antibiotics are needed that efficiently eradicate C. difficile from the intestinal tract. Since C. difficile was shown to be substantially sensitive to membrane-active antibiotics, it has been proposed that membrane-active antibiotics might be promising for the therapy of C. difficile infections. Therefore, we studied the response of C. difficile to amidochelocardin, a membrane-active antibiotic dissipating the membrane potential. Interestingly, C. difficile's response to amidochelocardin indicates a role of aromatic metabolites in mediating stress caused by dissipation of the membrane potential.

Myxopyronin B inhibits growth of a Fidaxomicin-resistant Clostridioides difficile isolate and interferes with toxin synthesis.

The anaerobic, gastrointestinal pathogen Clostridioides difficile can cause severe forms of enterocolitis which is mainly mediated by the toxins it produces. The RNA polymerase inhibitor Fidaxomicin is the current gold standard for the therapy of C. difficile infections due to several beneficial features including its ability to suppress toxin synthesis in C. difficile. In contrast to the Rifamycins, Fidaxomicin binds to the RNA polymerase switch region, which is also the binding site for Myxopyronin B. Here, serial broth dilution assays were performed to test the susceptibility of C. difficile and other anaerobes to Myxopyronin B, proving that the natural product is considerably active against C. difficile and that there is no cross-resistance between Fidaxomicin and Myxopyronin B in a Fidaxomicin-resistant C. difficile strain. Moreover, mass spectrometry analysis indicated that Myxopyronin B is able to suppress early phase toxin synthesis in C. difficile to the same degree as Fidaxomicin. Conclusively, Myxopyronin B is proposed as a new lead structure for the design of novel antibiotics for the therapy of C. difficile infections.

Assays to Study Enzymatic and Non-Enzymatic Protein Lysine Acetylation In Vitro.

Proteins can be lysine-acetylated both enzymatically, by lysine acetyltransferases (KATs), and non-enzymatically, by acetyl-CoA and/or acetyl-phosphate. Such modification can be reversed by lysine deacetylases classified as NAD+ -dependent sirtuins or by classical Zn2+ -dependent deacetylases (KDACs). The regulation of protein lysine acetylation events by KATs and sirtuins/KDACs, or by non-enzymatic processes, is often assessed only indirectly by mass spectrometry or by mutational studies in cells. Mutational approaches to study lysine acetylation are limited, as these often poorly mimic lysine acetylation. Here, we describe protocols to assess the direct regulation of protein lysine acetylation by both sirtuins/KDACs and KATs, as well as non-enzymatically. We first describe a protocol for the production of site-specific lysine-acetylated proteins using a synthetic biological approach, the genetic code expansion concept (GCEC). These natively folded, lysine-acetylated proteins can then be used as direct substrates for sirtuins and KDACs. This approach addresses various limitations encountered with other methods. First, results from sirtuin/KDAC-catalyzed deacetylation assays obtained using acetylated peptides as substrates can vary considerably compared to experiments using natively folded substrate proteins. In addition, producing lysine-acetylated proteins for deacetylation assays by using recombinantly expressed KATs is difficult, as these often do not yield proteins that are homogeneously and quantitatively lysine acetylated. Moreover, KATs are often huge multi-domain proteins, which are difficult to recombinantly express and purify in soluble form. We also describe protocols to study the direct regulation of protein lysine acetylation, both enzymatically, by sirtuins/KDACs and KATs, and non-enzymatically, by acetyl-CoA and/or acetyl-phosphate. The latter protocol also includes a section that explains how specific lysine acetylation sites can be detected by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The protocols described here can be useful for providing a more detailed understanding of the enzymatic and non-enzymatic regulation of lysine acetylation sites, an important aspect to judge their physiological significance. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of N-(ε)-lysine-acetylated proteins using the genetic code expansion concept (GCEC) Basic Protocol 2: In vitro sirtuin (SIRT)-catalyzed deacetylation of lysine-acetylated proteins prepared by the GCEC Basic Protocol 3: In vitro KDAC/HDAC-catalyzed deacetylation of lysine-acetylated proteins Basic Protocol 4: In vitro lysine acetylation of recombinantly expressed proteins by lysine acetyltransferases (KATs) Basic Protocol 5: In vitro non-enzymatic lysine acetylation of proteins by acetyl-CoA and/or acetyl-phosphate.

Influenza A H1N1 Induced Disturbance of the Respiratory and Fecal Microbiome of German Landrace Pigs - a Multi-Omics Characterization.

Seasonal influenza outbreaks represent a large burden for the health care system as well as the economy. While the role of the microbiome has been elucidated in the context of various diseases, the impact of respiratory viral infections on the human microbiome is largely unknown. In this study, swine was used as an animal model to characterize the temporal dynamics of the respiratory and gastrointestinal microbiome in response to an influenza A virus (IAV) infection. A multi-omics approach was applied on fecal samples to identify alterations in microbiome composition and function during IAV infection. We observed significantly altered microbial richness and diversity in the gastrointestinal microbiome after IAV infection. In particular, increased abundances of Prevotellaceae were detected, while Clostridiaceae and Lachnospiraceae decreased. Moreover, our metaproteomics data indicated that the functional composition of the microbiome was heavily affected by the influenza infection. For instance, we identified decreased amounts of flagellin, correlating with reduced abundances of Lachnospiraceae and Clostridiaceae, possibly indicating involvement of a direct immune response toward flagellated Clostridia during IAV infection. Furthermore, enzymes involved in short-chain fatty acid (SCFA) synthesis were identified in higher abundances, while metabolome analyses revealed rather stable concentrations of SCFAs. In addition, 16S rRNA gene sequencing was used to characterize effects on the composition and natural development of the upper respiratory tract microbiome. Our results showed that IAV infection resulted in significant changes in the abundance of Moraxellaceae and Pasteurellaceae in the upper respiratory tract. Surprisingly, temporal development of the respiratory microbiome structure was not affected. IMPORTANCE Here, we used swine as a biomedical model to elucidate the impact of influenza A H1N1 infection on structure and function of the respiratory and gastrointestinal tract microbiome by employing a multi-omics analytical approach. To our knowledge, this is the first study to investigate the temporal development of the porcine microbiome and to provide insights into the functional capacity of the gastrointestinal microbiome during influenza A virus infection.

Metagenome-Assembled Genome Sequences from Different Wastewater Treatment Stages in Germany.

Metagenome-assembled genome sequences (MAGs) were generated from two wastewater treatment systems in two German cities (Göttingen and Greifswald), based on metagenomes derived from hospital effluent, different wastewater treatment stages, and adjacent water bodies. The MAGs mainly originated from bacterial members of Proteobacteria, Bacteroidota, Firmicutes, "Candidatus Patescibacteria," Actinobacteriota, Chloroflexota, Desulfobacterota, and Verrucomicrobiota.

The Lichens' Microbiota, Still a Mystery?

Lichens represent self-supporting symbioses, which occur in a wide range of terrestrial habitats and which contribute significantly to mineral cycling and energy flow at a global scale. Lichens usually grow much slower than higher plants. Nevertheless, lichens can contribute substantially to biomass production. This review focuses on the lichen symbiosis in general and especially on the model species Lobaria pulmonaria L. Hoffm., which is a large foliose lichen that occurs worldwide on tree trunks in undisturbed forests with long ecological continuity. In comparison to many other lichens, L. pulmonaria is less tolerant to desiccation and highly sensitive to air pollution. The name-giving mycobiont (belonging to the Ascomycota), provides a protective layer covering a layer of the green-algal photobiont (Dictyochloropsis reticulata) and interspersed cyanobacterial cell clusters (Nostoc spec.). Recently performed metaproteome analyses confirm the partition of functions in lichen partnerships. The ample functional diversity of the mycobiont contrasts the predominant function of the photobiont in production (and secretion) of energy-rich carbohydrates, and the cyanobiont's contribution by nitrogen fixation. In addition, high throughput and state-of-the-art metagenomics and community fingerprinting, metatranscriptomics, and MS-based metaproteomics identify the bacterial community present on L. pulmonaria as a surprisingly abundant and structurally integrated element of the lichen symbiosis. Comparative metaproteome analyses of lichens from different sampling sites suggest the presence of a relatively stable core microbiome and a sampling site-specific portion of the microbiome. Moreover, these studies indicate how the microbiota may contribute to the symbiotic system, to improve its health, growth and fitness.

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.

Biofilm and Pathogenesis-Related Proteins in the Foodborne P. fluorescens ITEM 17298 With Distinctive Phenotypes During Cold Storage.

In food chain, Pseudomonas spp. cause spoilage by reducing shelf life of fresh products, especially during cold storage, with a high economic burden for industries. However, recent studies have shed new light on health risks occurring when they colonize immunocompromised patient tissues. Likewise to P. aeruginosa, they exhibit antibiotic resistance and biofilm formation, responsible for their spread and persistence in the environment. Biofilm formation might be induced by environmental stresses, such as temperature fluctuations causing physiological and metabolic changes exacerbating food spoilage (by protease and pigment synthesis), and the production of adhesion molecules, chemotactic or underestimated virulence factors. In order to provide a new insight into phenotypic biodiversity of Pseudomonas spoilers isolated from cold stored cheese, in this work 19 Pseudomonas spp. were investigated for biofilm, pigments, exopolysaccharide production and motility at low temperature. Only nine strains showed these phenotypic traits and the blue pigmenting cheese strain P. fluorescens ITEM 17298 was the most distinctive. In addition, this strain decreased the survival probability of infected Galleria mellonella larvae, showing, for the first time, a pathogenic potential. Genomic and proteomic analyses performed on the ITEM 17298 planktonic cells treated or not with lactoferrin derived antibiofilm peptides allowed to reveal specific biofilm related-pathways as well as proteins involved in pathogenesis. Indeed, several genes were found related to signaling system by cGMP-dependent protein kinases, cellulose, rhamnolipid and alginate synthesis, antibiotic resistance, adhesion and virulence factors. The proteome of the untreated ITEM 17298, growing at low temperature, showed that most of the proteins associated with biofilm regulation, pigmentation motility, antibiotic resistance and pathogenecity were repressed, or decreased their levels in comparison to that of the untreated cultures. Thus, the results of this work shed light on the complex pathways network allowing psychrotrophic pseudomonads to adapt themselves to food-refrigerated conditions and enhance their spoilage. In addition, the discovery of virulence factors and antibiotic resistance determinants raises some questions about the need to deeper investigate these underestimated bacteria in order to increase awareness and provide input to update legislation on their detection limits in foods.

Impact of Different Trace Elements on the Growth and Proteome of Two Strains of Granulicella, Class "Acidobacteriia".

Acidobacteria represents one of the most dominant bacterial groups across diverse ecosystems. However, insight into their ecology and physiology has been hampered by difficulties in cultivating members of this phylum. Previous cultivation efforts have suggested an important role of trace elements for the proliferation of Acidobacteria, however, the impact of these metals on their growth and metabolism is not known. In order to gain insight into this relationship, we evaluated the effect of trace element solution SL10 on the growth of two strains (5B5 and WH15) of Acidobacteria belonging to the genus Granulicella and studied the proteomic responses to manganese (Mn). Granulicella species had highest growth with the addition of Mn, as well as higher tolerance to this metal compared to seven other metal salts. Variations in tolerance to metal salt concentrations suggests that Granulicella sp. strains possess different mechanisms to deal with metal ion homeostasis and stress. Furthermore, Granulicella sp. 5B5 might be more adapted to survive in an environment with higher concentration of several metal ions when compared to Granulicella sp. WH15. The proteomic profiles of both strains indicated that Mn was more important in enhancing enzymatic activity than to protein expression regulation. In the genomic analyses, we did not find the most common transcriptional regulation of Mn homeostasis, but we found candidate transporters that could be potentially involved in Mn homeostasis for Granulicella species. The presence of such transporters might be involved in tolerance to higher Mn concentrations, improving the adaptability of bacteria to metal enriched environments, such as the decaying wood-rich Mn environment from which these two Granulicella strains were isolated.

Complete Genome Sequence of Escherichia coli GW-AmxH19, Isolated from Hospital Wastewater in Greifswald, Germany.

The Gram-negative and rod-shaped Escherichia coli strain GW-AmxH19 was isolated from university hospital wastewater in Greifswald, Germany. The genome consists of two replicons, including one circular chromosome (5.04 Mb) and a circular plasmid (126.96 kb). The genome harbors 4,694 protein-coding genes, comprising multidrug resistance and a potential association with urogenital tract infections.

An optimized metaproteomics protocol for a holistic taxonomic and functional characterization of microbial communities from marine particles.

This study aimed to establish a robust and reliable metaproteomics protocol for an in-depth characterization of marine particle-associated (PA) bacteria. To this end, we compared six well-established protein extraction protocols together with different MS-sample preparation techniques using particles sampled during a North Sea spring algae bloom in 2009. In the final optimized workflow, proteins are extracted using a combination of SDS-containing lysis buffer and cell disruption by bead-beating, separated by SDS-PAGE, in-gel digested and analysed by LC-MS/MS, before MASCOT search against a metagenome-based database and data processing/visualization with the in-house-developed bioinformatics tools Prophane and Paver. As an application example, free-living (FL) and particulate communities sampled in April 2009 were analysed, resulting in an as yet unprecedented number of 9354 and 5034 identified protein groups for FL and PA bacteria, respectively. Our data suggest that FL and PA communities appeared similar in their taxonomic distribution, with notable exceptions: eukaryotic proteins and proteins assigned to Flavobacteriia, Cyanobacteria, and some proteobacterial genera were found more abundant on particles, whilst overall proteins belonging to Proteobacteria were more dominant in the FL fraction. Furthermore, our data points to functional differences including proteins involved in polysaccharide degradation, sugar- and phosphorus uptake, adhesion, motility, and stress response.

Metabolic Rearrangements Causing Elevated Proline and Polyhydroxybutyrate Accumulation During the Osmotic Adaptation Response of Bacillus megaterium.

For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.

Responses of Acidobacteria Granulicella sp. WH15 to High Carbon Revealed by Integrated Omics Analyses.

The phylum Acidobacteria is widely distributed in soils, but few representatives have been cultured. In general, Acidobacteria are oligotrophs and exhibit slow growth under laboratory conditions. We sequenced the genome of Granulicella sp. WH15, a strain obtained from decaying wood, and determined the bacterial transcriptome and proteome under growth in poor medium with a low or high concentration of sugar. We detected the presence of 217 carbohydrate-associated enzymes in the genome of strain WH15. Integrated analysis of the transcriptomic and proteomic profiles showed that high sugar triggered a stress response. As part of this response, transcripts related to cell wall stress, such as sigma factor σW and toxin-antitoxin (TA) systems, were upregulated, as were several proteins involved in detoxification and repair, including MdtA and OprM. KEGG metabolic pathway analysis indicated the repression of carbon metabolism (especially the pentose phosphate pathway) and the reduction of protein synthesis, carbohydrate metabolism, and cell division, suggesting the arrest of cell activity and growth. In summary, the stress response of Granulicella sp. WH15 induced by the presence of a high sugar concentration in the medium resulted in the intensification of secretion functions to eliminate toxic compounds and the reallocation of resources to cell maintenance instead of growth.