Ralph Bertram

Klinikum Nürnberg · Institut für Klinikhygiene, Medizinische Mikrobiologie und Klinische Infektiologie

Stenotrophomonas maltophilia is increasingly recognized as an important nosocomial pathogen among the Gram-negative bacteria. Intrinsic resistance to different classes of antibiotics makes treatment of infections challenging. A deeper understanding of S. maltophilia physiology and virulence requires molecular genetic tools. Here, we describe the im...

During 2022, a total of 9,515 asymptomatic healthcare workers of a large hospital in Germany underwent SARS-CoV-2 PCR screening twice weekly. Of 398,784 saliva samples, 3,555 (0.89%) were PCR positive (median cycle threshold value 30). Early identification of infected healthcare workers can help reduce SARS-CoV-2 transmission in the hospital enviro...

Purpose: The spectrum of causative organisms in infective endocarditis (IE) has changed significantly in the last decades. Reliable pathogen detection is crucial for appropriate antimicrobial therapy for IE. The aim of the study was to evaluate the diagnostic value of microbiological methods for detecting the causative microorganism of IE and to a...

Purpose: Community-acquired methicillin-resistant Staphylococcus aureus strains (CA-MRSA) are spread worldwide and often cause recurring and persistent infections in humans. CA-MRSA strains frequently carry Panton-Valentine leukocidin (PVL) as a distinctive virulence factor. This study investigates the molecular epidemiology, antibiotic resistance...

Background: The broad-spectrum triazole isavuconazole is used for the treatment of invasive aspergillosis and mucormycosis. Data regarding human plasma concentrations in clinical routine of the drug are rare. Objectives: Plasma concentrations of isavuconazole were determined in critically ill ICU patients while considering different patients' ch...

In early 2022, the Coronavirus disease 2019 (COVID-19) remains a global challenge. COVID-19 is caused by an increasing number of variants of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we report an outbreak of SARS-CoV-2 breakthrough infections related to a student festive event with 100 mostly vaccinated guests, which t...

We report an outbreak with SARS-CoV-2 breakthrough infections related to a festive event in Northern Bavaria, Germany in October 2021, with 24 of 95 participants infected. Correlation analyses among 15 interrogated variables revealed that duration at the event and conversation with the supposed index person were significant risk factors. Article S...

The tetracycline repressor (TetR) belongs to the most popular, versatile and efficient transcriptional regulators used in bacterial genetics. In the tetracycline (Tc) resistance determinant tet(B) of transposon Tn10, tetR regulates the expression of a divergently oriented tetA gene that encodes a Tc antiporter. These components of Tn10 and of other...

The capability of Pseudomonas aeruginosa and Staphylococcus aureus to form biofilm on varying CI component materials differs in the presence and absence of bioactive glass (BAG). The application of BAG induces significant changes in biofilm morphology which can be visualized via scanning electron microscopy (SEM). Bacterial biofilm formation on med...

Invasion and persistence of bacteria within host cells requires that they adapt to life in an intracellular environment. This adaptation induces bacterial stress through events such as phagocytosis and enhanced nutrient-restriction. During stress, bacteria synthesize a family of proteins known as heat shock proteins (HSPs) to facilitate adaptation...

To enable specific and tightly controlled gene expression both in-vitro and during the intracellular lifecycle of the pathogen Listeria monocytogenes, a TetR-dependent genetic induction system was developed. Highest concentration of cytoplasmic TetR and best repression of tetO-controlled genes was obtained by tetR expression from the synthetic prom...

Representation of pMX6. The relevant features are indicated with boxes (rep, replicon in S. aureus; cole1, replicon in E. coli), genes with arrows (tetR, TET repressor; bla, ampicillin resistant gene; cat, chloramphenicol resistance gene), promoters with triangles, and paired termini with rectangles.

Detection of asRNAyycG and its effects on yycG mRNA. Total RNA of cultures grown for 6 or 12 h was extracted and expression levels of asRNA (A) and mRNAs (B) were examined by qRT-PCR.

Alignment of amino acid sequences of YycF from S. epidermidis RP62A, S. aureus RN4220, and B. subtilis str. 168. Shading indicates the conserved residues, and the two boxes indicate the DNA-binding winged helix-turn-helix domains. The amino acids at identity positions are marked with black backgrounds and the consensus positions are marked in gray.

DNase I footprinting assay of YycF binding property to promoters of qoxB (A) and pitR (B).

Impacts of asRNAyycF on relative expression of genes in the yyc operon. The yycF coding sequence were divided into two parts yycF-5 and yycF-3, in which specific RT primers were located. The endogenous gene gyrB was used as an internal control. Similar results were obtained from three independent experiments.

Effects of asRNAyycF on expression of regulon genes. The selected genes in the list of YycF target genes were detected by qRT-PCR, using gyrB as an internal control. Genes showing significant changes (cutoff = 2-fold) on expression level were shown. Similar results were obtained from three independent experiments.

Impacts of asRNAyycF on relative expression of icaA in SE1457 and arlRS mutant strain. The endogenous gene gyrB was used as an internal control. Similar results were obtained from three independent experiments.

Genes directly regulated by YycF with atypical promoter sequences.

Predicted regulon genes of YycF based on the new motif in S. epidermidis.

Biofilms play a crucial role in the pathogenicity of Staphylococcus epidermidis, while little is known about whether the essential YycFG two-component signal transduction system (TCS) is involved in biofilm formation. We used antisense RNA (asRNA) to silence the yycFG TCS in order to study its regulatory functions in S. epidermidis. Strain 1457 exp...

Inducible gene expression systems are very useful to analyze cellular processes. The ability to switch the expression state of genes of interest may even be crucial if essential traits or genetic instability are involved. An integrative plasmid, pTEX2, was designed using the (anhydro)tetracycline-inducible promoter Pxyl/tet from staphylococcal plas...

Toxin-antitoxin (TA) systems are small genetic elements found in the majority of prokaryotes. They encode toxin proteins that interfere with vital cellular functions and are counteracted by antitoxins. Dependent on the chemical nature of the antitoxins (protein or RNA) and how they control the activity of the toxin, TA systems are currently divided...

Multidrug tolerant bacterial persister cells frequently arise in response to the activation of toxin–antitoxin systems. However, this prevailing view may be less general than assumed. ATP depletion may mediate another route to the persister state for the Gram-positive pathogen Staphylococcus aureus.

Treatment of Staphylococcus aureus in stationary growth phase with high doses of the antibiotic daptomycin (DAP) eradicates the vast majority of the culture and leaves persister cells behind. Despite resting in a drug-tolerant and dormant state, persister cells exhibit metabolic activity which might be exploited for their elimination. We here repor...

Penicillin and vancomycin treatment of SA113 ± glucose. Stationary phase SA113 cells were treated with 100-fold the MIC of penicillin (square) or 100-fold the MIC of vancomycin (triangle) at t = 0h. Glucose was added at t = 3h (arrow and filled symbols). (TIF)

pH and acetate/glucose measurement. Cultures were treated with 100-fold the MIC of DAP at t = 0h. A) Glucose was added (filled squares) at t = 3h (arrow) and pH values were determined over time. B) Acetate (triangle) and glucose (square) measurement of a culture with glucose added at t = 3h. (TIF)

Effect of CCCP on growth of SA113. SA113 was grown in TSB supplemented with glucose (t = 0h, squares), 100 μM CCCP (t = 0h, diamonds), glucose and CCCP (t = 0h, triangles), or glucose (t = 0h) and CCCP (t = 3h) (circles), respectively. (TIF)

Influence of the glucose concentration on the efficiency of the Glc-DAP effect. Stationary phase SA113 cells were treated with 100-fold the MIC of DAP at t = 0h. At t = 3h, different amounts of glucose were added and CFU values were determined after another four hours. Pearson’s r coefficient: r = -0,704. (TIF)

Time-dependent killing of SA113 cells in PBS. Stationary phase SA113 cells grown in TSB were harvested and resuspended in PBS. At t = 0h, 100-fold the MIC of DAP was added to the cell suspensions. At t = 3h, one cell suspension was supplemented with glucose (filled square), the other was left unaffected (open square) and CFU concentrations were det...

Investigation of enzymatic branch points in glycolysis and TCA cycle. Time dependent killing of stationary phase cultures with 250-fold the MIC of DAP. NE427 (fumC-, fumarate hydratase, diamonds), NE476 (fba-, fructose bisphosphate aldolase, squares), NE491 (icd-, isocitrate dehydrogenase, triangles), NE1003 (mqo-, malate-quinone oxidoreductase, x-...

Understand the mechanisms how bacteria become tolerant towards antibiotics during clinical therapy is a very important object. In a previous study we showed that increased daptomycin (DAP) tolerance of Staphylococcus aureus was due to a point mutation in pitA (inorganic phosphate transporter) that led to intracellular accumulation of both inorganic...

Recalcitrance of genetically susceptible bacteria to antibiotic killing is a hallmark of bacterial drug tolerance. This phenomenon is prevalent in biofilms, persisters and also planktonic cells and is associated with chronic or relapsing infections by pathogens such as Staphylococcus aureus. We here report the in vitro evolution of an S. aureus str...

Toxin-antitoxin (TA) systems are genetic elements of prokaryotes which encode a stable toxin and an unstable antitoxin that can counteract toxicity. TA systems residing on plasmids are often involved in episomal maintenance whereas those on chromosomes can have multiple functions. The opportunistic pathogen Staphylococcus aureus possesses at least...

A cornucopia of methods and molecular tools is available for genetic modification of staphylococci, as shown for at least ten different species to date (Prax et al. Microbiology 159:421-435, 2013). This chapter reviews a number of frequently used vectors for complementation purposes that usually replicate in E. coli and staphylococci and differ in...

Treatment of stationary growth phase Staphylococcus aureus SA113 with 100-fold of the MIC of the lipopeptide antibiotic daptomycin leaves alive a small fraction of drug tolerant albeit genetically susceptible bacteria. This study shows that cells of this subpopulation exhibit active metabolism even hours after the onset of the drug challenge. Isoto...

Persister cells form a multi-drug tolerant subpopulation within an isogenic culture of bacteria that are genetically susceptible to antibiotics. Studies with different Gram negative and Gram positive bacteria have identified a large number of genes associated with the persister state. In contrast, the revelation of persister metabolism has only bee...

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel elec...

Background Persister cells constitute a subpopulation of dormant cells within a microbial population which are genetically identical but phenotypically different to regular cells. Notably, persister cells show an elevated tolerance to antimicrobial agents. Thus, they are considered to represent a microbial ‘bet-hedging’ strategy and are of particul...

Toxin-antitoxin (TA) systems are small genetic elements ubiquitous in prokaryotic genomes that encode toxic proteins targeting various vital cellular functions. Typically, toxin activity is controlled by adjacently encoded protein or RNA antitoxins and unleashed as a consequence of genetic fluctuations or stressful conditions. Whereas some TA syste...

The tricarboxylic acid cycle (TCA cycle) is a central metabolic pathway that provides energy, reducing potential, and biosynthetic intermediates. In Staphylococcus aureus, TCA cycle activity is controlled by several regulators (e.g. CcpA, CodY, and RpiRc) in response to the availability of sugars, amino acids, and environmental stress. Developing a...

The metabolic properties of Campylobacter jejuni enabling an effective colonization of its hosts are not well characterized. Sugars are generally not utilized, whereas the amino acids asparagine, aspartate, glutamine, glutamate, proline and serine are ideal growth substrates for C. jejuni 81-176 in vitro and in a murine infection model. Their utili...

In this study, we attempted to adopt the auxotrophic mevalonate synthase mutant (ΔmvaS mutant) of Staphylococcus aureus to study whether a nongrowing but viable cell population is tolerant to bactericidal antibiotics. The mevalonate-depleted nongrowing ΔmvaS mutant was found tolerant to antibiotics. Surprisingly, after prolonged cultivation, we obt...

Toxin-Antitoxin (TA) systems are small genetic elements of prokaryotes associated with persister cell formation, phage defence, stress regulation and programmed cell arrest. In this study, we characterized two paralogues of the ribosome-dependent RNase YefM-YoeB TA-system from the Gram positive organism Staphylococcus equorum SE3. 5' RACE confirmed...

Bacterial persisters represent a small dynamic subpopulation of phenotypically variant, slowly or non-growing cells within an isogenic bacterial population. Persisters of pathogenic bacteria are causative of chronic or relapsing infections due to inherent tolerance to single or multiple antibiotics. Genes associated with the persister state are oft...

Staphylococci are Gram positive spherical bacteria of enormous clinical and biotechnological relevance. Staphylococcus aureus has been extensively studied as a model pathogen. A plethora of methods and molecular tools has been developed for genetic modification of at least ten different staphylococcal species to date. Here we review recent developm...

Population dynamics parameters of Staphylococcus aureus strain SA113 were quantified based on growth and killing experiments with batch culture cells in rich medium. Eradication kinetics and the concomitant isolation of a subpopulation of drug-tolerant SA113 persisters upon treatment with super-minimal inhibitory concentrations of antibiotics such...

Toxin-antitoxin (TA) systems are small genetic elements found on plasmids or chromosomes of countless bacteria, archaea and possibly also unicellular fungi. Under normal growth conditions the activity of the toxin protein or its translation is counteracted by an antitoxin protein or non-coding RNA. Five types of TA systems have been proposed which...

Toxin-antitoxin (TA) systems encoded in prokaryotic genomes fall into five types, typically composed of two distinct small molecules, an endotoxic protein and a cis-encoded antitoxin of ribonucleic or proteinaceous nature. In silico analysis revealed seven putative type I and three putative type II TA systems in the genome of the nonpathogenic spec...

Bacterial persister cells are non- or slow-growing reversible phenotypic variants of the wild type, tolerant to bactericidal antibiotics. We analyzed here Staphylococcus aureus persister levels by monitoring colony-forming unit counts of planktonically grown cells treated with six different antimicrobials over time. The model laboratory strains HG0...

An intracellular approach for monitoring protein production in Staphylococcus aureus is described. mCherry, fused to the dodecapeptide Tip, was capable of inducing tetracycline repressor (TetR). Time- and concentration-dependent production of mCherry could be correlated to TetR-controlled GFPmut2 activity. This approach can potentially be extended...

A set of vectors for improved tetracycline-dependent gene regulation in Staphylococcus aureus is presented. Plasmid pRAB11 was generated from pRMC2 by adding a second tet operator within the TetR-regulated promoter P(xyl/tet). Pronounced repression was observed in the absence of anhydrotetracycline (ATc) combined with high induction in the presence...

N-Acetylglucosamine (GlcNAc) is the most abundant carbon-nitrogen biocompound on earth and has been shown to be an important source of nutrients for both catabolic and anabolic purposes in Bacillus species. In this work we show that the GntR family regulator YvoA of Bacillus subtilis serves as a negative transcriptional regulator of GlcNAc cataboli...

Inducible expression is a valuable approach for the elucidation of gene functions. Here, we present new configurations of the tetracycline-dependent gene regulation (tet) system for Staphylococcus aureus. To provide improved and expanded modes of control, strains and plasmids were constructed for the constitutive expression of tetR or a variant all...

Previously it has been demonstrated that Staphylococcus aureus is sensitive toward Pseudomonas-secreted exotoxins, which preferentially target the electron transport chain in staphylococci. Here it is shown that a subpopulation of S. aureus survives these respiratory toxins of Pseudomonas aeruginosa by selection of the small-colony variant (SCV) ph...

Tetracycline repressor (TetR) bears an unstructured loop region between helices alpha8 and alpha9, which is moderately permissive to amino acid exchanges and length variations. Recognition sites for the site-specific recombinases Flp (FRT) or Cre (lox) were inserted in-frame into tetR, substituting some of this loop's codons. A number of the deduce...

The putative transcriptional regulator protein YvoA (BSU35030) from Bacillus subtilis was cloned and heterologously expressed in Escherichia coli. The protein was purified by immobilized metal-affinity chromatography and size-exclusion chromatography and subsequently crystallized. A complete native data set was collected to 2.50 A resolution. The c...

Allelic replacement in staphylococci is frequently aided by antibiotic resistance markers that replace the gene(s) of interest. In multiply modified strains, the number of mutated genes usually correlates with the number of selection markers in the strain's chromosome. Site-specific recombination systems are capable of eliminating such markers, if...

Inducible gene expression based upon Tet repressor (tet regulation) is a broadly applied tool in molecular genetics. In its original environment, Tet repressor (TetR) negatively controls tetracycline (tc) resistance in bacteria. In the presence of tc, TetR is induced and detaches from its cognate DNA sequence tetO, so that a tc antiporter protein i...

We report the construction and application of a novel insertion element for transposase-mediated mutagenesis in gram-negative bacteria. Besides Kmr as a selectable marker, the insertion element InsTetG−1 carries the anhydrotetracycline (atc)-regulated outward-directed PA promoter so that atc-dependent conditional gene knockouts or knockdowns are ge...

HPr kinase/phosphorylase (HPrK/P), a central metabolic regulator in many Gram-positive bacteria, reversibly phosphorylates HPr and Crh, thus controlling their activities as effectors of CcpA predominantly in carbon catabolite repression (CCR). We have placed the constitutively expressed hprK in its native chromosomal locus under anhydrotetracycline...

In this article we report the in vivo and in vitro characterization of single chain tetracycline repressor (scTetR) variants in Escherichia coli. ScTetR is genetically and proteolytically stable and exhibits the same regulatory properties as dimeric TetR in E.coli. Urea-dependent denaturation of scTetR is independent of the protein concentration an...

TetR-basierte Transkriptionskontrolle ist eine in zahlreichen Organismen angewandte Methode zur konditionalen Genexpression. Der Anwendungsbereich des tet-Systems wurde durch Veränderungen des Regulators und dessen Liganden erweitert, indem TetR Varianten mit veränderter Induktor- oder Operatorerkennung generiert wurden (TetRi, TetRo), sowie Mutant...

To learn about the correlation between allostery and ligand binding of the Tet repressor (TetR) we analyzed the effect of mutations in the DNA reading head-core interface on the effector specific TetR(i2) variant. The same mutations in these subdomains can lead to completely different activities, e.g. the V99G exchange in the wild-type leads to cor...

Reversible tetracycline-dependent gene regulation allows induction of expression with the tetracycline repressor (TetR) or gene silencing with the newly developed reverse mutant revTetR. We report here the implementation of both approaches with full regulatory range in gram-positive bacteria as exemplified in Bacillus subtilis. A chromosomally loca...

We describe the construction and application of elements for random insertion of promoter containing DNA into the genome of Bacillus subtilis. The outward-facing promoter of these integrative elements termed InsTetG+ is inducible by tetracycline so that conditional mutants are generated. We constructed three InsTetG+ variants using different regula...