Interbacterial macromolecular transfer by the Campylobacter fetus subsp. venerealis type IV secretion system

We report here the first demonstration of intra- and interspecies conjugative plasmid DNA transfer for Campylobacter fetus. Gene regions carried by a Campylobacter coli plasmid were identified that are sufficient for conjugative mobilization to Escherichia coli and C. fetus recipients. A broader functional range is predicted. Efficient DNA transfer involves the virB9 and virD4 genes of the type IV bacterial secretion system encoded by a pathogenicity island of C. fetus subsp. venerealis. Complementation of these phenotypes from expression constructions based on the promoter of the C. fetus surface antigen protein (sap) locus was temperature dependent, and a temperature regulation of the sap promoter was subsequently confirmed under laboratory conditions. Gene transfer was sensitive to surface or entry exclusion functions in potential recipient cells carrying IncPα plasmid RP4 implying functional relatedness to C. fetus proteins. The virB/virD4 locus is also known to be involved in bacterial invasion and killing of cultured human cells in vitro. Whether specifically secreted effector proteins contribute to host colonization and infection activities is currently unknown. Two putative effector proteins carrying an FIC domain conserved in a few bacterial type III and type IV secreted proteins of pathogens were analyzed for secretion by the C. fetus or heterologous conjugative systems. No evidence for interbacterial translocation of the Fic proteins was found.     

MenD as a versatile catalyst for asymmetric synthesis

The thiamine diphosphate (ThDP)-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (MenD) from Escherichia coli K12, formerly known as SHCHC-synthase, catalyses the decarboxylation of α-ketoglutarate and the subsequent addition of the resulting succinyl-THDP to isochorismate. Here, the enzyme is tested for unphysiologial C–C bond-forming reactions.Condensation of α-ketoglutarate after decarboxylation to a broad range of aldehydes gave α-hydroxyketones with isolated yields from 26 to 87% and 94 to 98% ee for addition to aromatic aldehydes. MenD accepts a wide range of aldehydes as acceptor substrates to produce chiral α-hydroxyketones with conserved regioselectivity where the activated succinylsemialdehyde serves selectively as the donor. Regioselectivity is inverted only for condensation of α-ketoglutarate with pyruvate (activated acetaldehyde) as donor. Besides α-ketoglutarate, pyruvate and oxalacetate are accepted as donors in combination with benzaldehyde and 2-fluorobenzaldehyde as acceptors, however with decreased activity of C–C bond formation.The physiological 1,4-addition of α-ketoglutarate to isochorismate was investigated for acceptor substrate variability. (2S,3S)-2,3-Dihydroxy-2,3-dihydrobenzoate (2,3-CHD), which lacks the pyruvyl found in isochorismate, is converted to (5S,6S)-2-succinyl-5,6-dihydroxycyclohex-2-enecarboxylate. In contrast to the addition to carbonyls, the active site of MenD does appear to impose specific constraints on the acceptor substrate for 1,4-addition with α,β-unsaturated carboxylic acids.     

Expression of an L-alanine dehydrogenase gene in Zymomonas mobilis and excretion of L-alanine.

An approach to broaden the product range of the ethanologenic, gram-negative bacterium Zymomonas mobilis by means of genetic engineering is presented. Gene alaD for L-alanine dehydrogenase (EC 1.4.1.1.) from Bacillus sphaericus was cloned and introduced into Z. mobilis. Under the control of the strong promoter of the pyruvate decarboxylase (pdc) gene, the enzyme was expressed up to a specific activity of nearly 1 mu mol . min -1 . mg of protein -1 in recombinant cells. As a results of this high L-alanine dehydrogenase activity, growing cells excreted up to 10 mmol of alanine per 280 mmol of glucose utilized into a mineral salts medium. By the addition of 85 mM NH4+ to the medium, growth of the recombinant cells stopped, and up to 41 mmol alanine was secreted. As alanine dehydrogenase competed with pyruvate decarboxylase (PDC) (EC 4.1.1.1.) for the same substrate (pyruvate), PDC activity was reduced by starvation for the essential PDC cofactor thiamine PPi. A thiamine auxotrophy mutant of Z. mobilis which carried the alaD gene was starved for 40 h in glucose-supplemented mineral salts medium and then shifted to mineral salts medium with 85 mM NH4+ and 280 mmol of glucose. The recombinants excreted up to 84 mmol of alanine (7.5 g/liter) over 25 h. Alanine excretion proceeded at an initial velocity of 238 nmol . min-1 . mg [dry weight]-1. Despite this high activity, the excretion rate seemed to be a limiting factor, as the intracellular concentration of alanine was as high as 260 mM at the beginning of the excretion phase and decreased to 80 to 90 mM over 24 h.     

Altered velocity processing in schizophrenia during pursuit eye tracking

Smooth pursuit eye movements (SPEM) are needed to keep the retinal image of slowly moving objects within the fovea. Depending on the task, about 50%-80% of patients with schizophrenia have difficulties in maintaining SPEM. We designed a study that comprised different target velocities as well as testing for internal (extraretinal) guidance of SPEM in the absence of a visual target. We applied event-related fMRI by presenting four velocities (5, 10, 15, 20°/s) both with and without intervals of target blanking. 17 patients and 16 healthy participants were included. Eye movements were registered during scanning sessions. Statistical analysis included mixed ANOVAs and regression analyses of the target velocity on the Blood Oxygen Level Dependency (BOLD) signal. The main effect group and the interaction of velocity×group revealed reduced activation in V5 and putamen but increased activation of cerebellar regions in patients. Regression analysis showed that activation in supplementary eye field, putamen, and cerebellum was not correlated to target velocity in patients in contrast to controls. Furthermore, activation in V5 and in intraparietal sulcus (putative LIP) bilaterally was less strongly correlated to target velocity in patients than controls. Altered correlation of target velocity and neural activation in the cortical network supporting SPEM (V5, SEF, LIP, putamen) implies impaired transformation of the visual motion signal into an adequate motor command in patients. Cerebellar regions seem to be involved in compensatory mechanisms although cerebellar activity in patients was not related to target velocity.     

Der Einfluß von Metachromasie und Photodekomposition auf die quantitative Feulgen-DNS Fluoreszenzcytophotometrie

Zusammenfassung Zur quantitativen Bestimmung der Feulgen-DNS-Mengen wurden Leberzellkerne mit Acriflavin fluorochromiert. Durch fluoreszenzmikrospektrographische Untersuchung konnte gezeigt werden, daß die Meßergebnisse nicht durch Metachromasie beeinträchtigt werden. Bei der Photodekomposition kommt es zu keiner Verschiebung der relativen spektralen Intensitätsverteilung. Die Histogramme der Feulgen-DNS-Mengen von di- und tetraploiden Leberzellen zeigen die exakte Einhaltung der Ploidiestufen und eine geringe Streuung der Meßwerte.

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Influence of metachromasia and photofading on the quantitative Feulgen-Dna fluorescence cytophotometry

Summary For the purpose of quantitative determination of Feulgen-DNA liver nuclei had been stained with fluorescent Schiff-type dye Acriflavin according to Böhm and Sprenger (1968). Fluorescence microspectrophotometric investigations revealed that the quantitative measuring results are not falsified by metachromasia and that photofading does not cause a shift in the relative spectral distribution of fluorescence intensity.The Feulgen-DNA histograms of di- and tetraploid liver nuclei confirm the accuracy of quantitative measurements by exact reproduction of ploidy classes and low variation range in each class.

     

Fairy wrasses perceive and respond to their deep red fluorescent coloration

Fluorescence enables the display of wavelengths that are absent in the natural environment, offering the potential to generate conspicuous colour contrasts. The marine fairy wrasse Cirrhilabrus solorensis displays prominent fluorescence in the deep red range (650–700 nm). This is remarkable because marine fishes are generally assumed to have poor sensitivity in this part of the visual spectrum. Here, we investigated whether C. solorensis males can perceive the fluorescence featured in this species by testing whether the presence or absence of red fluorescence affects male–male interactions under exclusive blue illumination. Given that males respond aggressively towards mirror-image stimuli, we quantified agonistic behaviour against mirrors covered with filters that did or did not absorb long (i.e. red) wavelengths. Males showed significantly fewer agonistic responses when their fluorescent signal was masked, independent of brightness differences. Our results unequivocally show that C. solorensis can see its deep red fluorescent coloration and that this pattern affects male–male interactions. This is the first study to demonstrate that deep red fluorescent body coloration can be perceived and has behavioural significance in a reef fish.     

Fructan of the inulin neoseries is synthesized in transgenic chicory plants (Cichorium intybus L.) harbouring onion (Allium cepa L.) fructan:fructan 6G-fructosyltransferase

Fructan (polyfructosylsucrose) is an important storage carbohydrate in many plant families. fructan:fructan 6G-fructosyltransferase (6G-FFT) is a key enzyme in the formation of the inulin neoseries, a type of fructan accumulated by members of the Liliales. We have cloned the 6G-FFT from onion by screening a cDNA library using barley sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. The deduced amino acid sequence showed a high homology with plant invertases and 6-SFT. Incubation of protein extracts from transgenic tobacco plants with the trisaccharide 1-kestose and sucrose resulted in the formation of neokestose and fructans of the inulin neoseries with a degree of polymerization up to six. Introduction of the onion 6G-FFT into chicory resulted in the synthesis of fructan of the inulin neoseries, in addition to the synthesis of linear inulin.