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Background Microbially induced calcite precipitation (MICP) is an ancient property of bacteria, which has recently gained considerable attention for biotechnological applications. It occurs as a by-product of bacterial metabolism and involves a combination of chemical changes in the extracellular environment, e.g. pH increase, and presence of nucleation sites on the cell surface or extracellular substances produced by the bacteria. However, the molecular mechanisms underpinning MICP and the interplay between the contributing factors remain poorly understood, thus placing barriers to the full biotechnological and synthetic biology exploitation of bacterial biomineralisation. Results In this study, we adopted a bottom-up approach of systematically engineering Bacillus subtilis, which has no detectable intrinsic MICP activity, for biomineralisation. We showed that heterologous production of urease can induce MICP by local increases in extracellular pH, and this can be enhanced by co-expression of urease accessory genes for urea and nickel uptake, depending on environmental conditions. MICP can be strongly enhanced by biofilm-promoting conditions, which appeared to be mainly driven by production of exopolysaccharide, while the protein component of the biofilm matrix was dispensable. Attempts to modulate the cell surface charge of B. subtilis had surprisingly minor effects, and our results suggest this organism may intrinsically have a very negative cell surface, potentially predisposing it for MICP activity. Conclusions Our findings give insights into the molecular mechanisms driving MICP in an application-relevant chassis organism and the genetic elements that can be used to engineer de novo or enhanced biomineralisation. This study also highlights mutual influences between the genetic drivers and the chemical composition of the surrounding environment in determining the speed, spatial distribution and resulting mineral crystals of MICP. Taken together, these data pave the way for future rational design of synthetic precipitator strains optimised for specific applications.

The use of bacteria for self-healing cement-based materials has shown great potential in recent years. Microbially induced calcium carbonate precipitation (MICP) is a novel technology that relies on the metabolic activity of bacteria, principally from the genus Bacillus and close relatives, to precipitate calcium carbonate (CaCO3 ) inside the cracks to heal them. Among the different bacteria that can be used for this purpose, aerobic non-ureolytic bacteria have shown promising results to improve healing efficiencies and engineering properties of self-healing cementitious composites in a more sustainable way. Unfortunately, research results involving these specific bacteria species are scattered throughout the literature. Therefore, this review aims to present in one place the state-of-the-art knowledge relevant to the development of self-healing cementitious composites that rely on aerobic non-ureolytic bacteria. In this review, the most recent advances regarding the various aerobic non-ureolytic bacteria species normally used (e.g., B. cohnii, B. pseudofirmus , etc.), the methods for embedding these bacteria, the effects of these bacteria on the healing performance of cementitious composites, the results from various outdoors trials and the economic feasibility of these systems are reported, and the principal findings discussed and summarised at the end of each section. Finally, research gaps and future research work are identified and presented in the last section.

Micro-organisms contribute to Earth’s mineral deposits through a process known as bacteria-induced mineral precipitation (BIMP). It is a complex phenomenon that can occur as a result of a variety of physiological activities that influence the supersaturation state and nucleation catalysis of mineral precipitation in the environment. There is a good understanding of BIMP induced by bacterial metabolism through the control of metal redox states and enzyme-mediated reactions such as ureolysis. However, other forms of BIMP often cannot be attributed to a single pathway but rather appear to be a passive result of bacterial activity, where minerals form as a result of metabolic by-products and surface interactions within the surrounding environment. BIMP from such processes has formed the basis of many new innovative biotechnologies, such as soil consolidation, heavy metal remediation, restoration of historic buildings and even self-healing concrete. However, these applications to date have primarily incorporated BIMP-capable bacteria sampled from the environment, while detailed investigations of the underpinning mechanisms have been lagging behind. This review covers our current mechanistic understanding of bacterial activities that indirectly influence BIMP and highlights the complexity and connectivity between the different cellular and metabolic processes involved. Ultimately, detailed insights will facilitate the rational design of application-specific BIMP technologies and deepen our understanding of how bacteria are shaping our world.

Cell wall antibiotics are important tools in our fight against Gram-positive pathogens, but many strains become increasingly resistant against existing drugs. Laspartomycin C is a novel antibiotic that targets undecaprenyl phosphate (UP), a key intermediate in the lipid II cycle of cell wall biosynthesis. While laspartomycin C has been thoroughly examined biochemically, detailed knowledge about potential resistance mechanisms in bacteria is lacking. Here, we use reporter strains to monitor the activity of central resistance modules in the Bacillus subtilis cell envelope stress response network during laspartomycin C attack and determine the impact on the resistance of these modules using knock-out strains. In contrast to the closely related UP-binding antibiotic friulimicin B, which only activates ECF &sigma, factor-controlled stress response modules, we find that laspartomycin C additionally triggers activation of stress response systems reacting to membrane perturbation and blockage of other lipid II cycle intermediates. Interestingly, none of the studied resistance genes conferred any kind of protection against laspartomycin C. While this appears promising for therapeutic use of laspartomycin C, it raises concerns that existing cell envelope stress response networks may already be poised for spontaneous development of resistance during prolonged or repeated exposure to this new antibiotic.

SUMMARYBacteria closely control gene expression to ensure optimal physiological responses to their environment. Such careful gene expression can minimize the fitness cost associated with antibiotic resistance. We previously described a novel regulatory logic in Bacillus subtilis enabling the cell to directly monitor its need for detoxification. This cost-effective strategy is achieved via a two-component regulatory system (BceRS) working in a sensory complex with an ABC-transporter (BceAB), together acting as a flux-sensor where signaling is proportional to transport activity. How this is realized at the molecular level has remained unknown. Using experimentation and computation we here show that the histidine kinase is activated by piston-like displacements in the membrane, which are converted to helical rotations in the catalytic core via an intervening HAMP-like domain. Intriguingly, the transporter was not only required for kinase activation, but also to actively maintain the kinase in its inactive state in the absence of antibiotics. Such coupling of kinase activity to that of the transporter ensures the complete control required for transport flux-dependent signaling. Moreover, we show that the transporter likely conserves energy by signaling with sub-maximal sensitivity. These results provide the first mechanistic insights into transport flux-dependent signaling, a unique strategy for energy-efficient decision making.

Antibiotic resistance poses a major threat to global health, and systematic studies to understand the underlying resistance mechanisms are urgently needed. Although significant progress has been made in deciphering the mechanistic basis of individual resistance determinants, many bacterial species rely on the induction of a whole battery of resistance modules, and the complex regulatory networks controlling these modules in response to antibiotic stress are often poorly understood. In this work we combined experiments and theoretical modeling to decipher the resistance network of Bacillus subtilis against bacitracin, which inhibits cell wall biosynthesis in Gram-positive bacteria. We found a high level of cross-regulation between the two major resistance modules in response to bacitracin stress and quantified their effects on bacterial resistance. To rationalize our experimental data, we expanded a previously established computational model for the lipid II cycle through incorporating the quantitative action of the resistance modules. This led us to a systems-level description of the bacitracin stress response network that captures the complex interplay between resistance modules and the essential lipid II cycle of cell wall biosynthesis and accurately predicts the minimal inhibitory bacitracin concentration in all the studied mutants. With this, our study highlights how bacterial resistance emerges from an interlaced network of redundant homeostasis and stress response modules., Bacterial resistance against antibiotics often involves multiple mechanisms that are interconnected to ensure robust protection. So far, the knowledge about underlying regulatory features of those resistance networks is sparse, since they can hardly be determined by experimentation alone. Here, we present the first computational approach to elucidate the interplay between multiple resistance modules against a single antibiotic and how regulatory network structure allows the cell to respond to and compensate for perturbations of resistance. Based on the response of Bacillus subtilis toward the cell wall synthesis-inhibiting antibiotic bacitracin, we developed a mathematical model that comprehensively describes the protective effect of two well-studied resistance modules (BceAB and BcrC) on the progression of the lipid II cycle. By integrating experimental measurements of expression levels, the model accurately predicts the efficacy of bacitracin against the B. subtilis wild type as well as mutant strains lacking one or both of the resistance modules. Our study reveals that bacitracin-induced changes in the properties of the lipid II cycle itself control the interplay between the two resistance modules. In particular, variations in the concentrations of UPP, the lipid II cycle intermediate that is targeted by bacitracin, connect the effect of the BceAB transporter and the homeostatic response via BcrC to an overall resistance response. We propose that monitoring changes in pathway properties caused by a stressor allows the cell to fine-tune deployment of multiple resistance systems and may serve as a cost-beneficial strategy to control the overall response toward this stressor. IMPORTANCE Antibiotic resistance poses a major threat to global health, and systematic studies to understand the underlying resistance mechanisms are urgently needed. Although significant progress has been made in deciphering the mechanistic basis of individual resistance determinants, many bacterial species rely on the induction of a whole battery of resistance modules, and the complex regulatory networks controlling these modules in response to antibiotic stress are often poorly understood. In this work we combined experiments and theoretical modeling to decipher the resistance network of Bacillus subtilis against bacitracin, which inhibits cell wall biosynthesis in Gram-positive bacteria. We found a high level of cross-regulation between the two major resistance modules in response to bacitracin stress and quantified their effects on bacterial resistance. To rationalize our experimental data, we expanded a previously established computational model for the lipid II cycle through incorporating the quantitative action of the resistance modules. This led us to a systems-level description of the bacitracin stress response network that captures the complex interplay between resistance modules and the essential lipid II cycle of cell wall biosynthesis and accurately predicts the minimal inhibitory bacitracin concentration in all the studied mutants. With this, our study highlights how bacterial resistance emerges from an interlaced network of redundant homeostasis and stress response modules.

// Kei Ishibashi 1, 2 , Tobias Haber 2 , Ines Breuksch 3 , Susanne Gebhard 3 , Takashi Sugino 4 , Hitoshi Kubo 5 , Junya Hata 1 , Tomoyuki Koguchi 1 , Michihiro Yabe 1 , Masao Kataoka 1 , Soichiro Ogawa 1 , Hiroyuki Hiraki 1 , Tomohiko Yanagida 1 , Nobuhiro Haga 1 , Joachim W. Thuroff 2 , Dirk Prawitt 6, * , Walburgis Brenner 2, 3, * and Yoshiyuki Kojima 1, * 1 Department of Urology, Fukushima Medical University, Fukushima, Japan 2 Department of Urology, Johannes Gutenberg University Medical Center, Mainz, Germany 3 Department of Gynecology and Obstetrics, Johannes Gutenberg University Medical Center, Mainz, Germany 4 Department of Pathology, Shizuoka Cancer Center, Shizuoka, Japan 5 Advanced Clinical Research Center, Fukushima Medical University, Fukushima, Japan 6 Center for Pediatrics and Adolescent Medicine, Johannes Gutenberg University Medical Center, Mainz, Germany * These authors contributed equally to this work Correspondence to: Walburgis Brenner, email: brenner@uni-mainz.de Kei Ishibashi, email: keikun@fmu.ac.jp Keywords: renal cell carcinoma, tyrosine kinase inhibitor, resistance, IL-6, tocilizumab Received: March 01, 2017 Accepted: July 12, 2017 Published: July 21, 2017 ABSTRACT Metastatic renal cell carcinoma (RCC) is a tumor entity with poor prognosis due to limited therapy options. Tyrosine kinase inhibitors (TKI) represent the standard of care for RCCs, however a significant proportion of RCC patients develop resistance to this therapy. Interleukin-6 (IL-6) is considered to be associated with poor prognosis in RCCs. We therefore hypothesized that TKI resistance and IL-6 secretion are causally connected. We first analyzed IL-6 expression after TKI treatment in RCC cells and RCC tumor specimens. Cell proliferation and signal transduction activity were then quantified after co-treatment with tocilizumab, an IL-6R inhibitor, in vitro and in vivo . 786-O RCC cells secrete high IL-6 levels after low dose stimulation with the TKIs sorafenib, sunitinib and pazopanib, inducing activation of AKT-mTOR pathway, NFκB, HIF-2α and VEGF expression. Tocilizumab neutralizes the AKT-mTOR pathway activation and results in reduced proliferation. Using a mouse xenograft model we can show that a combination therapy with tocilizumab and low dosage of sorafenib suppresses 786-O tumor growth, reduces AKT-mTOR pathway and inhibits angiogenesis in vivo more efficient than sorafenib alone. Furthermore FDG-PET imaging detected early decrease of maximum standardized uptake values prior to extended central necrosis. Our findings suggest that a combination therapy of IL-6R inhibitors and TKIs may represent a novel therapeutic approach for RCC treatment.

Biomineralization triggered by bacteria is important in the natural environment and has many applications in industry and in civil and geotechnical engineering. The diversity in biomineralization capabilities of environmental bacteria is, however, not well understood. This study surveyed environmental bacteria for their ability to precipitate calcium carbonate minerals and investigated both the mechanisms and the resulting crystals. We show that while urease activity leads to the fastest precipitation, it is by no means essential. Importantly, the same quantities of calcium carbonate are produced by nonureolytic bacteria, and the resulting crystals appear to have larger volumes and more organic components, which are likely beneficial in specific applications. Testing both precipitation mechanisms in a self-healing concrete application showed that nonureolytic bacteria delivered more robust results. Here, we performed a systematic study of the fundamental differences in biomineralization between environmental bacteria, and we provide important information for the design of bacterially based engineering solutions., Microbially induced calcite precipitation (MICP) has not only helped to shape our planet’s geological features but is also a promising technology to address environmental concerns in civil engineering applications. However, limited understanding of the biomineralization capacity of environmental bacteria impedes application. We therefore surveyed the environment for different mechanisms of precipitation across bacteria. The most fundamental difference was in ureolytic ability, where urease-positive bacteria caused rapid, widespread increases in pH, whereas nonureolytic strains produced such changes slowly and locally. These pH shifts correlated well with patterns of precipitation on solid medium. Strikingly, while both mechanisms led to high levels of precipitation, we observed clear differences in the precipitate. Ureolytic bacteria produced homogenous, inorganic fine crystals, whereas the crystals of nonureolytic strains were larger and had a mixed organic/inorganic composition. When representative strains were tested in application for crack healing in cement mortars, nonureolytic bacteria gave robust results, while ureolytic strains showed more variation. This may be explained by our observation that urease activity differed between growth conditions or by the different natures and therefore different material performances of the precipitates. Our results shed light on the breadth of biomineralization activity among environmental bacteria, an important step toward the rational design of bacterially based engineering solutions. IMPORTANCE Biomineralization triggered by bacteria is important in the natural environment and has many applications in industry and in civil and geotechnical engineering. The diversity in biomineralization capabilities of environmental bacteria is, however, not well understood. This study surveyed environmental bacteria for their ability to precipitate calcium carbonate minerals and investigated both the mechanisms and the resulting crystals. We show that while urease activity leads to the fastest precipitation, it is by no means essential. Importantly, the same quantities of calcium carbonate are produced by nonureolytic bacteria, and the resulting crystals appear to have larger volumes and more organic components, which are likely beneficial in specific applications. Testing both precipitation mechanisms in a self-healing concrete application showed that nonureolytic bacteria delivered more robust results. Here, we performed a systematic study of the fundamental differences in biomineralization between environmental bacteria, and we provide important information for the design of bacterially based engineering solutions.

SUMMARY Microbial-induced calcite precipitation (MICP) has not only helped to shape our planet’s geological features, but is also a promising technology to address environmental concerns in civil engineering applications. However, limited understanding of the biomineralization capacity of environmental bacteria impedes application. We therefore surveyed the environment for different mechanisms of precipitation across bacteria. The most fundamental difference was ureolytic ability, where urease-positive bacteria caused rapid, widespread increases in pH, while non-ureolytic strains produced such changes slowly and locally. These pH shifts correlated well with patterns of precipitation on solid media. Strikingly, while both mechanisms led to high levels of precipitation, we observed clear differences in the precipitate. Ureolytic bacteria produced homogenous, inorganic fine crystals, whereas the crystals of non-ureolytic strains were larger with a mixed organic/inorganic composition. When representative strains were tested in application for crack healing in cement mortars, non-ureolytic bacteria gave robust results, while ureolytic strains showed more variation. This may be explained by our observation that urease activity varied between growth conditions, or by the different nature and therefore material performance of the precipitate. Our results shed light on the breadth of biomineralization activity among environmental bacteria, an important step towards the rational design of bacteria-based engineering solutions. ORIGINALITY-SIGNIFICANCE STATEMENT Biomineralisation triggered by bacteria is important in the natural environment but also has many applications in industry, civil and geotechnical engineering. The diversity of biomineralization capability of environmental bacteria is, however, not well understood and the full potential for exploitation might therefore remain underestimated. This study surveyed environmental bacteria for their ability to precipitate calcium carbonate minerals and investigated both the mechanism and the resulting crystals. We show that while urease activity leads to the fastest precipitation, it is by no means essential. Importantly, the same quantities of calcium carbonate are produced by non-ureolytic bacteria, and the resulting crystals appear to have larger volumes and more organic components, which is likely beneficial in specific applications. This study also compares the performance of both precipitation mechanisms in a self-healing concrete application, with non-ureolytic bacteria delivering the more robust results. To our knowledge, this is the first systematic study into the fundamental differences in biomineralization between environmental bacteria and provides important information for the design of bacteria-based engineering solutions.

Resistance against cell wall-active antimicrobial peptides in bacteria is often mediated by transporters. In low-GC-content Gram-positive bacteria, a common type of such transporters is BceAB-like systems, which frequently provide high-level resistance against peptide antibiotics that target intermediates of the lipid II cycle of cell wall synthesis. How a transporter can offer protection from drugs that are active on the cell surface, however, has presented researchers with a conundrum., Resistance against cell wall-active antimicrobial peptides in bacteria is often mediated by transporters. In low-GC-content Gram-positive bacteria, a common type of such transporters is BceAB-like systems, which frequently provide high-level resistance against peptide antibiotics that target intermediates of the lipid II cycle of cell wall synthesis. How a transporter can offer protection from drugs that are active on the cell surface, however, has presented researchers with a conundrum. Multiple theories have been discussed, ranging from removal of the peptides from the membrane and internalization of the drug for degradation to removal of the cellular target rather than the drug itself. To resolve this much-debated question, we here investigated the mode of action of the transporter BceAB of Bacillus subtilis. We show that it does not inactivate or import its substrate antibiotic bacitracin. Moreover, we present evidence that the critical factor driving transport activity is not the drug itself but instead the concentration of drug-target complexes in the cell. Our results, together with previously reported findings, lead us to propose that BceAB-type transporters act by transiently freeing lipid II cycle intermediates from the inhibitory grip of antimicrobial peptides and thus provide resistance through target protection of cell wall synthesis. Target protection has so far only been reported for resistance against antibiotics with intracellular targets, such as the ribosome. However, this mechanism offers a plausible explanation for the use of transporters as resistance determinants against cell wall-active antibiotics in Gram-positive bacteria where cell wall synthesis lacks the additional protection of an outer membrane.

Bacitracin is a cell wall targeting antimicrobial with clinical and agricultural applications. With the growing mismatch between antimicrobial resistance and development, it is essential we understand the molecular mechanisms of resistance in order to prioritize and generate new effective antimicrobials. BcrR is a unique membrane-bound one-component system that regulates high-level bacitracin resistance in Enterococcus faecalis. In the presence of bacitracin, BcrR activates transcription of the bcrABD operon conferring resistance through a putative ATP-binding cassette (ABC) transporter (BcrAB). BcrR has three putative functional domains, an N-terminal helix-turn-helix DNA-binding domain, an intermediate oligomerization domain and a C-terminal transmembrane domain. However, the molecular mechanisms of signal transduction remain unknown. Random mutagenesis of bcrR was performed to generate loss- and gain-of-function mutants using transcriptional reporters fused to the target promoter PbcrA. Fifteen unique mutants were isolated across all three proposed functional domains, comprising 14 loss-of-function and one gain-of-function mutant. The gain-of-function variant (G64D) mapped to the putative dimerization domain of BcrR, and functional analyses indicated that the G64D mutant constitutively expresses the PbcrA-luxABCDE reporter. DNA-binding and membrane insertion were not affected in the five mutants chosen for further characterization. Homology modelling revealed putative roles for two key residues (R11 and S33) in BcrR activation. Here we present a new model of BcrR activation and signal transduction, providing valuable insight into the functional characterization of membrane-bound one-component systems and how they can coordinate critical bacterial responses, such as antimicrobial resistance.

Microbially-induced calcite precipitation (MICP) is ubiquitous in nature and has become an area of interest for environmental, geotechnical, and civil engineering applications. These include bioremediation, soil engineering, and self-healing of cementitious materials. To date, ureolytic bacteria have been favoured due to their ability to rapidly increase the pH of the environment through the hydrolysis of urea and thereby induce precipitation of calcite. However, the requirement for urea can contribute to nitrogen-loading in the environment and prove to be incompatible in certain applications, such as in self-healing concrete where it delays setting. Non-ureolytic bacteria are thought to be less efficient at MICP as they lack the ability to hydrolyze urea and thus to induce rapid increases in pH. Profiling of environmental bacteria has revealed the fundamentally different mechanisms that ureolytic and non-ureolytic bacteria utilize to precipitate calcite. These affect the timing of MICP and morphology of the crystals, but not necessarily the overall quantity of calcite precipitated. Furthermore, we show that MICP facilitated by non-ureolytic bacteria results in precipitates that contain significant organic components. These precipitates appear to have increased volume and cohesiveness, which may prove advantageous in application. Our findings offer important new insights into the use of MICP for geotechnical and environmental engineering and will enable us to create a toolbox of microbial precipitators tailored for specific applications.

The genetic enhancement of Surfactin production increasingly gained attention in the last years, since relatively low product yields limit the industrial application of this biosurfactant. The natural quorum sensing regulation of the srfA operon (coding for the Surfactin synthetase) can reasonably be assumed to be the bottleneck of Surfactin synthesis. Therefore, the replacement of the naturally quorum sensing regulated, and herewith cell density dependent, promoter PsrfA against the Bacillus subtilis endogenous and constitutive promoter Pveg was hypothesized to generally enhance Surfactin yields. The markerless promoter replacement was conducted in the two B. subtilis Surfactin producer strains 3A38 and DSM 10T. The promoter substitution led to an enhancement of Surfactin concentrations in the producer strain 3A38, initially producing only minor amounts of Surfactin (0.07 g/L increased to 0.26 g/L). In contrast, promoter exchange in B. subtilis DSM 10T (wild-type strain producing 0.62 g/L Surfactin) did not achieve an enhancement of Surfactin concentrations (detrimental reduction to 0.04 g/L). These findings implicate that Surfactin synthesis is differently regulated in minor and strong Surfactin producer strains. The hypothesized general enhancement of Surfactin yields after substitution of the native promoter was therefore not confirmed.

In their habitats, microorganisms are often in competition for limited nutrients, and many Gram-positive bacteria resort to production of peptide antibiotics to succeed. Consequently, resistance against these compounds is essential. The first step in ensuring survival is the detection of the toxic peptides. Interestingly, many of the sensory proteins lack obvious binding domains. We investigate how such proteins are able to detect their substrates and regulate resistance.

Whole-cell biosensors, based on the visualization of a reporter strain's response to a particular stimulus, are a robust and cost-effective means to monitor defined environmental conditions or the presence of chemical compounds. One specific field in which such biosensors are frequently applied is drug discovery, i.e., the screening of large numbers of bacterial or fungal strains for the production of antimicrobial compounds. We here describe the application of a luminescence-based Bacillus subtilis biosensor for the discovery of cell wall active substances. The system is based on the well-characterized promoter P liaI , which is induced in response to a wide range of conditions that cause cell envelope stress, particularly antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis. A simple "spot-on-lawn" assay, where colonies of potential producer strains are grown directly on a lawn of the reporter strain, allows for quantitative and time-resolved detection of antimicrobial compounds. Due to the very low technical demands of this procedure, we expect it to be easily applicable to a large variety of candidate producer strains and growth conditions.

Purpose: Epithelial cell adhesion molecule (Ep-CAM) recently received increased attention not only as a prognostic factor in breast cancer but also as a potential target for immunotherapy. We examined Ep-CAM expression in 402 consecutive node-negative breast cancer patients with long-term follow-up not treated in the adjuvant setting. Experimental Design: Ep-CAM expression was evaluated by immunostaining. Its prognostic effect was estimated relative to overexpression/amplification of HER-2, histologic grade, tumor size, age, and hormone receptor expression. Results: Ep-CAM status was positive in 106 (26.4%) patients. In multivariate analysis, Ep-CAM status was associated with disease-free survival independent of age, pT stage, histologic grade, estrogen receptor (ER), progesterone receptor (PR), as well as HER2 status (P = 0.028; hazard ratio, 1.60; 95% confidence interval, 1.05-2.44). Recently, so-called triple-negative (HER-2, ER, and PR) breast cancer has received increased attention. We noticed a similar association of Ep-CAM with disease-free survival in the triple-negative group as for the entire cohort. Conclusion: In this study of untreated breast cancer patients, Ep-CAM overexpression was associated with poor survival in the entire cohort and in the subgroup of triple-negative breast cancer. This suggests that Ep-CAM may be a well-suited target for specific therapies particularly in HER-2–, ER-, and PR-negative tumors.

BcrR has been identified as a novel regulatory protein of high level bacitracin resistance encoded by the bcrABD operon in Enterococcus faecalis. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNA-binding proteins, and topological modeling predicts that the C-terminal domain contains four transmembrane alpha-helices. These data have led to the hypothesis that BcrR functions as both a membrane-bound sensor and transducer of bacitracin availability to regulate bcrABD expression. To characterize the bcrABD promoter and identify the promoter elements to which BcrR binds, a series of bcrA-lacZ fusions were constructed. A 69-bp region was identified that was essential for bacitracin-dependent bcrA-lacZ expression. Mutations that targeted this region were used to identify two inverted repeat sequences, each with the sequence 5'-GACA(N)(7)TGTC-3', on the bcrABD promoter that were required for bcrA-lacZ expression. To study BcrR binding to this region, we over-produced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-beta-d-maltoside, and subsequently purified it via Ni(2+)-nitrilotriacetic acid and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes, and BcrR binding to bcrABD promoter DNA was analyzed using electrophoretic mobility shift assays. Both inverted repeat sequences were required for BcrR binding, both in the presence and absence of bacitracin. These data demonstrate that membrane-bound BcrR binds specifically to the bcrABD promoter, irrespective of bacitracin concentration. We therefore propose that bacitracin-dependent induction of bcrABD expression by BcrR occurs after DNA binding.

The growth of Pyrobaculum aerophilum on yeast extract and nitrate was stimulated by the addition of maltose. Extracts of maltose/yeast extract/nitrate-grown cells contained all enzyme activities of a modified Embden–Meyerhof (EM) pathway, including ATP-dependent glucokinase, phosphoglucose isomerase, ATP-dependent 6-phosphofructokinase, fructose-1,6-phosphate aldolase, triose-phosphate isomerase, GAPOR, phosphoglycerate mutase, enolase and pyruvate kinase. The activity of GAPOR was stimulated about fourfold by maltose, indicating a role in sugar degradation. GAPOR was purified 200-fold to homogeneity and characterized as a 67 kDa monomeric, extremely thermostable protein. The enzyme showed high specificity for glyceraldehyde-3-phosphate and did not use glyceraldehyde, acetaldehyde or formaldehyde as substrates. By matrix-assisted laser desorption/ionization-time of flight analysis of the purified enzyme, ORF PA1029 was identified as a coding gene, gapor , in the sequenced genome of Pyrobaculum aerophilum . The data indicate that the (micro)aerophilic Pyrobaculum aerophilum contains a functional GAPOR as part of a modified EM pathway. Cells of the strictly aerobic crenarchaeon Aeropyrum pernix also contain enzyme activities of a modified EM pathway similar to that of Pyrobaculum aerophilum , except that a GAPN activity replaces GAPOR activity.

Co-exposure to cadmium, cobalt, lead and other heavy metals occurs in many occupational settings, such as pigment and batteries production, galvanization and recycling of electric tools. However, little is known about interactions between several heavy metals. In the present study we determined DNA single strand break (DNA-SSB) induction and repair capacity for 8-oxoguanine in mononuclear blood cells of 78 individuals co-exposed to cadmium (range of concentrations in air: 0.05-138.00 micro g/m(3)), cobalt (range: 0-10 micro g/m(3)) and lead (range: 0-125 micro g/m(3)). Exposure to heavy metals was determined in air, blood and urine. Non-parametric correlation analysis showed a correlation between cadmium concentrations in air with DNA-SSB (P = 0.001, R = 0.371). Surprisingly, cobalt air concentrations correlated even better (P < 0.001, R = 0.401), whereas lead did not correlate with DNA-SSB. Logistic regression analysis including 11 possible parameters of influence resulted in a model showing that cobalt in air, cadmium in air, cadmium in blood and lead in blood influence the level of DNA-SSB. The positive result with cobalt was surprising, since exposure levels were much lower compared with the TRK-value of 100 micro g/m(3). To examine, whether the positive result with cobalt is stable, we applied several logistic regression models with two blocks, where all factors except cobalt were considered preferentially. All strategies resulted in the model described above. Logistic regression analysis considering also all possible interactions between the relevant parameters of influence finally resulted in the following model: Odds ratio = 1.286(Co in air) x 1.040(Cd in air) x 3.111(Cd in blood) x 0.861(Pb in air) x 1.023(Co in air x Pb in air). This model correctly predicts an increased level of DNA-SSB in 91% of the subjects in our study. One conclusion from this model is the existence of more than multiplicative effects for co-exposures of cadmium, cobalt and lead. For instance increasing lead air concentrations from 1.6 to 50 micro g/m(3) in the presence of constant exposures to cobalt and cadmium (8 micro g/m(3) and 3.8 micro g/m(3)) leads to an almost 5-fold increase in the odds ratio, although lead alone does not increase DNA-SSB. The mechanism behind these interactions might be repair inhibition of oxidative DNA damage, since a decrease in repair capacity will increase susceptibility to reactive oxygen species generated by cadmium or cobalt. Indeed, repair of 8-oxoguanine decreased with increasing exposures and inversely correlated with the level of DNA-SSB (P = 0.001, R = -0.427). Protein expression patterns of individuals exposed to cobalt concentrations of approximately 10 micro g/m(3) were compared with those of unexposed individuals using two-dimensional gel electrophoresis. Qualitative and apparent quantitative alterations in protein expression were selective and certainly occurred in

A recent study reported that exposure of student embalmers in Cincinnati to high concentrations of formaldehyde (2 mg/m3) reduced the activity of the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT). Reduction in a DNA repair enzyme may strongly increase the cancer risk not only with respect to the repair-enzyme causing agent but with respect to all carcinogens causing lesions subject to repair by the enzyme in question. Thus, we examined whether formaldehyde exposure of 57 medical students during their anatomy course at two different Universities in Germany influenced MGMT activity in mononuclear blood cells. Mean formaldehyde exposure of 41 students was 0.2 +/- 0.05 mg/m3 for 6 h per week. MGMT activity was 133.2 +/- 14.9 fmol MGMT/10(6) cells before the beginning of the formaldehyde exposure, 131.1 +/- 15.8 fmol MGMT/10(6) cells after 50 days (P = 0.56) and 128.2 +/- 19.0 fmol MGMT/10(6) cells after 111 days of exposure (P = 0.92). Similarly, no significant influence of formaldehyde exposure was observed, when smoking habits, alcohol consumption, allergic disease and sex of students were considered. In addition no significant difference was obtained in MGMT activity between 16 students with mean formaldehyde exposure of 0.8 +/- 0.6 mg/m3 and students without formaldehyde exposure (n = 51; P = 0.37). In conclusion, exposure of the medical students in western Europe to formaldehyde did not decrease MGMT activity in mononuclear blood cells.

Undecaprenyl pyrophosphate phosphatases (UppPs) have been implicated in bacitracin resistance in some bacterial genera and the aim of this study was to determine the role of UppPs in mediating low-level bacitracin resistance in Enterococcus faecalis.The uppP gene was identified in the genomes of laboratory (JH2-2) and clinical (V583) strains of E. faecalis. Gene fusions (uppP-lacZ) and 5'-RACE were used to study uppP expression. The uppP gene in both strains was inactivated and mutants were studied for antimicrobial susceptibility and their susceptibilities to various stress agents.The UppP protein from E. faecalis showed high sequence identity to the Escherichia coli BacA-type UppP and was predicted to be a hydrophobic protein with eight transmembrane helices. The expression of uppP-lacZ was constitutive and not affected by bacitracin or cell wall-active antimicrobials. E. faecalis uppP mutants showed no significant changes in growth rate, colony morphology and biofilm formation. The uppP mutants exhibited increased susceptibility to bacitracin (MICs=3-6 mg/L) relative to the isogenic wild-type (MICs=32-48 mg/L). When uppP was expressed in a wild-type background, the MIC of bacitracin increased to 128-≥256 mg/L. The MICs of cefoxitin, teicoplanin, vancomycin, gentamicin, enrofloxacin and d-cycloserine were unaltered in the uppP mutant relative to the wild-type, as were susceptibilities to other stress agents (glycine, lysozyme, NaCl, SDS, low and high pH, oxidative stress and ethanol).The results demonstrate that low-level bacitracin resistance in E. faecalis is mediated by a BacA-type UppP.

After in vitro incubation of human peripheral mononuclear blood cells with glycolaldehyde (a putative metabolite of ethylene oxide) for 2 h at 37 degrees C, a dose-dependent increase in DNA crosslinks was observed in a dose range between 1 and 10 mM using the alkaline filter elution technique. The elution rate of mononuclear blood cells after treatment with ionizing radiation (600 cGy) was reduced more than 5-fold if cells were incubated with 10 mM glycolaldehyde for 2 h. After treatment with proteinase K DNA crosslinks were no longer detected in cells incubated with glycolaldehyde. Therefore the crosslinks produced by glycolaldehyde could clearly be identified as DNA-protein crosslinks. Additionally glycolaldehyde induced DNA single-strand breaks in a dose range between 1 and 10 mM. The elution rate of mononuclear blood cells was increased about 18-fold if cells were incubated with 5 mM glycolaldehyde for 2 h using an elution procedure with proteinase K. In vitro incubation of mononuclear cells with ethylene oxide for 2 h at 37 degrees resulted in a dose-dependent increase in DNA single-strand breaks between 0.5 and 10 mM ethylene oxide. Moreover, a time-dependent increase in DNA single-strand breaks after incubation with 1.5 mM ethylene oxide was observed with an increased number of single-strand breaks already detectable after 15 min and a maximum level which was detected after 2 h of incubation. However, no DNA-DNA or DNA-protein crosslinks could be detected although a wide concentration range and many different incubation times were tested. Therefore DNA crosslinks, for which evidence was found in mononuclear blood cells of humans occupationally exposed to ethylene oxide, are possibly generated by glycolaldehyde, a putative intermediate in the metabolism of ethylene oxide to glycolic acid.

International audience; Epithelial cell adhesion molecule (Ep-CAM) recently received increased attention as a prognostic factor in breast cancer. We aimed to validate the influence of Ep-CAM RNA expression in untreated node-negative breast cancer. Ep-CAM RNA expression was evaluated utilizing microarray-based gene-expression profiling in 194 consecutive node-negative breast cancer patients with long-term follow-up not treated in the adjuvant setting. The prognostic significance of Ep-CAM RNA expression for disease-free survival (DFS), metastasis-free survival (MFS), and breast cancer-specific overall survival (OS) was evaluated in univariate and multivariate analysis adjusted for age, grading, pTstage, ER as well as PR receptor and HER-2 status. Additionally, Ep-CAM RNA expression was compared with immunohistochemistry (IHC) for Ep-CAM in 194 patients. The prognostic impact of Ep-CAM gene expression was validated in further 588 node-negative breast cancer patients. Levels of Ep-CAM RNA expression showed a significant correlation with IHC ( = 0.001) and predicted in univariate analysis DFS ( = 0.001, HR = 2.4), MFS ( = 0.003, HR = 2.5), and OS ( = 0.002, HR = 3.1) accurately. The prognostic influence of Ep-CAM RNA was significant also in multivariate analysis for DFS ( = 0.017, HR = 2.0), MFS ( = 0.049, HR = 1.9), and OS ( = 0.042, HR = 2.3), respectively. The association with MFS was confirmed in an independent validation cohort in univariate ( = 0.006, HR = 1.9) and multivariate ( = 0.035, HR = 1.7) analysis. Ep-CAM RNA correlated with the proliferation metagene (

Summary Few membrane proteins with a role in transcriptional regulation have been studied, and none are able to perceive their respective stimuli and activate transcription of their regulons without the aid of auxiliary proteins. The bacitracin resistance regulator, BcrR, of Enterococcus faecalis is a membrane-bound DNA binding protein and is required for bacitracin-dependent expression of the bacitracin resistance genes, bcrABD. Here, we show that BcrR interacts directly with Zn2+ bacitracin (Kd = 2–5 μM), but not metal-free bacitracin. A solution-based DNA binding assay demonstrated that the affinity of BcrR for its target DNA is much higher (Kd = 40 nM) than previously found for transmembrane regulators and is comparable to that of soluble DNA binding proteins. A construct of BcrR that lacked the transmembrane domain was unable to bind to DNA, indicating that membrane localization was important for DNA binding. Bacitracin did not cause a change in the DNaseI footprint of BcrR on the bcrA promoter, but in vitro transcription assays with BcrR proteoliposomes showed bacitracin-dependent activation of transcription. These findings demonstrate that BcrR is a bona fide one-component transmembrane signal transduction system, which perceives an extracellular stimulus (presence of bacitracin) and relays it to an intracellular transcriptional response independent of any auxiliary proteins.

Background Mycobacteria have been shown to contain an apparent redundancy of high-affinity phosphate uptake systems, with two to four copies of such systems encoded in all mycobacterial genomes sequenced to date. In addition, all mycobacteria also contain at least one gene encoding the low-affinity phosphate transporter, Pit. No information is available on a Pit system from a Gram-positive microorganism, and the importance of this system in a background of multiple other phosphate transporters is unclear. Results The aim of this study was to determine the physiological role of the PitA phosphate transporter in Mycobacterium smegmatis. Expression of pitA was found to be constitutive under a variety of growth conditions. An unmarked deletion mutant in pitA of M. smegmatis was created. The deletion did not affect in vitro growth or phosphate uptake of M. smegmatis. Expression of the high-affinity transporters, PstSCAB and PhnDCE, was increased in the pitA deletion strain. Conclusion PitA is the only low-affinity phosphate transport system annotated in the genome of M. smegmatis. The lack of phenotype of the pitA deletion strain shows that this system is dispensable for in vitro growth of this organism. However, increased expression of the remaining phosphate transporters in the mutant indicates a compensatory mechanism and implies that PitA is indeed used for the uptake of phosphate in M. smegmatis.

Uptake of inorganic phosphate, an essential but often limiting nutrient, in bacteria is usually accomplished by the high-affinity ABC-transport system Pst. Pathogenic species of mycobacteria contain several copies of the genes encoding the Pst system (pstSCAB), and two of the encoded proteins, PstS1 and PstS2, have been shown to be virulence factors inMycobacterium tuberculosis. The fast-growingMycobacterium smegmatiscontains only a single copy of thepstoperon. This study reports the biochemical and molecular characterization of a second high-affinity phosphate transport system, designated Phn. The Phn system is encoded by a three-gene operon that constitutes the components of a putative ABC-type phosphonate/phosphate transport system. Expression studies usingphnD–andpstS–lacZtranscriptional fusions showed that both operons were induced when the culture entered phosphate limitation, indicating a role for both systems in phosphate uptake at low extracellular concentrations. Deletion mutants in eitherphnDorpstSfailed to grow in minimal medium with a 10 mM phosphate concentration, while the isogenic wild-type strain mc2155 grew at micromolar phosphate concentrations. Analysis of the kinetics of phosphate transport in the wild-type and mutant strains led to the proposal that the Phn and Pst systems are both high-affinity phosphate transporters with similar affinities for phosphate (i.e. apparentKmvalues between 40 and 90 μM Pi). The Phn system ofM. smegmatisappears to be unique in that, unlike previously identified Phn systems, it does not recognize phosphonates or phosphite as substrates.

Mycobacterium smegmatis is able to grow and survive at acidic pH, and exhibits intracellular pH homeostasis under these conditions. In this study, the authors have identified low proton permeability of the cytoplasmic membrane, and high cytoplasmic buffering capacity, as determinants of intrinsic acid resistance of M. smegmatis. To identify genes encoding proteins involved in protecting cells from acid stress, a screening method was developed using the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP was used to suppress intrinsic acid resistance of M. smegmatis. The screen involved exposing cells to pH 5·0 in the presence of CCCP, and survivors were rescued at various time intervals on solid medium at pH 7·5. Cells capable of responding to intracellular acidification (due to CCCP-induced proton equilibration) will survive longer under these conditions than acid-sensitive cells. From a total pool of 5000 transposon (Tn611) insertion mutants screened, eight acid-sensitive M. smegmatis mutants were isolated. These acid-sensitive mutants were unable to grow at pH 5·0 in the presence of 1–5 μM CCCP, a concentration not lethal to the wild-type strain mc2155. The DNA flanking the site of Tn611 was identified using marker rescue in Escherichia coli, and DNA sequencing to identify the disrupted locus. Acid-sensitive mutants of M. smegmatis were disrupted in genes involved in phosphonate/phosphite assimilation, methionine biosynthesis, the PPE multigene family, xenobiotic-response regulation and lipid biosynthesis. Several of the acid-sensitive mutants were also defective in stationary-phase survival, suggesting that overlapping stress protection systems exist in M. smegmatis.

The purpose of this study was to examine whether exposure to magnetic fields (MFs) relevant for magnetic resonance imaging (MRI) in clinical routine influences cell cycle progression in two tumor cell lines in vitro. HL60 and EA2 cells were exposed to four types of MFs: (i) static MF of 1.5 and 7.05 T, (ii) extremely low frequency magnetic gradient fields (ELFMGFs) with ± 10 mT/m and 100 Hz, as well as ± 100 mT/m and 100 Hz, (iii) pulsed high frequency MF in the radiofrequency (RF) range (63.6 MHz, 5.8 μT), and (iv) a combination of (i–iii). Exposure periods ranged from 1 to 24 h. Cell cycle distribution (G0/G1, S, and G2/M phases) was analyzed by flow cytometry. Cell cycle analysis did not reveal differences between the exposed and the control cells. As expected, positive controls with irradiated (8 Gy) HL60 and EA2 cells showed a strong G2/M arrest. Using conditions that are relevant for patients during MRI, no influence of MFs on cell cycle progression was observed in these cell lines. Care was taken to control secondary parameters of influence, such as vibration by the MR scanner or temperature to avoid false positive results. Bioelectromagnetics 24:241-250, 2003. © 2003 Wiley-Liss, Inc.

The biochemical inhibition by Cu2+ on eight phylogenetically and biochemically different phosphofructokinases (PFKs) was investigated. The enzymes screened included representatives from thermophilic and mesophilic bacteria, a hyperthermophilic archaeon and a eukaryote, covering all three phosphoryl donor subtypes (ATP, ADP and pyrophosphate). The sensitivities of the enzymes to Cu2+ varied greatly, with the archaeal ADP-PFK being the least and the eukaryote ATP-PFK being the most sensitive. The bacterial ATP- and pyrophosphate-dependent PFKs showed intermediate sensitivity with the exception of the Spirochaeta thermophila enzyme (pyrophosphate-dependent) which was relatively resistant.

The radionuclides (131)I and (99m)Tc are frequently used for therapy of benign and malignant thyroid disease ((131)I) and for diagnosis of thyroid and other diseases ((99m)Tc). However, the levels of DNA single-strand breaks (SSBs) induced in cells of patients after administration of (131)I and (99m)Tc are not known. In this study, we measured the number of SSBs per cell induced by (131)I and (99m)Tc in vitro, extrapolated the results to the clinical situation, and assessed their biological relevance by comparing levels of SSBs induced after therapeutic administration of (131)I and (99m)Tc to those induced by endogenous processes or by occupational exposure to genotoxic substances. A linear dose-response relationship between the radioactivity concentrations of (131)I and (99m)Tc and SSBs in human mononuclear blood cells (determined by alkaline elution) was obtained after incubation at 4 and 37 degrees C. At 4 degrees C, where almost no repair of SSBs takes place, (131)I and (99m)Tc induced 81 and 7 SSBs per cell per hour/(MBq/ml), respectively. At 37 degrees C, only 20 and 1.6 SSBs per cell per hour/(MBq/ml) were observed after incubation with (131)I and (99m)Tc. To estimate the induction of SSBs in vivo in cells of patients after administration of 3700 MBq (131)I (oral) or 60 MBq (99m)Tc (i.v.), the rates of induction of SSBs obtained in vitro were extrapolated to the concentrations of (131)I and (99m)Tc measured in blood of patients. The total number of SSBs (mean +/- standard deviation) accumulated after oral administration of 3700 MBq (131)I up to 70 h after administration was calculated as 200 +/- 59 SSBs/cell. After administration of 60 MBq (99m)Tc (i.v.), 0.032 +/- 0.009 SSBs per cell (total SSBs up to 2 h after administration) were cumulated. The induction of SSBs by endogenous processes (estimated 2,000 SSBs per cell per hour) and by occupational exposure to genotoxic substances (125-430 SSBs per cell) has been estimated in earlier studies. In conclusion, the frequency of SSBs induced by thyroid diagnosis with 60 MBq (99m)Tc is approximately 5 orders of magnitude smaller than the frequency of spontaneous SSBs and thus is most probably without biological relevance. Since the frequency of induction of SSBs by therapy with (131)I (3700 MBq) is about 6000-fold higher compared to thyroid diagnosis by (99m)Tc, its biological relevance is more difficult to assess. Nevertheless, the number of SSBs induced by therapy with (131)I is substantially lower than that induced by endogenous processes.