Studies on polar cell wall growth and antibiotic susceptibility of Corynebacterium glutamicum

Corynebacterium glutamicum is a Gram positive soil bacterium with high industrial importance in ton scale production of amino acids. Apart from that, it becomes more and more important for medical studies, where it serves as model organism due to its close relation to bacteria causing several pathogens such as tuberculosis, diphtheria and leprosy. C. glutamicum, like Mycobacterium tuberculosis, has a distinct cell wall which is composed of a peptidoglycan layer (murein) with covalently bound polysaccharide layers that are capped with mycolic acids. In addition, both organisms have a polar cell wall synthesis machinery which is spatially regulated by DivIVA (Wag31 in M. tuberculosis). The present study shows that DivIVA regulates cell wall synthesis upon direct interaction with the lipid II flippase RodA. RodA determines morphology and growth in C. glutamicum and is localized to the poles and septa. The absence of rodA results in growth defects and cell shape alterations as well as altered lipid II proliferation of the poles (polar cell growth is sustained). DivIVA is furthermore involved in chromosome segregation upon direct interaction with the partitioning ParB protein, which binds to parS sites on the chromosome, thus tethering the replicated nucleoids to the cell poles. Interactions of DivIVA with ParB and RodA were identified in a synthetic in vivo protein-protein interaction assay where fluorescently labeled proteins of interest are expressed in E. coli cells and interaction is analyzed microscopically. A decisive improvement of this assay is the application of FRET, which is more sensitive and allows quantification of interaction. In order to test whether ParB and RodA compete for the same interaction site in DivIVA, we mapped interaction sites of both proteins. It turned out that ParB binds to a middle region of DivIVA, whereas RodA binds to the N-terminal domain of DivIVA where one lysine residue is essential for interaction. To fight bacterial infections, that cause thousands of casualties each year, it is mandatory to understand mechanisms in cellular processes, such as cell division and growth, to find new targets for antibiotic intervention. Unfortunately, bacteria are able to develop resistances against many antibiotics. The mycolic acid or arabinan layer and synthesis machinery are good candidates for new antibiotics. Amongst others, two of them have emerged as useful drugs against M. tuberculosis, ethambutol (EMB) and BTZ043. In this study, we investigated the modes of action and antibiotic susceptibility of C. glutamicum after EMB and BTZ043 treatment. We found that both antibiotics, which target the arabinan synthesis pathway, affect exclusively polar elongation growth, as demonstrated in different staining assays. Interestingly, only 10% of the cells were killed and cells in stationary phase were not affected by EMB or BTZ043. Moreover, we used a chromosomal DivIVA-mCherry fusion and found that DivIVA protein level is drastically increased. The cells show asymmetric recovery after treatment, in which one daughter cell acquires the excess DivIVA whereas the other daughter cell exhibits normal cell growth.Corynebacterium glutamicum ist ein Gram-positives Bodenbakterium mit großer industrieller Bedeutung für die Herstellung von Aminosäuren im Tonnenmaßstab. Des Weiteren bekommt es zunehmende Bedeutung für die medizinische Forschung, wo es aufgrund seiner engen Verwandtschaft zu den pathogenen Erregern von Tuberkulose, Diphtherie und Lepra als idealer Modellorganismus dient. Besonders die Zellwand von C. glutamicum hat große Ähnlichkeit zu der vieler pathogener Vertreter wie Mykobakterium tuberculosis. Sie besteht aus einer Peptidoglycan-Schicht (Murein), an der über weitere Polysaccharid-Schichten die charakteristischen Mycolsäuren gebunden sind. Darüber hinaus besitzen beide Organismen eine polare Zellwandsynthese, die von DivIVA (Wag31 in M. tuberculosis) räumlich reguliert wird. Die Rolle von DivIVA am Zellwachstum wurde vor Jahren erstmals beschrieben, jedoch war seine exakte Funktion bis zuletzt unbekannt. In dieser Studie wird erstmals die Funktion von DivIVA am polaren Zellwachstum durch Interaktion mit der Lipid II-Flippase RodA gezeigt. RodA beeinflusst die Morphologie und das Wachstum von C. glutamicum und wird von DivIVA an die Zellpole lokalisiert. Deletion von rodA resultiert in reduziertem Wachstum und veränderter Morphologie, sowie einer alternativen Lipid II Versorgung der Zellpole, da das polare Zellwachstum erhalten bleibt. DivIVA ist darüber hinaus an der Chromosomensegregation beteiligt, wo es direkt mit ParB interagiert, das über parS-Seiten an die replizierten Chromosomen bindet um sie an die Zellpole zu fixieren. Die Interaktionen zwischen DivIVA und ParB bzw. RodA wurden mit Hilfe eines synthetischen in vivo Assays identifiziert, worin die zu untersuchenden Gene an Fluorophore gekoppelt und in E. coli Zellen exprimiert werden. Somit lässt sich eine Co-Lokalisation nach individueller und Co-Expression der Fusionsproteine mikroskopisch analysieren. Eine entscheidende Verbesserung dieses Assays ist die Verwendung von FRET, das sensitiver ist und eine Quantifizierung der Interaktion ermöglicht. Um herauszufinden, ob ParB und RodA um die gleiche Bindungsstelle an DivIVA konkurrieren, wurden die Interaktionsdomänen beider Proteine ermittelt. Während ParB an eine mittlere Region in DivIVA bindet, bindet RodA an die N-terminale Domäne von DivIVA, in der ein Lysin-Rest für die Bindung essenziell ist. Für den Kampf gegen bakterielle Infektionskrankheiten, die jährlich tausende Todesfälle verursachen, ist es dringend notwendig zelluläre Mechanismen, beispielsweise der Zellteilung und des Wachstums, zu entschlüsseln um Targets für neue Antibiotika zu finden. Insbesondere die kontinuierliche Entstehung neue Resistenzen macht diese Aufgabe wichtiger denn je. Die Mykolsäureschicht und ihre Synthese sind vielversprechende Targets, da bisher nur wenige Antibiotika, wie Ethambutol (EMB) oder BTZ043, dagegen existieren. Wir haben die Wirkungsweise und antibiotische Suszeptibilität von C. glutamicum nach EMB und BTZ043 Behandlung untersucht. Beide Antibiotika, die in die Arabinogalactan-Synthese eingreifen, beeinflussen ausschließlich das polare Zellwachstum, wie in mehrerer Färbeassays gezeigt. Lediglich 10% der Zellen wurden getötet. Zellen, die sich in der stationären Phase befanden, wurde nicht beeinflusst. Darüber hinaus zeigte die Verwendung eines Stammes mit chromosomaler DivIVA-mCherry Fusion, dass das DivIVA Protein Level stark erhöht ist. Erholungsexperimente nach Antibiotikazugabe zeigten, dass die Zellen asymmetrisch reagieren, wobei eine Tochterzelle das überschüssige DivIVA übernimmt, während die andere Zelle normales Wachstum erfährt

Shelf ice-associated cryo-benthos and environmental features

Incidences of cryo-benthic communities beneath ice shelves are rare and recent discoveries. Combined seal- and ROV-borne imagery and novel sampling technologies allowed for a re-assessment and augmentation of earlier findings on a cryo-benthic isopod community (Antarcturus cf. spinacoronatus), being attached head-down to the underside of floating shelf ice at depths of around 80-150m. The shelf ice-associated cryo-benthos was discovered at Drescher Inlet (-72.83667 -19.15300), Riiser-Larsen Ice Shelf (eastern Weddell Sea). The inlet constitutes a 25km long and between 2 and 4km wide crack in the surrounding shelf ice, which is associated with certain environmental features. Here we compile all available local physical, biological, and biogeochemical data and discuss their relevance in the wider regional context for this faunal hotspot. These include data on shelf, sea and platelet ice, seafloor topography, hydrography and water chemistry, as well as associated pelagic and benthic marine life, in particular affinities of the cryo-benthic isopod community to related fauna occurring in nearby seabed communities using molecular barcoding

The Antituberculosis Drug Ethambutol Selectively Blocks Apical Growth in CMN Group Bacteria

Members of the genus Mycobacterium are the most prevalent cause of infectious diseases. Mycobacteria have a complex cell envelope containing a peptidoglycan layer and an additional arabinogalactan polymer to which a mycolic acid bilayer is linked;this complex, multilayered cell wall composition (mAGP) is conserved among all CMN group bacteria. The arabinogalactan and mycolic acid synthesis pathways constitute effective drug targets for tuberculosis treatment. Ethambutol (EMB), a classical antituberculosis drug, inhibits the synthesis of the arabinose polymer. Although EMB acts bacteriostatically, its underlying molecular mechanism remains unclear. Here, we used Corynebacterium glutamicum and Mycobacterium phlei as model organisms to study the effects of EMB at the single-cell level. Our results demonstrate that EMB specifically blocks apical cell wall synthesis, but not cell division, explaining the bacteriostatic effect of EMB. Furthermore, the data suggest that members of the family Corynebacterineae have two dedicated machineries for cell elongation (elongasome) and cytokinesis (divisome). IMPORTANCE Antibiotic treatment of bacterial pathogens has contributed enormously to the increase in human health. Despite the apparent importance of antibiotic treatment of bacterial infections, surprisingly little is known about the molecular functions of antibiotic actions in the bacterial cell. Here, we analyzed the molecular effects of ethambutol, a first-line antibiotic against infections caused by members of the genus Mycobacterium. We find that this drug selectively blocks apical cell growth but still allows for effective cytokinesis. As a consequence, cells survive ethambutol treatment and adopt a pneumococcal cell growth mode with cell wall synthesis only at the site of cell division. However, combined treatment of ethambutol and beta-lactam antibiotics acts synergistically and effectively stops cell proliferation

From pole to pole : 33 years of physical oceanography onboard R/V Polarstern

Measuring temperature and salinity profiles in the world's oceans is crucial to understanding ocean dynamics and its influence on the heat budget, the water cycle, the marine environment and on our climate. Since 1983 the German research vessel and icebreaker Polarstern has been the platform of numerous CTD (conductivity, temperature, depth instrument) deployments in the Arctic and the Antarctic. We report on a unique data collection spanning 33 years of polar CTD data. In total 131 data sets (1 data set per cruise leg) containing data from 10 063 CTD casts are now freely available at doi: 10.1594/PANGAEA.860066. During this long period five CTD types with different characteristics and accuracies have been used. Therefore the instruments and processing procedures (sensor calibration, data validation, etc.) are described in detail. This compilation is special not only with regard to the quantity but also the quality of the data -the latter indicated for each data set using defined quality codes. The complete data collection includes a number of repeated sections for which the quality code can be used to investigate and evaluate long-term changes. Beginning with 2010, the salinity measurements presented here are of the highest quality possible in this field owing to the introduction of the OPTIMARE Precision Salinometer.Peer reviewe

Interaction sites of DivIVA and RodA from Corynebacterium glutamicum

Elongation growth in actinobacteria is localized at the cell poles. This is in contrast to many classical model organisms where insertion of new cell wall material is localized around the lateral site. We previously described a role of RodA from Corynebacterium glutamicum in apical cell growth and morphogenesis. Deletion of rodA had drastic effects on morphology and growth, likely a result from misregulation of penicillin-binding proteins and cell wall precursor delivery. We identified the interaction of RodA with the polar scaffold protein DivIVA, thus explaining subcellular localization of RodA to the cell poles. In this study, we describe this interaction in detail and map the interaction sites of DivIVA and RodA. A single amino acid residue in the N-terminal domain of DivIVA was found to be crucial for the interaction with RodA. The interaction site of RodA was mapped to its cytoplasmic, C-terminal domain, in a region encompassing the last 10 amino acids (AAs). Deletion of these 10 AAs significantly decreased the interaction efficiency with DivIVA. Our results corroborate the interaction of DivIVA and RodA, underscoring the important role of DivIVA as a spatial organizer of the elongation machinery in Corynebacterineae

Towards on-chip mid-infrared sensors

This Feature highlights recent advances on mid-infrared thin-film waveguide technology and on-chip photonics facilitating next-generation label-free chem/bio sensor and assay platforms. Complemented by more recent advancements toward on-chip semiconductor waveguides, it is anticipated that label-free integrated mid-infrared sensing schemes will readily complement existing chem/bio sensor technologies in applications ranging from process monitoring and environmental analysis to biomedical diagnostics and point-of-care device

Sensing chlorinated hydrocarbons via miniaturized GaAs/AlGaAs thin-film waveguide flow cells coupled to quantum cascade lasers

Mid-infrared (MIR) sensors based on attenuated total reflection (ATR) spectroscopy provide robust, rapid and sensitive platforms for the detection of low levels of organic molecules and pollutants. Nowadays, MIR (3–15 μm) spectroscopy has evolved into a versatile sensing technique providing inherent molecular selectivity for the detection of organic and inorganic molecules. The excitation of vibrational and rotational transitions enables the qualitative and quantitative analysis of molecular constituents in solid, liquid, and vapor phases, which facilitates the application of MIR chem/bio sensors for on-site environmental analysis in scenarios such as trace pollutant monitoring or spill detection. This report presents the first integration of thin-film gallium arsenide/aluminum gallium arsenide (GaAs/AlGaAs) into a miniaturized liquid flow cell designed for continuous trace analysis of chlorinated hydrocarbons (CHCs) in water coupled to a broadly tunable quantum cascade laser (QCL), which facilitates in-field deployment of QCL-based sensing devices ensuring water quality and water safety

Portable infrared laser spectroscopy for on-site mycotoxin analysis

Mycotoxins are toxic secondary metabolites of fungi that spoil food, and severely impact human health (e.g., causing cancer). Therefore, the rapid determination of mycotoxin contamination includingdeoxynivalenolandaflatoxinB1infoodandfeedsamplesisofprimeinterestforcommodity importers and processors. While chromatography-based techniques are well established in laboratory environments, only very few (i.e., mostly immunochemical) techniques exist enabling direct on-site analysis for traders and manufacturers. In this study, we present MYCOSPEC - an innovative approach for spectroscopic mycotoxin contamination analysis at EU regulatory limits for the first time utilizing mid-infrared tunable quantum cascade laser (QCL) spectroscopy. This analysis technique facilitates on-site mycotoxin analysis by combining QCL technology with GaAs/AlGaAs thin-film waveguides. Multivariate data mining strategies (i.e., principal component analysis) enabled the classification of deoxynivalenol-contaminated maize and wheat samples, and of aflatoxin B1 affected peanuts at EU regulatory limits of 1250 μg kg−1 and 8 μg kg−1, respectively

Infrared Attenuated Total Reflection Spectroscopy for the Characterization of Gold Nanoparticles in Solution

In situ synthesis of bare gold nanoparticles mediated by stainless steel as reducing agent was monitored via infrared attenuated total reflection (IR-ATR) spectroscopy. Gold nanoparticles were directly synthesized within the liquid cell of the ATR unit taking immediate advantage of the stainless steel walls of the ATR cell. As nanoparticles were formed, a layer of particles was deposited at the SiO₂ ATR waveguide surface. Incidentally, the absorption bands of water increased resulting from surface-enhanced infrared absorption (SEIRA) effects arising from the presence of the gold nanoparticles within the evanescent field. Next to the influence of the Au­(III) precursor concentration and the temperature, the suitability of IR-ATR spectroscopy as an innovative tool for investigating changes of nanoparticles in solution, including their aggregation promoted by an increase of the ionic strength or via a pH decrease, and for detailing the sedimentation process of gold nanoparticles was confirmed

Mid-infrared thin-film diamond waveguides combined with tunable quantum cascade lasers for analyzing the secondary structure of proteins

Diamond has excellent optical properties including broadband transmissivity, low self- absorption and a high refractive index, which have prompted its use in for optical sensing applications. Thin-film diamond strip waveguides (DSWGs) combined with tunable quantum cascade lasers (tQCLs) providing an emission wavelength range of 5.78-6.35 μm (1735-1570 cm-1) have been used to obtain mid-infrared (MIR) spectra of proteins, thereby enabling the analysis of their secondary structure via the amide I band. Three different proteins were analyzed, namely bovine serum albumin (BSA), myoglobin, and γ-globulin. The secondary structure of BSA and myoglobin has a major contribution of -helices, whereas γ-globulins are rich in β-sheet structures, which is reflected in the amide I band. A comparison of the spectra obtained via the combination of the tQCL and DSWG with spectra obtained using conventional Fourier transform infrared (FTIR) spectroscopy and a commercial diamond attenuated total reflection (ATR) element has been performed. It is shown that the main features evident in FTIR-ATR spectra are also obtained using tQCL-DSWG sensors