@article{SchroederBernhardtMarincolaetal.2014, author = {Schr{\"o}der, Wiebke and Bernhardt, J{\"o}rg and Marincola, Gabriella and Klein-Hitpass, Ludger and Herbig, Alexander and Krupp, Guido and Nieselt, Kay and Wolz, Christiane}, title = {Altering gene expression by aminocoumarins: the role of DNA supercoiling in Staphylococcus aureus}, series = {BMC Genomics}, volume = {15}, journal = {BMC Genomics}, number = {291}, issn = {1471-2164}, doi = {10.1186/1471-2164-15-291}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121609}, year = {2014}, abstract = {BACKGROUND: It has been shown previously that aminocoumarin antibiotics such as novobiocin lead to immediate downregulation of recA expression and thereby inhibit the SOS response, mutation frequency and recombination capacity in Staphylococcus aureus. Aminocoumarins function by inhibiting the ATPase activity of DNA gyrase subunit B with a severe impact on DNA supercoiling. RESULTS: Here, we have analysed the global impact of the DNA relaxing agent novobiocin on gene expression in S. aureus. Using a novobiocin-resistant mutant, it became evident that the change in recA expression is due to gyrase inhibition. Microarray analysis and northern blot hybridisation revealed that the expression levels of a distinct set of genes were increased (e.g., recF-gyrB-gyrA, the rib operon and the ure operon) or decreased (e.g., arlRS, recA, lukA, hlgC and fnbA) by novobiocin. The two-component ArlRS system was previously found to decrease the level of supercoiling in S. aureus. Thus, downregulation of arlRS might partially compensate for the relaxing effect of novobiocin. Global analysis and gene mapping of supercoiling-sensitive genes did not provide any indication that they are clustered in the genome. Promoter fusion assays confirmed that the responsiveness of a given gene is intrinsic to the promoter region but independent of the chromosomal location. CONCLUSIONS: The results indicate that the molecular properties of a given promoter, rather than the chromosomal topology, dictate the responsiveness to changes in supercoiling in the pathogen Staphylococcus aureus.}, language = {en} } @article{SchoenfelderMarincolaGeigeretal.2013, author = {Schoenfelder, Sonja M. K. and Marincola, Gabriella and Geiger, Tobias and Goerke, Christiane and Wolz, Christiane and Ziebuhr, Wilma}, title = {Methionine Biosynthesis in Staphylococcus aureus Is Tightly Controlled by a Hierarchical Network Involving an Initiator tRNA-Specific T-box Riboswitch}, series = {PLoS Pathogens}, volume = {9}, journal = {PLoS Pathogens}, number = {9}, doi = {10.1371/journal.ppat.1003606}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130365}, pages = {e1003606}, year = {2013}, abstract = {Abstract In line with the key role of methionine in protein biosynthesis initiation and many cellular processes most microorganisms have evolved mechanisms to synthesize methionine de novo. Here we demonstrate that, in the bacterial pathogen Staphylococcus aureus, a rare combination of stringent response-controlled CodY activity, T-box riboswitch and mRNA decay mechanisms regulate the synthesis and stability of methionine biosynthesis metICFE-mdh mRNA. In contrast to other Bacillales which employ S-box riboswitches to control methionine biosynthesis, the S. aureus metICFE-mdh mRNA is preceded by a 5′-untranslated met leader RNA harboring a T-box riboswitch. Interestingly, this T-box riboswitch is revealed to specifically interact with uncharged initiator formylmethionyl-tRNA \((tRNA_i^{fMet})\)while binding of elongator \(tRNA^{Met}\) proved to be weak, suggesting a putative additional function of the system in translation initiation control. met leader RNA/metICFE-mdh operon expression is under the control of the repressor CodY which binds upstream of the met leader RNA promoter. As part of the metabolic emergency circuit of the stringent response, methionine depletion activates RelA-dependent (p)ppGpp alarmone synthesis, releasing CodY from its binding site and thereby activating the met leader promoter. Our data further suggest that subsequent steps in metICFE-mdh transcription are tightly controlled by the 5′ met leader-associated T-box riboswitch which mediates premature transcription termination when methionine is present. If methionine supply is limited, and hence \((tRNA_i^{fMet})\) becomes uncharged, full-length met leader/metICFE-mdh mRNA is transcribed which is rapidly degraded by nucleases involving RNase J2. Together, the data demonstrate that staphylococci have evolved special mechanisms to prevent the accumulation of excess methionine. We hypothesize that this strict control might reflect the limited metabolic capacities of staphylococci to reuse methionine as, other than Bacillus, staphylococci lack both the methionine salvage and polyamine synthesis pathways. Thus, methionine metabolism might represent a metabolic Achilles' heel making the pathway an interesting target for future anti-staphylococcal drug development. Author Summary Prokaryote metabolism is key for our understanding of bacterial virulence and pathogenesis and it is also an area with huge opportunity to identify novel targets for antibiotic drugs. Here, we have addressed the so far poorly characterized regulation of methionine biosynthesis in S. aureus. We demonstrate that methionine biosynthesis control in staphylococci significantly differs from that predicted for other Bacillales. Notably, involvement of a T-box instead of an S-box riboswitch separates staphylococci from other bacteria in the order. We provide, for the first time, direct experimental proof for an interaction of a methionyl-tRNA-specific T-box with its cognate tRNA, and the identification of initiator \((tRNA_i^{fMet})\) as the specific binding partner is an unexpected finding whose exact function in Staphylococcus metabolism remains to be established. The data further suggest that in staphylococci a range of regulatory elements are integrated to form a hierarchical network that elegantly limits costly (excess) methionine biosynthesis and, at the same time, reliably ensures production of the amino acid in a highly selective manner. Our findings open a perspective to exploit methionine biosynthesis and especially its T-box-mediated control as putative target(s) for the development of future anti-staphylococcal therapeutics.}, language = {en} } @article{MakgotlhoMarincolaSchaeferetal.2013, author = {Makgotlho, Phuti E. and Marincola, Gabriella and Sch{\"a}fer, Daniel and Liu, Quian and Bae, Taeok and Geiger, Tobias and Wasserman, Elizabeth and Wolz, Christine and Ziebuhr, Wilma and Sinha, Bhanu}, title = {SDS Interferes with SaeS Signaling of Staphylococcus aureus Independently of SaePQ}, series = {PLOS ONE}, volume = {8}, journal = {PLOS ONE}, number = {8}, issn = {1932-6203}, doi = {10.1371/journal.pone.0071644}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-128469}, pages = {e71644}, year = {2013}, abstract = {The Staphylococcus aureus regulatory saePQRS system controls the expression of numerous virulence factors, including extracellular adherence protein (Eap), which amongst others facilitates invasion of host cells. The saePQRS operon codes for 4 proteins: the histidine kinase SaeS, the response regulator SaeR, the lipoprotein SaeP and the transmembrane protein SaeQ. S. aureus strain Newman has a single amino acid substitution in the transmembrane domain of SaeS (L18P) which results in constitutive kinase activity. SDS was shown to be one of the signals interfering with SaeS activity leading to inhibition of the sae target gene eap in strains with SaeS(L) but causing activation in strains containing SaeS(P). Here, we analyzed the possible involvement of the SaeP protein and saePQ region in SDS-mediated sae/eap expression. We found that SaePQ is not needed for SDS-mediated SaeS signaling. Furthermore, we could show that SaeS activity is closely linked to the expression of Eap and the capacity to invade host cells in a number of clinical isolates. This suggests that SaeS activity might be directly modulated by structurally non-complex environmental signals, as SDS, which possibly altering its kinase/phosphatase activity.}, language = {en} } @article{MarincolaLiongSchoenetal.2021, author = {Marincola, Gabriella and Liong, Olivia and Schoen, Christoph and Abouelfetouh, Alaa and Hamdy, Aisha and Wencker, Freya D. R. and Marciniak, Tessa and Becker, Karsten and K{\"o}ck, Robin and Ziebuhr, Wilma}, title = {Antimicrobial Resistance Profiles of Coagulase-Negative Staphylococci in Community-Based Healthy Individuals in Germany}, series = {Frontiers in Public Health}, volume = {9}, journal = {Frontiers in Public Health}, issn = {2296-2565}, doi = {10.3389/fpubh.2021.684456}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-240881}, year = {2021}, abstract = {Coagulase-negative staphylococci (CoNS) are common opportunistic pathogens, but also ubiquitous human and animal commensals. Infection-associated CoNS from healthcare environments are typically characterized by pronounced antimicrobial resistance (AMR) including both methicillin- and multidrug-resistant isolates. Less is known about AMR patterns of CoNS colonizing the general population. Here we report on AMR in commensal CoNS recovered from 117 non-hospitalized volunteers in a region of Germany with a high livestock density. Among the 69 individuals colonized with CoNS, 29 had reported contacts to either companion or farm animals. CoNS were selectively cultivated from nasal swabs, followed by species definition by 16S rDNA sequencing and routine antibiotic susceptibility testing. Isolates displaying phenotypic AMR were further tested by PCR for presence of selected AMR genes. A total of 127 CoNS were isolated and Staphylococcus epidermidis (75\%) was the most common CoNS species identified. Nine isolates (7\%) were methicillin-resistant (MR) and carried the mecA gene, with seven individuals (10\%) being colonized with at least one MR-CoNS isolate. While resistance against gentamicin, phenicols and spectinomycin was rare, high resistance rates were found against tetracycline (39\%), erythromycin (33\%) and fusidic acid (24\%). In the majority of isolates, phenotypic resistance could be associated with corresponding AMR gene detection. Multidrug-resistance (MDR) was observed in 23\% (29/127) of the isolates, with 33\% (23/69) of the individuals being colonized with MDR-CoNS. The combined data suggest that MR- and MDR-CoNS are present in the community, with previous animal contact not significantly influencing the risk of becoming colonized with such isolates.}, language = {en} } @article{WenckerMarincolaSchoenfelderetal.2021, author = {Wencker, Freya D. R and Marincola, Gabriella and Schoenfelder, Sonja M. K. and Maaß, Sandra and Becher, D{\"o}rte and Ziebuhr, Wilma}, title = {Another layer of complexity in Staphylococcus aureus methionine biosynthesis control: unusual RNase III-driven T-box riboswitch cleavage determines met operon mRNA stability and decay}, series = {Nucleic Acids Research}, volume = {49}, journal = {Nucleic Acids Research}, number = {4}, doi = {10.1093/nar/gkaa1277}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259029}, pages = {2192-2212}, year = {2021}, abstract = {In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5′ untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5′-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3′-end. The obtained data suggest that coordinated RNA decay represents another checkpoint in a complex regulatory network that adjusts costly methionine biosynthesis to current metabolic requirements.}, language = {en} } @article{MarincolaJaschkowitzKieningeretal.2021, author = {Marincola, Gabriella and Jaschkowitz, Greta and Kieninger, Ann-Katrin and Wencker, Freya D.R. and Feßler, Andrea T. and Schwarz, Stefan and Ziebuhr, Wilma}, title = {Plasmid-Chromosome Crosstalk in Staphylococcus aureus: A Horizontally Acquired Transcription Regulator Controls Polysaccharide Intercellular Adhesin-Mediated Biofilm Formation}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {11}, journal = {Frontiers in Cellular and Infection Microbiology}, issn = {2235-2988}, doi = {10.3389/fcimb.2021.660702}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-232903}, year = {2021}, abstract = {Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of clonal complex CC398 typically carry various antimicrobial resistance genes, many of them located on plasmids. In the bovine LA-MRSA isolate Rd11, we previously identified plasmid pAFS11 in which resistance genes are co-localized with a novel ica-like gene cluster, harboring genes required for polysaccharide intercellular adhesin (PIA)-mediated biofilm formation. The ica genes on pAFS11 were acquired in addition to a pre-existing ica locus on the S. aureus Rd11 chromosomal DNA. Both loci consist of an icaADBC operon and icaR, encoding a corresponding icaADBC repressor. Despite carrying two biofilm gene copies, strain Rd11 did not produce PIA and transformation of pAFS11 into another S. aureus strain even slightly diminished PIA-mediated biofilm formation. By focusing on the molecular background of the biofilm-negative phenotype of pAFS11-carrying S. aureus, we identified the pAFS11-borne ica locus copy as functionally fully active. However, transcription of both plasmid- and core genome-derived icaADBC operons were efficiently suppressed involving IcaR. Surprisingly, although being different on the amino acid sequence level, the two IcaR repressor proteins are mutually replaceable and are able to interact with the icaA promoter region of the other copy. We speculate that this regulatory crosstalk causes the biofilm-negative phenotype in S. aureus Rd11. The data shed light on an unexpected regulatory interplay between pre-existing and newly acquired DNA traits in S. aureus. This also raises interesting general questions regarding functional consequences of gene transfer events and their putative implications for the adaptation and evolution of bacterial pathogens.}, language = {en} } @article{SoundararajanMarincolaLiongetal.2023, author = {Soundararajan, Manonmani and Marincola, Gabriella and Liong, Olivia and Marciniak, Tessa and Wencker, Freya D. R. and Hofmann, Franka and Schollenbruch, Hannah and Kobusch, Iris and Linnemann, Sabrina and Wolf, Silver A. and Helal, Mustafa and Semmler, Torsten and Walther, Birgit and Schoen, Christoph and Nyasinga, Justin and Revathi, Gunturu and Boelhauve, Marc and Ziebuhr, Wilma}, title = {Farming practice influences antimicrobial resistance burden of non-aureus staphylococci in pig husbandries}, series = {Microorganisms}, volume = {11}, journal = {Microorganisms}, number = {1}, issn = {2076-2607}, doi = {10.3390/microorganisms11010031}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-312750}, year = {2023}, abstract = {Non-aureus staphylococci (NAS) are ubiquitous bacteria in livestock-associated environments where they may act as reservoirs of antimicrobial resistance (AMR) genes for pathogens such as Staphylococcus aureus. Here, we tested whether housing conditions in pig farms could influence the overall AMR-NAS burden. Two hundred and forty porcine commensal and environmental NAS isolates from three different farm types (conventional, alternative, and organic) were tested for phenotypic antimicrobial susceptibility and subjected to whole genome sequencing. Genomic data were analysed regarding species identity and AMR gene carriage. Seventeen different NAS species were identified across all farm types. In contrast to conventional farms, no AMR genes were detectable towards methicillin, aminoglycosides, and phenicols in organic farms. Additionally, AMR genes to macrolides and tetracycline were rare among NAS in organic farms, while such genes were common in conventional husbandries. No differences in AMR detection existed between farm types regarding fosfomycin, lincosamides, fusidic acid, and heavy metal resistance gene presence. The combined data show that husbandry conditions influence the occurrence of resistant and multidrug-resistant bacteria in livestock, suggesting that changing husbandry practices may be an appropriate means of limiting the spread of AMR bacteria on farms.}, language = {en} } @article{BarthelsMarincolaMarciniaketal.2020, author = {Barthels, Fabian and Marincola, Gabriella and Marciniak, Tessa and Konh{\"a}user, Matthias and Hammerschmidt, Stefan and Bierlmeier, Jan and Distler, Ute and Wich, Peter R. and Tenzer, Stefan and Schwarzer, Dirk and Ziebuhr, Wilma and Schirmeister, Tanja}, title = {Asymmetric Disulfanylbenzamides as Irreversible and Selective Inhibitors of Staphylococcus aureus Sortase A}, series = {ChemMedChem}, volume = {15}, journal = {ChemMedChem}, number = {10}, doi = {10.1002/cmdc.201900687}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-214581}, pages = {839 -- 850}, year = {2020}, abstract = {Staphylococcus aureus is one of the most frequent causes of nosocomial and community-acquired infections, with drug-resistant strains being responsible for tens of thousands of deaths per year. S. aureus sortase A inhibitors are designed to interfere with virulence determinants. We have identified disulfanylbenzamides as a new class of potent inhibitors against sortase A that act by covalent modification of the active-site cysteine. A broad series of derivatives were synthesized to derive structure-activity relationships (SAR). In vitro and in silico methods allowed the experimentally observed binding affinities and selectivities to be rationalized. The most active compounds were found to have single-digit micromolar Ki values and caused up to a 66 \% reduction of S. aureus fibrinogen attachment at an effective inhibitor concentration of 10 μM. This new molecule class exhibited minimal cytotoxicity, low bacterial growth inhibition and impaired sortase-mediated adherence of S. aureus cells.}, language = {en} } @article{MielichSuessWagnerMietrachetal.2017, author = {Mielich-S{\"u}ss, Benjamin and Wagner, Rabea M. and Mietrach, Nicole and Hertlein, Tobias and Marincola, Gabriella and Ohlsen, Knut and Geibel, Sebastian and Lopez, Daniel}, title = {Flotillin scaffold activity contributes to type VII secretion system assembly in Staphylococcus aureus}, series = {PLoS Pathogens}, volume = {13}, journal = {PLoS Pathogens}, number = {11}, doi = {10.1371/journal.ppat.1006728}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170035}, pages = {e1006728}, year = {2017}, abstract = {Scaffold proteins are ubiquitous chaperones that promote efficient interactions between partners of multi-enzymatic protein complexes; although they are well studied in eukaryotes, their role in prokaryotic systems is poorly understood. Bacterial membranes have functional membrane microdomains (FMM), a structure homologous to eukaryotic lipid rafts. Similar to their eukaryotic counterparts, bacterial FMM harbor a scaffold protein termed flotillin that is thought to promote interactions between proteins spatially confined to the FMM. Here we used biochemical approaches to define the scaffold activity of the flotillin homolog FloA of the human pathogen Staphylococcus aureus, using assembly of interacting protein partners of the type VII secretion system (T7SS) as a case study. Staphylococcus aureus cells that lacked FloA showed reduced T7SS function, and thus reduced secretion of T7SS-related effectors, probably due to the supporting scaffold activity of flotillin. We found that the presence of flotillin mediates intermolecular interactions of T7SS proteins. We tested several small molecules that interfere with flotillin scaffold activity, which perturbed T7SS activity in vitro and in vivo. Our results suggest that flotillin assists in the assembly of S. aureus membrane components that participate in infection and influences the infective potential of this pathogen.}, language = {en} } @article{KoernerMeyerMarincolaetal.2023, author = {K{\"o}rner, Maria and Meyer, Susanne R. and Marincola, Gabriella and Kern, Maximilian J. and Grimm, Clemens and Schuelein-Voelk, Christina and Fischer, Utz and Hofmann, Kay and Buchberger, Alexander}, title = {The FAM104 proteins VCF1/2 promote the nuclear localization of p97/VCP}, series = {eLife}, volume = {12}, journal = {eLife}, doi = {10.7554/eLife.92409}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350222}, year = {2023}, abstract = {The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97-cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members VCP nuclear cofactor family member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.}, language = {en} }