Regulation of Riboflavin Biosynthesis in Bacillus subtilis Is Affected by the Activity of the Flavokinase/Flavin Adenine Dinucleotide Synthetase Encoded by ribC

This work shows that the ribC wild-type gene product has both flavokinase and flavin adenine dinucleotide synthetase (FAD-synthetase) activities. RibC plays an essential role in the flavin metabolism of Bacillus subtilis, as growth of a ribC deletion mutant strain was dependent on exogenous supply of FMN and the presence of a heterologous FAD-synthetase gene in its chromosome. Upon cultivation with growth-limiting amounts of FMN, this ribC deletion mutant strain overproduced riboflavin, while with elevated amounts of FMN in the culture medium, no riboflavin overproduction was observed. In a B. subtilis ribC820 mutant strain, the corresponding ribC820 gene product has reduced flavokinase/FAD-synthetase activity. In this strain, riboflavin overproduction was also repressed by exogenous FMN but not by riboflavin. Thus, flavin nucleotides, but not riboflavin, have an effector function for regulation of riboflavin biosynthesis in B. subtilis, and RibC seemingly is not directly involved in the riboflavin regulatory system. The mutation ribC820 leads to deregulation of riboflavin biosynthesis in B. subtilis, most likely by preventing the accumulation of the effector molecule FMN or FAD.     

Eye Movement Deficits Are Consistent with a Staging Model of pTDP-43 Pathology in Amyotrophic Lateral Sclerosis

Background

The neuropathological process underlying amyotrophic lateral sclerosis (ALS) can be traced as a four-stage progression scheme of sequential corticofugal axonal spread. The examination of eye movement control gains deep insights into brain network pathology and provides the opportunity to detect both disturbance of the brainstem oculomotor circuitry as well as executive deficits of oculomotor function associated with higher brain networks.

Objective

To study systematically oculomotor characteristics in ALS and its underlying network pathology in order to determine whether eye movement deterioration can be categorized within a staging system of oculomotor decline that corresponds to the neuropathological model.

Methods

Sixty-eight ALS patients and 31 controls underwent video-oculographic, clinical and neuropsychological assessments.

Results

Oculomotor examinations revealed increased anti- and delayed saccades’ errors, gaze-palsy and a cerebellary type of smooth pursuit disturbance. The oculomotor disturbances occurred in a sequential manner: Stage 1, only executive control of eye movements was affected. Stage 2 indicates disturbed executive control plus ‘genuine’ oculomotor dysfunctions such as gaze-paly. We found high correlations (p<0.001) between the oculomotor stages and both, the clinical presentation as assessed by the ALS Functional Rating Scale (ALSFRS) score, and cognitive scores from the Edinburgh Cognitive and Behavioral ALS Screen (ECAS).

Conclusions

Dysfunction of eye movement control in ALS can be characterized by a two-staged sequential pattern comprising executive deficits in Stage 1 and additional impaired infratentorial oculomotor control pathways in Stage 2. This pattern parallels the neuropathological staging of ALS and may serve as a technical marker of the neuropathological spreading.     

Mzabimyces algeriensis gen. nov., sp. nov., a halophilic filamentous actinobacterium isolated from a Saharan soil,and proposal of Mzabimycetaceae fam. nov.

Three halophilic mycelium-forming actinobacteria, strains H195^T, H150 and H151, were isolated from a Saharan soil sample collected from Béni-isguen in the Mzab region (Ghardaïa, South of Algeria) and subjected to a polyphasic taxonomic characterisation. These strains were observed to show an aerial mycelium differentiated into coccoid spore chains and fragmented substrate mycelium. Comparative analysis of the 16S rRNA gene sequences revealed that the highest sequence similarities were to Saccharopolyspora qijiaojingensis YIM 91168^T (92.02 % to H195^T). Phylogenetic analyses showed that the strains H195^T, H150 and H151 represent a distinct phylogenetic lineage. The cell-wall hydrolysate was found to contain meso-diaminopimelic acid, and the diagnostic whole-cell sugars were identified as arabinose and galactose. The major cellular fatty acids were identified as iso-C_15:0, iso-C_16:0, iso-C_17:0 and anteiso-C_17:0. The diagnostic phospholipid detected was phosphatidylcholine and MK-9 (H₄) was found to be the predominant menaquinone. The genomic DNA G+C content of strain H195^T was 68.2 mol%. On the basis of its phenotypic features and phylogenetic position, we propose that strain H195^T represents a novel genus and species, Mzabimyces algeriensis gen. nov., sp. nov., within a new family, Mzabimycetaceae fam. nov. The type strain of M. algeriensis is strain H195^T (=DSM 46680^T = MTCC 12101^T).     

Nocardia vulneris sp. nov., isolated from wounds of human patients in North America

Nocardia species are ubiquitous in the environment with an increasing number of species isolated from clinical sources. From 2005 to 2009, eight isolates (W9042, W9247, W9290, W9319, W9846, W9851^T, W9865, and W9908) were obtained from eight patients from three states in the United States and Canada; all were from males ranging in age from 47 to 81 years old; and all were obtained from finger (n = 5) or leg (n = 3) wounds. Isolates were characterized by polyphasic analysis using molecular, phenotypic, morphologic and chemotaxonomic methods. Sequence analysis of 16S rRNA gene sequences showed the eight isolates are 100 % identical to each other and belong in the genus Nocardia. The nearest phylogenetically related neighbours were found to be the type strains for Nocardia altamirensis (99.33 % sequence similarity), Nocardia brasiliensis (99.37 %), Nocardia iowensis (98.95 %) and Nocardia tenerifensis (98.44 %). The G+C content of isolate W9851^T was determined to be 68.4 mol %. The DNA–DNA relatedness between strain W9851^T and the N. brasiliensis type strain was 72.8 % and 65.8 % when measured in the laboratory and in silico from genome sequences, respectively, and 95.6 % ANI. Whole-cell peptidoglycan was found to contain meso-diaminopimelic acid; MK-8-(H₄)_ω-cyc was identified as the major menaquinone; the major fatty acids were identified as C_16:0, 10 Me C_18:0, and C_{18:1 w9c}, the predominant phospholipids were found to include diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides; whole-cell sugars detected were arabinose and galactose; and mycolic acids ranging from 38 to 60 carbon atoms were found to be present. These chemotaxonomic analyses are consistent with assignment of the isolates to the genus Nocardia. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra of the clinical isolates showed genus and species level profiles that were different from other Nocardia species. All isolates were resistant to ciprofloxacin, clarithromycin and imipenem but were susceptible to amikacin, amoxicillin/clavulanate, linezolid and trimethoprim/sulfamethoxazole. The results of our polyphasic analysis suggest the new isolates obtained from wound infections represent a novel species within the genus Nocardia, for which the name Nocardia vulneris sp. nov. is proposed, with strain W9851^T (= DSM 45737^T = CCUG 62683^T = NBRC 108936^T) as the type strain.     

Critical Evaluation of the Volumetric “Bottle Effect” on Microbial Batch Growth

We have analyzed the impact of surface-to-volume ratio on final bacterial concentrations after batch growth. We examined six bottle sizes (20 to 1,000 ml) using three independent enumeration methods to quantify growth. We found no evidence of a so-called volumetric bottle effect, thus contradicting numerous previous reports.Microbial batch growth during confined incubation in bottles of various sizes is used daily in a broad variety of microbiological studies and methods, including bioassays such as the assimilable organic carbon (AOC) assay (6, 10, 18) and the analysis of pure culture or microbial community growth in freshwater (3, 11, 19, 20). In this context, “bottle effect” or “volume effect” is a term that has cropped up frequently in aquatic microbiology papers (e.g., references 12, 13, and 21) during the last 100 years to explain inexplicable phenomena and variations in results obtained from such batch growth studies. The uncertainty surrounding this apparent effect was clearly summarized in a recent paper by Pernthaler and Amann (16): “Such investigations are often plagued by the mysterious ‘bottle effect’, a hard-to-define concept that reflects the worry of whether phenomena observed in confined assemblages are nonspecific consequences of the confinement rather than a result of the planned manipulation.” The “bottle effect” alludes to an apparent reaction of bacteria to batchwise incubation in a confined environment, and this concept has intermittently been linked to influences on final cell concentrations (3) and grazing/bacterivory (13), a change in viability/activity parameters (9), a change in cultivability (5), and a change in population composition (1).The fact that microbiological processes during confined incubation differ from those in the environment is indisputable. However, a particular section of “bottle effect” literature focuses specifically on a volumetric “bottle effect”, where the above-mentioned effects are linked specifically to the size (or surface-to-volume ratio) of the incubation vessel (3, 8, 11-13, 15, 21). One of the oldest and best-known studies summarized clearly: “It will be observed that the densest bacterial populations appear in the bottles of water which offer the largest area of glass surface per unit volume of water” (21). This idea has established itself as dogma during the last century, with only a few differing opinions (4). However, precious little empirical data that actually quantify and explain the volumetric “bottle effect” are ever presented. In one example, Bischofberger et al. (3) observed that incubation of groundwater led to significantly more growth (about 2 log units) in small bottles (100 ml) than in big ones (10 liters). More often, however, the “bottle effect” is merely mentioned, as if it is self-explanatory and indisputable (2, 11, 12). In the present study, we took a simple but detailed look at the effect of bottle size on the outcome of short-term (<5-day) batch growth assays and compared the data critically to information in the literature and current opinion on this topic.Three batch growth experiments were conducted to assess the volumetric bottle effect on final cell concentrations after growth into stationary phase. Six different bottle sizes were used, covering the ranges most often reported in “bottle effect” literature. All glassware and Teflon-coated caps were cleaned comprehensively as described elsewhere (6) to remove any traces of organic carbon that might have been present on surfaces. The bottle sizes were as follows (water volumes and surface area-to-volume ratios [square centimeters to milliliters] are respectively included in parentheses): 1,000 ml (900 ml, 0.3:1), 500 ml (400 ml, 0.4:1), 250 ml (200 ml, 0.6:1), 100 ml (90 ml, 0.8:1), 40 ml (35 ml, 1.5:1), and 20 ml (15 ml, 2.4:1). In the first experiment, a sample of natural river water (dissolved organic carbon [DOC], 3.8 mg/liter; AOC, 0.3 mg/liter) from a small oligotrophic stream was obtained, filter sterilized with a 50-kDa dialysis filter (Fresenius Medical Care), and inoculated (at 10³ cells/ml) with a microbial community used for AOC assays (19). In the second experiment, a sample of the effluent (DOC, 1.2 mg/liter; AOC, 0.03 mg/liter; total cell concentration [TCC], 3 × 10⁵ cells/ml) from a granulated active carbon filter situated in a drinking water pilot plant (7) was collected and used directly for the experiment without additional treatment or inoculation. For the third experiment, sterile Luria-Bertani (LB) medium (diluted 1:10,000; DOC, 0.7 mg/liter; AOC, 0.46 mg/liter) was inoculated with Vibrio cholerae O1 (10³ cells/ml) as described previously (19). The water from each experiment was distributed into triplicate flasks of each size and incubated (at 30°C) until stationary phase was reached. Stationary phase was indicated by no significant increase in the TCC (measured after 3, 4, and 5 days) on consecutive days. Samples from all experiments were analyzed (i) for TCCs after being stained with SYBR green I and subjected to flow cytometry (7, 19), (ii) for ATP by using a commercial luciferin-luciferase assay (Promega Corporation) (7), and (iii) for heterotrophic plate counts (HPC) on R2A agar by a pour plate method with incubation at 30°C for 10 days. Possible biofilm growth was checked by applying sonication to selected samples. However, no wall growth in bottles of any size was observed.Growth was observed in all three experiments. The results show the net growth after subtraction of the initial cell/ATP/HPC concentrations from the final concentrations (Fig. ​(Fig.1).1). The proposed concept of the volumetric bottle effect implies that more growth should occur in smaller bottles. All data sets were subjected to regression analysis, and we observed no significant correlation (P < 0.01) between bottle size and final growth in any of the experiments by any of the three independent methods used for quantification. Figure ​Figure1A1A shows the batch growth results for a natural microbial community in prefiltered river water. This experimental setup is reflective of a typical AOC assay (6) or batch cultivation of natural microbial communities (20). Figure ​Figure1B1B shows the results for direct incubation of a treated drinking water sample. This sample and experimental setup were chosen specifically to assess any potential volumetric “bottle effect” on an indigenous microbial community in a biologically stable water sample, where only limited growth is expected. Indeed, the final cell concentration in the sample was only about 25% higher than the original cell concentration. The cultivability (HPC/TCC × 100) at day 0 was 0.4%, and at the end of the experimental period it had increased to 2.5%. This points to increased cultivability as a result of growth during confinement (5), yet it does not relate at all to the size of the incubation vessel. Figure ​Figure1C1C shows the data for V. cholerae grown in sterile LB medium (diluted 1:10,000) to stationary phase. Again, this particular setup is of specific relevance since a recently published paper on the growth of V. cholerae referred directly to the volumetric “bottle effect” to explain rather large differences between growth results from two separate studies (11, 19). The data from Fig. ​Fig.1C1C suggest at least that a “bottle effect” should be ruled out as an interfering factor in this case.Open in a separate windowFIG. 1.Effects of bottle size on bacterial batch growth of a natural microbial community in filter-sterilized surface water (A), growth of bacteria during direct incubation of water from a drinking water treatment plant (B), and batch growth of a V. cholerae pure culture in diluted LB medium (C). Growth (expressed as the net growth) was quantified by flow cytometric total cell counting (circles), total ATP analysis (diamonds), and conventional plating (squares). All data points represent averages of triplicate measurements.The results presented in this study clearly dispute the concept of a volumetric “bottle effect” on the outcome of short-term batch growth assays, be it for pure cultures or natural microbial communities. These findings contradict evidence reported by many other researchers (3, 8, 11-13, 15, 21). Although the volumetric “bottle effect” is often cited as a somewhat mysterious occurrence, it is imperative that clear experimental data are required for the critical appraisal thereof. The main experimental theory behind the phenomenon is that organic carbon adsorbs to clean glass surfaces, thus locally concentrating the carbon and creating more favorable growth conditions (2, 14). This adsorption and the fact that bacteria can utilize such adsorbed carbon have been demonstrated experimentally (14). What has, in our opinion, not been shown conclusively is that these effects can be so dramatic that they would alter the growth of samples to the extent that different sizes of bottles would render different final cell numbers after growth. Since we have not observed any volumetric “bottle effect” in our work, we can only speculate on the possible reasons why this has been observed previously. One explanation may be that glassware contaminated with organic carbon can contribute to the perception of a volumetric “bottle effect,” as large surface-to-volume ratios (found in small bottles) would account for increased contamination compared to that in bottles with smaller ratios. Hence, more additional available carbon would be introduced into smaller bottles, giving rise to higher final cell numbers after growth. In this context, it is essential that a comprehensive glassware-cleaning protocol be followed, including heating to a high temperature (>500°C) and storage away from volatile organics (6). In addition, it is important that such experiments at low carbon concentrations are complemented with the inclusion of correct and sensitive controls to assess potential organic carbon contamination. For example, the use of deionized water as a negative control should be avoided, since the absence of inorganic nutrients is bound to lead to no growth and thus false-negative results (10). A good negative control would be water that is only carbon limited, e.g., bottled drinking water (17). Moreover, the use of multiple tools for analyzing growth, including cultivation-independent methods, is encouraged.In conclusion, we did not observe evidence of a volumetric bottle effect on short-term (<5-day) batch incubations. The findings of this study suggest that reference to the so-called volumetric bottle effect should be considered carefully unless supported by clear experimental data. This study does not dispute the fact that many authors have observed results implying apparent bottle effects during growth studies, but it questions the interpretation and understanding of this concept and the random use of the term “bottle effect” to explain uncertainty in results, specifically in relation to bottle size. Hopefully, these data will assist with experimental setups and comparison of data among different groups and stimulate discussion of and future research on this interesting, but slightly controversial, topic.     

Estimates of dietary threshold levels for crude protein and amino acids to obtain plateau values of apparent ileal crude protein and amino acid digestibilities in newly weaned pigs

This study was conducted to estimate dietary threshold levels for crude protein (CP) and amino acids (AA) at which apparent ileal digestibilities (AID) of CP and AA in assay diets for newly weaned pigs reach plateaus. A total of 14 (12 + 2 for replacement) three-week old barrows were fitted with simple T cannulas at the distal ileum. Corn starch-based diets containing six graded levels of CP from casein, 90, 155, 220, 285, 350, or 415 g CP/kg assay diet (as-fed), were formulated. At 28 days of age, the pigs were randomly allocated to the six dietary treatments with two pigs per CP level in four weekly repeated measurement periods. They were fed twice daily a total of 30 g (as-fed) per kg of individual body weight at 8:00 and 20:00 h. The dietary CP and AA levels affected AID of CP and most AA (p = 0.005 to p = 0.040) in the assay diets. The AID of CP and AA were higher at 155 and 220 compared to 90 g CP/kg assay diet (p < 0.001 to p = 0.047). Initially, the AID of CP and AA increased sharply then gradually reached, at individual break points (p < 0.001 to p = 0.047), their plateaus (p < 0.001), which did not change up to dietary CP levels of 415 g/kg assay diet and the AID values became independent of the dietary AA levels. The piglets' capacity to digest CP and absorb AA was not limiting under these experimental conditions. There was no effect of age on AID of CP and AA (p = 0.056 to p = 0.899) except for a linear increase (p = 0.045) in AID of glycine from Period 1 to 4. Segmented quadratic with plateau relationships between the AID of CP and AA and their dietary contents were fitted for CP and each AA. The lower end points of 95% confidence intervals of the plateau AID values were defined to represent the initial plateau AID. The dietary CP and AA contents, corresponding to the initial plateau AID values, represent the dietary threshold levels. For CP and the indispensable amino acids, the plateau AID [%] and the dietary threshold levels [g/kg DM], respectively, in casein were: CP, 94.2 and 176; arginine, 95.1 and 7; histidine, 96.0 and 5; isoleucine, 96.4 and 8; leucine, 96.8 and 16; lysine, 96.8 and 12; methionine, 97.9 and 5; phenylalanine, 96.2 and 10; threonine, 93.4 and 9; tryptophan, 94.3 and 2; valine, 95.9 and 11. For the determination of plateau AID in piglets, the crude protein and amino acid contents in the assay diets should meet or exceed the corresponding dietary threshold levels.     

Background correction of two-colour cDNA microarray data using spatial smoothing methods

The analysis of two-colour cDNA microarray data usually involves subtracting background values from foreground values prior to normalization and further analysis. This approach has the advantage of reducing bias and the disadvantage of blowing up the variance of lower abundant spots. Whenever background subtraction is considered, it implicitly assumes locally constant background values. In practice, this assumption is often not met, which casts doubts on the usefulness of simple background subtraction. In order to improve background correction, we propose local background smoothing within the pre-processing pipeline of cDNA microarray data prior to background correction. For this purpose, we employ a geostatistical framework with ordinary kriging using both isotropic and anisotropic models of spatial correlation and 2-D locally weighted regression. We show that application of local background smoothing prior to background correction is beneficial in comparison to using raw background estimates. This is done using data of a self-versus-self experiment in Arabidopsis where subsets of differentially expressed genes were simulated. Using locally smoothed background values in conjunction with existing background correction methods increases the power, increases the accuracy and decreases the number of false positive results.     

Nonadditive protein accumulation patterns in Maize (Zea mays L.) hybrids during embryo development

Heterosis describes the superior performance of heterozygous F(1)-hybrid plants compared to their homozygous parental inbred lines. In the present study, heterosis was detected for length, weight, and the time point of seminal root primordia initiation in maize (Zea mays L.) embryos of the reciprocal F(1)-hybrids UH005xUH250 and UH250xUH005. A two-dimensional gel electrophoresis (2-DE) proteome survey of the most abundant proteins of the reciprocal hybrids and their parental inbred lines 25 and 35 days after pollination revealed that 141 of 597 detected proteins (24%) exhibited nonadditive accumulation in at least one hybrid. Approximately 44% of all nonadditively accumulated proteins displayed an expression pattern that was not distinguishable from the low parent value. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) analyses and subsequent functional classification of the 141 proteins revealed that development, protein metabolism, redox-regulation, glycolysis, and amino acid metabolism were the most prominent functional classes among nonadditively accumulated proteins. In 35-day-old embryos of the hybrid UH250xUH005, a significant up-regulation of enzymes related to glucose metabolism which often exceeded the best parent values was observed. A comparison of nonadditive protein accumulation between rice and maize embryo data sets revealed a significant overlap of nonadditively accumulated proteins suggesting conserved organ- or tissue-specific regulatory mechanisms in monocots related to heterosis.     

d-Ribulokinase from Klebsiella pneumoniae for continuous production of d-(−)-ribulose-5-phosphate

Summary The production of d-ribulose-5-phosphate in an enzyme membrane reactor was examined. Phosphoryl transfer from ATP to d-ribulose was catalysed by d-ribulokinase isolated from Klebsiella pneumoniae. For production of d-ribulose-5-phosphate the phosphoryl donor ATP was used either in stoichiometric or in catalytic amounts. Using catalytic amounts of ATP requires a second enzyme, e.g. pyruvate kinase, to regenerate ATP. The kinetic parameters for d-ribulokinase and pyruvate kinase were determined to calculate the performance of an enzyme membrane reactor for continuous production of d-ribulose-5-phosphate. Both processes operated for more than 200 h. Regardless of whether ATP was used in catalytic or stoichiometric amounts, about the same production parameters were determined. In continuous production space/time yields of 117 g (with ATP regeneration) and 103 g (without ATP regeneration) of d-ribulose-5-phosphate 1^–1 per day were reached.Offprint requests to: D. Gygax