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Abstract
Aerated topsoils are important sinks for atmospheric methane (CH4) via oxidation by CH4‐oxidizing bacteria (MOB). However, intensified management of grasslands and forests may reduce the CH4 sink capacity of soils. We investigated the influence of grassland land‐use intensity (150 sites) and forest management type (149 sites) on potential atmospheric CH4 oxidation rates (PMORs) and the abundance and diversity of MOB (with qPCR) in topsoils of three temperate regions in Germany. PMORs measurements in microcosms under defined conditions yielded approximately twice as much CH4 oxidation in forest than in grassland soils. High land‐use intensity of grasslands had a negative effect on PMORs (−40%) in almost all regions and fertilization was the predominant factor of grassland land‐use intensity leading to PMOR reduction by 20%. In contrast, forest management did not affect PMORs in forest soils. Upland soil cluster (USC)‐α was the dominant group of MOBs in the forests. In contrast, USC‐γ was absent in more than half of the forest soils but present in almost all grassland soils. USC‐α abundance had a direct positive effect on PMOR in forest, while in grasslands USC‐α and USC‐γ abundance affected PMOR positively with a more pronounced contribution of USC‐γ than USC‐α. Soil bulk density negatively influenced PMOR in both forests and grasslands. We further found that the response of the PMORs to pH, soil texture, soil water holding capacity and organic carbon and nitrogen content differ between temperate forest and grassland soils. pH had no direct effects on PMOR, but indirect ones via the MOB abundances, showing a negative effect on USC‐α, and a positive on USC‐γ abundance. We conclude that reduction in grassland land‐use intensity and afforestation has the potential to increase the CH4 sink function of soils and that different parameters determine the microbial methane sink in forest and grassland soils.
Editorial: Streptococci in infectious diseases – pathogenic mechanisms and host immune responses
(2022)
Background: Hepatitis E virus (HEV) is the etiological agent of an acute self-limiting hepatitis in humans worldwide. The main route of infection is by ingestion of food or water contaminated with the virus. In Germany, several hundred human cases are reported each year, while preliminary studies suggest a high infestation rate of herds of domestic pig (Sus scrofa domesticus) and sounders of wild boar (Sus scrofa). Autochthonous cases are originating mainly from zoonotic transmission from domestic pig and wild boar, but other animals may also be involved. Recently, a novel strain of HEV (ratHEV) had been found in Norway rats (Rattus norvegicus) in Germany, that could contribute to human epidemiology. Therefore, the aim of this study was to assess the seroprevalence of both HEV and the novel ratHEV in human, domestic pig and rat. For each of the three mammal species, an indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was established, that based on an Escherichia coli-expressed carboxy-terminal segment (GT3-Ctr, amino acid (aa) 326–608) of the capsid protein of the autochthonous genotype 3 (GT3), derived from a wild boar from Germany. In parallel, a segment from ratHEV homologous to GT3-Ctr was also expressed in E. coli (ratHEV-Ctr, aa315–599) and was used in the ELISA. Hence, the established tests detect antibodies directed against HEV GT3 when using GT3-Ctr as antigen and ratHEV when using ratHEV-Ctr. Results: The GT3-based in-house human IgG test was validated using a commercial assay and showed high specificity and sensitivity. The average human population (represented by a panel of blood donors from Berlin and Brandenburg) reached a seroprevalence of 12.3% (37/301) with the in-house ELISA. A panel of forestry workers from Brandenburg had an even higher seroprevalence of 21.4% (119/555). Furthermore, ratHEV-specific antibodies could be detected in several sera of forestry workers. The novel ratHEV-based rat IgG ELISA could not be compared to similar tests, however, parallel testing with GT3-Ctr and statistical inference allowed conclusion of a seroprevalence. Rats trapped from several sites in Germany had an overall seroprevalence of 24.5% (36/147). The sera were reactive exclusively with ratHEV-Ctr. As with the in-house ELISA for human sera, the porcine IgG test was validated using a commercial assay, yielding high specificity and sensitivity. A panel of domestic pigs from ten federal states of Germany showed a seroprevalence of 42.7% (383/898) when tested with the in-house ELISA. Reactivity with ratHEV was present, but seemed to be caused mostly by cross-reactivity to GT3-Ctr. Conclusion: The HEV seroprevalence observed for human sera of the average population of Germany is among the highest in Europe and has been confirmed recently by other authors. The high seroprevalence found in forestry workers suggests that they should be counted as a risk group for HEV infection. Populations of rats have been shown to be infested heavily with ratHEV, as rats from all trapping sites situated within cities had a high prevalence for ratHEV exclusively and no serum reacted exclusively with GT3-Ctr. Seroprevalence in domestic pigs was demonstrated to be distributed evenly across federal states and districts. However, a vast difference of infestation could be detected in different herds, suggesting either differences in husbandry conditions, or an external source of infection that acts locally only. The rare but exclusive reactivity of human sera with ratHEV as well as the high cross-reactivity of swine sera with ratHEV suggests that viral strains other than the ones already known may contribute to cases of hepatitis E.
Recently, we engineered a tunable rhamnose promoter-based setup for the production of recombinant proteins in E. coli. This setup enabled us to show that being able to precisely set the production rate of a secretory recombinant protein is critical to enhance protein production yields in the periplasm. It is assumed that precisely setting the production rate of a secretory recombinant protein is required to harmonize its production rate with the protein translocation capacity of the cell. Here, using proteome analysis we show that enhancing periplasmic production of human Growth Hormone (hGH) using the tunable rhamnose promoter-based setup is accompanied by increased accumulation levels of at least three key players in protein translocation; the peripheral motor of the Sec-translocon (SecA), leader peptidase (LepB), and the cytoplasmic membrane protein integrase/chaperone (YidC). Thus, enhancing periplasmic hGH production leads to increased Sec-translocon capacity, increased capacity to cleave signal peptides from secretory proteins and an increased capacity of an alternative membrane protein biogenesis pathway, which frees up Sec-translocon capacity for protein secretion. When cells with enhanced periplasmic hGH production yields were harvested and subsequently cultured in the absence of inducer, SecA, LepB, and YidC levels went down again. This indicates that when using the tunable rhamnose-promoter system to enhance the production of a protein in the periplasm, E. coli can adapt its protein translocation machinery for enhanced recombinant protein production in the periplasm.
Feasible Cluster Model Method for Simulating the Redox Potentials of Laccase CueO and Its Variant
(2022)
Laccases are regarded as versatile green biocatalysts, and recent scientific research has focused on improving their redox potential for broader industrial and environmental applications. The density functional theory (DFT) quantum mechanics approach, sufficiently rigorous and efficient for the calculation of electronic structures, is conducted to better comprehend the connection between the redox potential and the atomic structural feature of laccases. According to the crystal structure of wild type laccase CueO and its variant, a truncated miniature cluster model method was established in this research. On the basic of thermodynamic cycle, the overall Gibbs free energy variations before and after the one-electron reduction were calculated. It turned out that the trends of redox potentials to increase after variant predicted by the theoretical calculations correlated well with those obtained by experiments, thereby validating the feasibility of this cluster model method for simulating the redox potentials of laccases.
The full genome of a Methanomassiliicoccales strain, U3.2.1, was obtained from enrichment cultures of percolation fen peat soil under methanogenic conditions, with methanol and hydrogen as the electron acceptor and donor, respectively. Metagenomic assembly of combined long-read and short-read sequences resulted in a 1.51-Mbp circular genome.
Streptococcus pneumoniae (pneumococci) are lancet-shaped, Gram-positive, alpha-hemolytic, facultative anaerobic human specific commensals of the upper and lower respiratory tract. Pneumococci may convert to pathogenic bacteria and spread to the lungs and blood. In different population groups, such as children, the elderly and immunocompromised individuals, pneumococci can cause local infections such as bronchitis, rhinitis, acute sinusitis, and otitis media as well as life-threatening invasive diseases such as community-acquired pneumonia, sepsis and meningitis. Pneumococci are surrounded by a rigid and complex exoskeleton, the peptidoglycan, also referred to as murein sacculus. The peptidoglycan (PNG) protects the cells from rupture by osmotic pressure and maintains their characteristic shape. The PNG is a heteropolymer made up of glycan strands that are cross-linked by short peptides and during growth the existing murein is continuously hydrolyzed by specific lytic enzymes to enable the insertion of new peptidoglycan. Bacterial cell-wall hydrolases are essential for peptidoglycan turnover and crucial to preserve cell shape. The D,D-carboxypeptidase DacA and L,D-carboxypeptidase DacB of Streptococcus pneumoniae function in a sequential manner. This study determined the crystal structure of the surface-exposed lipoprotein DacB, which differs considerably from the DacA structure. DacB contains a Zn2+ ion in its catalytic center located in the middle of a fully exposed, large groove. Two different conformations with differently arranged active site topology were identified. In addition the critical residues for catalysis and substrate specificity were identified. Deficiency in DacA or DacB resulted in a modified peptidoglycan peptide composition and led to an altered cell shape of the dac-mutants. In contrast, lgt-mutant lacking lipoprotein diacylglyceryl transferase activity required for proper lipoprotein maturation retained L,D-carboxypeptidase activity and showed an intact murein sacculus. Furthermore, this study demonstrated the pathophysiological effects of disordered DacA or DacB activities. Real-time bioimaging of intranasally infected mice indicated a substantially attenuated virulence of dacB- and dacAdacB-mutants pneumococci, while loss of function of DacA had no significant effect. In addition, uptake of these mutants by professional phagocytes was enhanced, while their adherence to lung epithelial cells was decreased. The second part of this study focused on the functional and structure determination of the soluble dimeric pneumococcal lipoprotein PccL. Because of its calycin fold and structural homology with the lipocalin YxeF from Bacillus subtilis, PccL was introduced as the first member of the lipocalin protein family in pneumococci and named “PccL” (Pneumococcal calycin fold containing Lipoprotein). Similar to other lipocalins, the distinct beta-barrel, which is open at one end, is significantly conserved in PccL. Moreover, the application of the in vivo acute pneumonia mouse infection model and the in vitro phagocytosis as well as adherence invasion studies revealed considerable differences in colonization and invasive infection between the wild-type D39 and the pccL-mutant. In conclusion, this study characterized the crucial role of pneumococcal carboxypeptidases DacA and DacB for PGN architecture, bacterial shape and pathogenesis. By applying in vivo and in vitro approaches, a close relationship between PGN metabolism and pathophysiological effects was discovered. In addition, the high resolution structure of DacB has been solved and analyzed and a structure model with a resolution of 2.0 Å is provided. Furthermore, analysis of the PGN composition was applied to indicate the impact of an impaired lipoprotein biogenesis pathway on localization and activity of DacB. The major impact of carboxypeptidases on cell shape and virulence proposes DacB as a promising target for the development of novel drugs or due to its surface exposition also as a promising vaccine candidate. PccL is the first pneumococcal lipocalin-like protein and this study indicated its contribution to pneumococcal virulence. However, the mechanism and the mode of action of PccL are still unknown and have to be deciphered in further studies.
Genome-wide responses and regulatory mechanisms to thiol-specific electrophiles in Bacillus subtilis
(2008)
The soil-dwelling bacterium Bacillus subtilis is regarded as model organism for functional genomic research of low GC Gram-positive bacteria. Recently, the group of Haike Antelmann has monitored the expression profile of B. subtilis after exposure to phenolic compounds. Interestingly, proteome and transcriptome analyses showed a strong overlap in the expression profile after exposure to catechol, MHQ that auto-oxidized to quinones and the thiol-reactive electrophile diamide. The response to electrophilic quinones and diamide is governed by a complex network of transcription factors, including Spx, CtsR, PerR, CymR and the novel MarR-type repressors MhqR (YkvE), YodB and YvaP. The regulatory mechanisms of these novel thiol-stress sensors YodB and YvaP are studied as part of this thesis in collaboration with the group of Peter Zuber (Oregon). YodB negatively regulates the expression of the nitroreductase YodC and the azoreductase YocJ (AzoR1) after exposure to electrophilic quinones and diamide. The azoreductase AzoR1 is a paralog of AzoR2 that is under control of MhqR. Both paralogous azoreductases (AzoR1 and AzoR2) have common functions in quinone and azo-compound reduction to protect cells against the thiol reactivity of electrophiles. DNA binding activity of YodB is directly inhibited by thiol-reactive compounds in vitro. Mass spectrometry approaches suggested that YodB is regulated by a thiol-(S)-alkylation mechanism in response to quinones. Mutational analyses revealed that the conserved Cys6 residue of YodB is required for optimal repression in vivo and in vitro. Recent studies further suggest that YodB is redox-regulated by intersubunit disulfide formation in vivo by diamide. In addition to the azoreductases, several thiol-dependent dioxygenases confer resistance to quinones. In collaboration with Kazuo Kobayashi (Nara), the YodB-paralogous MarR/DUF24-family regulator, YvaP was identified as repressor of the catechol-2,3-dioxygenase encoding yfiDE (catDE) operon. DNA binding activity of YvaP was also directly inhibited by quinones and diamide in vitro indicating that also YvaP is regulated via post-translational modifications. Mutational analyses showed that the conserved Cys7 is essential for YvaP regulation in vivo and serves as sensor for thiol-reactive compounds. In addition, also the basic amino acids K19, R20 are essential for YvaP repression in vivo as well as conserved basic arginine and lysine residues located in the DNA binding helix-turn-helix (HTH) motif. Non-reducing PAGE analysis suggests the formation of an intersubunit disulfide bond in a YvaP dimer upon treatment with quinones and diamide in vitro. Besides quinones, also aldehydes are electrophilic compounds which react via the thiol-(S)-alkylation reaction with thiols. Thus, we were also interested in the response of B. subtilis to the toxic electrophiles methylglyoxal (MG) and formaldehyde (FA). We analyzed the changes in the transcriptome and proteome of B. subtilis after exposure to MG and FA. Like quinone compounds, both MG and FA induce the thiol-specific stress response. Metabolomic approaches confirmed that these reactive aldehydes deplete the cellular thiol pool and thus act like quinones as another class of thiol-reactive electrophiles. Additionally, MG and FA also triggered responses to overcome DNA damage. Our studies further revealed the specific induction of two FA detoxification pathways regulated by the MarR/DUF24 family repressor HxlR, and the novel MerR/NmlR-type regulator YraB (AdhR). HxlR positively regulates the hxlAB operon encoding the ribulose monophosphate pathway. AdhR positively regulates an adhA-yraA operon that encodes the thiol-dependent formaldehyde dehydrogenase (AdhA) and the DJ1/PfpI-like cysteine proteinase (YraA), and the yraC gene that encodes a γ-carboxymuconolactone decarboxylase. Thus, the AdhR regulon is involved in the detoxification of FA to formate via the formaldehyde dehydrogenase AdhA which catalyzes the cleavage of S-hydroxymethylcysteine adducts. In addition, the cysteine proteinase YraA could be involved in the degradation of S-hydroxymethylcysteine-modified and damaged protein thiols. In collaboration with the group of John Helmann (Ithaca), it was shown that AdhR binds in vitro to a conserved inverted repeat between the -10 and -35 promoter elements upstream of adhA, yraB and yraC. In addition, we showed that the conserved Cys52 of AdhR is essential for aldehyde sensing and activation of adhA-yraA transcription in vivo. Thus, we speculate that redox regulation of AdhR involves thiol-(S)-alkylation of this Cys52 residue by aldehydes as another novel mechanism of bacterial physiology.
Hantaviruses (family Bunyaviridae) are enveloped viruses with a segmented RNA genome of negative polarity. They can cause two different diseases in humans, the hemorrhagic fever with renal syndrome in Europe and Asia and the hantavirus cardiopulmonary syndrome in America. The transmission to humans is mainly indirect by inhalation of aerosolized virus-contaminated rodent excreta. In contrast to the initial assumption that hantaviruses are mainly carried by rodents, during the last years many novel hantaviruses were detected in shrews, moles and recently in bats. These findings raise important questions about the evolutionary history of hantaviruses, their host association and adaptation, the role and frequency of spillover infections and host switch events. This study aims to prove the presence, geographical distribution and host association of the rodent-borne Tula virus (TULV) and the shrew-associated Seewis virus (SWSV) in Central Europe. For this purpose, novel laboratory techniques for molecular and serological hantavirus detection were developed. Initially, a broad-spectrum molecular assay to identify small mammal species from Central Europe was developed. This novel assay is based on PCR amplification using degenerated primers targeting the cytochrome b (cyt b) gene, nucleotide sequence analysis of the amplified cyt b gene portion and followed by pairwise sequence comparison to published sequences using the BLAST function of GenBank. Different small mammal species prevalent in Central Europe could be determined by this new approach, including not only representatives of various Rodentia and Soricomorpha, but also representatives of the orders Erinaceomorpha, Lagomorpha, Carnivora and Chiroptera. For characterization of insectivore-borne hantavirus Thottapalayam virus (TPMV), specific monoclonal antibodies were generated that detect native virus in infected mammalian cells. For the detection of TPMV-specific antibodies, Asian house shrew Suncus murinus immunoglobulin G (IgG)-specific antibodies were produced in laboratory mice and rabbit. Using this anti-shrew IgG and recombinant TPMV nucleocapsid (N) protein, an indirect enzyme-linked immunosorbent assay (ELISA) was developed allowing the detection of TPMV N protein-specific antibodies in immunized and experimentally TPMV infected shrews. A Pan-Hantavirus SYBR-Green RT-qPCR was developed for the search to novel hantaviruses. By this novel RT-qPCR and other conventional RT-PCR approaches, TULV infections were identified for the first time in the Eurasian water vole Arvicola amphibius from different regions in Germany and Switzerland. The phylogenetic analyses of the different partial TULV small (S)-, medium (M)- and large (L)-genome segment sequences from A. amphibius, with those of Microtus arvalis- and M. agrestis-derived TULV lineages, revealed a geographical, but host-independent clustering and may suggest multiple TULV spillover or a potential host switch from M. arvalis or M. agrestis to A. amphibius. In a further comprehensive study, different shrew species (Sorex araneus, S. minutus, S. coronatus, and S. alpinus) were collected in Germany, Czech Republic, and Slovakia and screened by another L-segment-targeting Pan-Hantavirus RT-PCR approach. This screening revealed hantavirus L-segment sequences in a large number of S. araneus and a few S. minutus indicating a broad geographical distribution of this hantavirus. For detailed analyses, S-segment sequences were obtained, from S. araneus and S. minutus. The sequences demonstrated their similarity to SWSV sequences from Hungary, Finland, Austria and Germany. A detailed phylogenetic analysis showed low intra-cluster sequence variability, but high inter-cluster divergence suggesting a long-term SWSV evolution in local shrew populations. In conclusion, the investigations demonstrated a broad geographical distribution and multiple spillover infections of rodent-borne TULV and shrew-borne SWSV in Europe. The finding of putative spillover transmissions described here and in other studies underline the current problem of the hantavirus reservoir host definition. In contrast to the hypothesis of a long-standing hantavirus–rodent (small mammal) host coevolution, the investigations support a more dynamic evolutionary history of hantavirus diversification including spillover infections and host-switch events. In future in vitro and in vivo infection studies as well as field studies has to define factors determining the host specificity of these hantaviruses.