Novel Genes Required for Surface-Associated Motility in Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic and increasingly multi-drug resistant human pathogen rated as a critical priority one pathogen for the development of new antibiotics by the WHO in 2017. Despite the lack of flagella, A. baumannii can move along wet surfaces in two different ways: via twitching motility and surface-associated motility. While twitching motility is known to depend on type IV pili, the mechanism of surface-associated motility is poorly understood. In this study, we established a library of 30 A. baumannii ATCC® 17978™ mutants that displayed deficiency in surface-associated motility. By making use of natural competence, we also introduced these mutations into strain 29D2 to differentiate strain-specific versus species-specific effects of mutations. Mutated genes were associated with purine/pyrimidine/folate biosynthesis (e.g. purH, purF, purM, purE), alarmone/stress metabolism (e.g. Ap4A hydrolase), RNA modification/regulation (e.g. methionyl-tRNA synthetase), outer membrane proteins (e.g. ompA), and genes involved in natural competence (comEC). All tested mutants originally identified as motility-deficient in strain ATCC® 17978™ also displayed a motility-deficient phenotype in 29D2. By contrast, further comparative characterization of the mutant sets of both strains regarding pellicle biofilm formation, antibiotic resistance, and virulence in the Galleria mellonella infection model revealed numerous strain-specific mutant phenotypes. Our studies highlight the need for comparative analyses to characterize gene functions in A. baumannii and for further studies on the mechanisms underlying surface-associated motility.Peer Reviewe

Complete Genome Sequencing of Acinetobacter sp. Strain LoGeW2-3, Isolated from the Pellet of a White Stork, Reveals a Novel Class D Beta-Lactamase Gene

Whole-genome sequencing of Acinetobacter sp. strain LoGeW2-3, isolated from the pellet of a white stork (Ciconia ciconia), reveals the presence of a plasmid of 179,399 bp encoding a CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated genes) system of the I-F type, and the chromosomally encoded novel class D beta-lactamase OXA-568

SHOX2 DNA Methylation is a Biomarker for the diagnosis of lung cancer based on bronchial aspirates

<p>Abstract</p> <p>Background</p> <p>This study aimed to show that SHOX2 DNA methylation is a tumor marker in patients with suspected lung cancer by using bronchial fluid aspirated during bronchoscopy. Such a biomarker would be clinically valuable, especially when, following the first bronchoscopy, a final diagnosis cannot be established by histology or cytology. A test with a low false positive rate can reduce the need for further invasive and costly procedures and ensure early treatment.</p> <p>Methods</p> <p>Marker discovery was carried out by differential methylation hybridization (DMH) and real-time PCR. The real-time PCR based HeavyMethyl technology was used for quantitative analysis of DNA methylation of SHOX2 using bronchial aspirates from two clinical centres in a case-control study. Fresh-frozen and Saccomanno-fixed samples were used to show the tumor marker performance in different sample types of clinical relevance.</p> <p>Results</p> <p>Valid measurements were obtained from a total of 523 patient samples (242 controls, 281 cases). DNA methylation of SHOX2 allowed to distinguish between malignant and benign lung disease, i.e. abscesses, infections, obstructive lung diseases, sarcoidosis, scleroderma, stenoses, at high specificity (68% sensitivity [95% CI 62-73%], 95% specificity [95% CI 91-97%]).</p> <p>Conclusions</p> <p>Hypermethylation of SHOX2 in bronchial aspirates appears to be a clinically useful tumor marker for identifying subjects with lung carcinoma, especially if histological and cytological findings after bronchoscopy are ambiguous.</p

Complete Genome Sequence of Acinetobacter sp. Strain NCu2D-2 Isolated from a Mouse

Whole-genome sequencing of Acinetobacter sp. strain NCu2D-2, isolated from the trachea of a mouse, revealed the presence of a plasmid of 309,964 bp with little overall similarity to known plasmids and enriched in insertion sequences (ISs) closely related to IS elements known from the nosocomial pathogen Acinetobacter baumannii

Surface-associated motility of Acinetobacter baumannii and its epigenetic regulation by AamA-specific DNA methylation

Trotz des Fehlens von Flagellen sind Bakterien der Gattung Acinetobacter in der Lage sich fortzubewegen. Dies geschieht zum einen durch die Typ IV Pilus-abhängige ‚twitching’-Motilität und zum anderen durch die mechanistisch unverstandene Oberflächen-assoziierte Motilität. So sollte die vorliegende Arbeit zur Aufklärung der Oberflächen-assoziierten Motilität von Acinetobacter baumannii beitragen, eines opportunistischen Krankheitserregers. Zunächst wurde eine A. baumannii ATCC 17978 Transposonmutanten-Bibliothek erstellt, aus welcher 30 Mutanten mit signifikanten Motilitätsdefekten identifiziert werden konnten. Diese betrafen vor allem die Genfunktionskategorien der Purin-/Pyrimidin-/Folat-Biosynthese, des Alarmon/Stress-Stoffwechsels, der RNA-Modifizierung/Regulation, der äußeren Membranproteine und der DNA-Modifikation. Um stamm- und artspezifische Merkmale zu unterscheiden, wurden ausgewählte Mutationen zusätzlich in den Stamm A. baumannii 29D2 eingeführt. Mit Hilfe der Mutantenbibliothek konnte eine DNA-Adenin-(N6)-Methyltransferase (A1S_0222) identifiziert werden, die nach ihrer Charakterisierung als AamA bezeichnet wurde. Die DNA-Methylierung in Bakterien erlaubt epigenetische Regulation und beschreibt einen reversiblen chemischen Prozess, in welchem die DNA durch das Anhängen einer Methylgruppe modifiziert wird. Die biochemische und strukturelle Charakterisierung der neuartigen A. baumannii Methyltransferase (MTase) AamA, sowie die Identifikation der spezifischen Erkennungssequenz lassen AamA als eine solitäre MTase mit distributivem Methylierungsmechanismus erscheinen. Dies steht im Gegensatz zur wohl bekanntesten solitären DNA-Adenin MTase Dam aus E. coli, welche hoch prozessiv ist und nahezu alle Erkennungsstellen methyliert. Darüber hinaus wurde eine vergleichende Multi-Omics Studie des Methyloms, Transkriptoms und Proteoms der aamA-Mutante und des Wildtyps durchgeführt. Es wurden, im Gegensatz zum planktonischen Wachstum, unter Oberflächen-assoziierter Motilität doppelt so viele m6A-methylierte AamA-spezifische Targetmotive detektiert. Auch zeigt sich eine deutliche Verbindung zwischen der Mutantenbibliothek insgesamt und den Genontologie-Gruppen der m6A-methylierten AamA-spezifischen Targetmotiven. Auf Transkriptom- und Proteom-Ebene offenbarten sich ebenfalls deutliche Einflüsse der aamA-Inaktivierung. Im Allgemeinen jedoch scheinen die Regulationsebenen Methylom, Transkriptom und Proteom, kaum miteinander gekoppelt zu sein. Diese Ergebnisse zeigen die Einbettung der Oberflächen-assoziierten Motilität von A. baumannii in ein komplexes Regulationsnetzwerk, bei dem die AamA-vermittelte differentielle DNA-Methylierung eine zentrale Rolle zu spielen scheint.Despite the absence of flagella, bacteria of the genus Acinetobacter are capable of locomotion. This occurs firstly through type IV pilus-dependent 'twitching' motility and secondly through surface-associated motility, which is mechanistically not understood. Thus, the present work was intended to contribute to the elucidation of the surface-associated motility of Acinetobacter baumannii, an opportunistic pathogen. First, an A. baumannii ATCC 17978 transposon mutant library was constructed, from which 30 mutants with significant motility defects were identified. These primarily affected the gene function categories of purine/pyrimidine/folate biosynthesis, alarmone/stress metabolism, RNA modification/regulation, outer membrane proteins, and DNA modification. In order to distinguish strain- and species-specific features, selected mutations were additionally introduced into A. baumannii strain 29D2. The mutant library was used to identify a DNA adenine-(N6)-methyltransferase (A1S_0222), which was named AamA after its characterization. DNA methylation in bacteria allows epigenetic regulation and describes a reversible chemical process in which DNA is modified by the addition of a methyl group. The biochemical and structural characterization of the novel A. baumannii methyltransferase (MTase) AamA, as well as the identification of its specific recognition sequence, suggest AamA to be an orphan MTase with a distributive methylation mechanism. This is in contrast to the well-established orphan DNA adenine MTase Dam of E. coli, which is highly processive and methylates almost all recognition sites. In addition, a comparative multi-omics study of the methylome, transcriptome and proteome of the aamA-mutant and wildtype strain was performed. Twice as many m6A-methylated AamA-specific target motifs were detected under surface-associated motility, in contrast to planktonic growth. Also, a clear link between the mutant library as a whole and the gene ontology groups of m6A-methylated AamA-specific target motifs is evident. At the transcriptome and proteome levels, significant influences of aamA-inactivation were also revealed. In general, however, the regulatory levels of methylome, transcriptome, and proteome, appear to be weakly coupled. These results demonstrate the embedding of the surface-associated motility of A. baumannii in a complex regulatory network in which AamA-mediated differential DNA methylation appears to play a central role

Seasonal and interannual dynamics of polyphenols in Myriophyllum verticillatum and their allelopathic activity on Anabaena variabilis

In 4 successive years, we investigated the seasonal and interannual variability of the total polyphenolic pool and of the individual polyphenolic compounds in Myriophyllum verticillatum, as well as their allelopathic activity in a small eutrophic lake. We tested whether nutrient availability explained interannual and seasonal changes in the production of polyphenols. There were no strong interannual variations in plant tissue carbon, nitrogen and phosphorus concentrations, while total phenolic compounds (TPC) significantly differed between years, especially in apical meristems (range: 38 122 mg g-1 dry weight (DW)). Seasonal patterns, with maxima between May and July, changed between years. Partially confirming the carbon-nutrient balance hypothesis sensu Bryant et al. [Bryant, J.P., Chapin III, F.S., Klein, D.R., 1983. Carbon/nutrient balance of boreal plants in relation to vertebrate herbivory. Oikos 40, 357 368], we found correlations between TPC and the C/N (carbon/nitrogen) ratio in some but not all years, especially in apical meristems. Plant tissue phosphorus content accounted also for the variability in TPC in some years. Crude extracts of apical meristems always inhibited the growth of Anabaena variabilis, used as a target cyanobacterium. Plant TPC concentration and allelopathic activity were significantly correlated in all years except in 2005. Bioassay-directed fractionation of M. verticillatum extracts coupled with LC MS analyses of the respective fractions revealed several isomers of HHDP-di- and -tri-galloylglucose apparently responsible for the allelopathic effects. The individual active compounds revealed a more distinct seasonal pattern compared to the pool of phenolic compounds in M. verticillatum, with a clear maximum in May, the ecologically most relevant period for inhibitory effects of submerged macrophytes on phytoplankton

Minocycline Mitigates the Gliogenic Effects of Proinflammatory Cytokines on Neural Stem Cells

Mobilizing endogenous neural stem cells (NSCs) in the adult brain is designed to enhance the brain's regenerative capacity after cerebral lesions, e.g., as a result of stroke. Cerebral ischemia elicits neuroinflammatory processes affecting NSCs in multiple ways, the precise mechanisms of which currently remain elusive. An inhibitory effect of minocycline on microglia activation, a hallmark of postischemic neuroinflammation, has already been demonstrated in clinical trials, showing minocycline to be safe and potentially effective in ischemic stroke. Here we investigate the direct effects of minocycline and of proinflammatory cytokines on the differentiation potential of NSCs in vitro and in vivo. Primary fetal rat NSCs were treated with minocycline plus a combination of the proinflammatory cytokines tumor necrosis factor-alpha, interleukin 1 beta, and interleukin 6. The differentiation fate of NSCs was assessed immunocytochemically. To investigate the effects of minocycline and inflammation in vivo, minocycline or lipopolysaccharides were injected intraperitoneally into adult rats, with subsequent immunohistochemistry. Minocycline alone did not affect the differentiation potential of NSCs in vivo or in vitro. In contrast, proinflammatory cytokines accelerated the differentiation of NSCs, promoting an astrocytic fate while inhibiting neurogenesis in vitro and in vivo. It is interesting to note that minocycline counteracted this cytokine-induced rapid astrocytic differentiation and restored the neurogenic and oligodendrogliogenic potential of NSCs. Data suggest that minocycline antagonizes the rapid glial differentiation induced by proinflammatory cytokines following cerebral ischemia but without having a direct effect on the differentiation potential of NSCs. Thus, minocycline constitutes a promising drug for stroke research, counteracting the detrimental effects of postischemic neuroinflammation in multiple ways. (c) 2015 Wiley Periodicals, Inc

Recombinant production of A1S_0222 from Acinetobacter baumannii ATCC 17978 and confirmation of its DNA-(adenine N6)-methyltransferase activity

Acinetobacter baumannii appears as an often multidrug-resistant nosocomial pathogen in hospitals worldwide. Its remarkable persistence in the hospital environment is probably due to intrinsic and acquired resistance to disinfectants and antibiotics, tolerance to desiccation stress, capability to form biofilms, and is possibly facilitated by surface-associated motility. Our attempts to elucidate surface-associated motility in A. baumannii revealed a mutant inactivated in a putative DNA-(adenine N6)-methyltransferase, designated A1S_0222 in strain ATCC 17978. We recombinantly produced A1S_0222 as a glutathione S-transferase (GST) fusion protein and purified it to near homogeneity through a combination of GST affinity chromatography, cation exchange chromatography and PD-10 desalting column. Furthermore we demonstrate A1S_0222-dependent adenine methylation at a GAATTC site. We propose the name AamA (Acinetobacteradenine methyltransferase A) in addition to the formal names M.AbaBGORF222P/M.Aba17978ORF8565P. Small angle X-ray scattering (SAXS) revealed that the protein is monomeric and has an extended and likely two-domain shape in solution