To enhance the production of isoprene, a volatile 5-carbon hydrocarbon, in the Gram-positive spore-forming rod-shaped bacterium Bacillus subtilis, 1-deoxy-D-xylulose-5-phosphate synthase (Dxs) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) were overexpressed in B. subtilis DSM 10. For the strain that overexpresses Dxs, the yield of isoprene was increased 40% over that by the wild-type strain. In the Dxr overexpression strain, the level of isoprene production was unchanged. Overexpression of Dxr together with Dxs showed an isoprene production level similar to that of the Dxs overproduction strain. The effects of external factors, such as stress factors including heat (48°C), salt (0.3 M NaCl), ethanol (1%), and oxidative (0.005% H 2 O 2 ) stress, on isoprene production were further examined. Heat, salt, and H 2 O 2 induced isoprene production; ethanol inhibited isoprene production. In addition, induction and repression effects are independent of SigB, which is the general stress-responsive alternative sigma factor of Gram-positive bacteria.Isoprene, also known as 2-methyl-1,3-butadiene, is a volatile 5-carbon pure hydrocarbon. Derived from biomass through a biochemical process, isoprene can be utilized as a fossil fuel alternative and a platform chemical for the production of highvalue biobased chemicals, such as rubber, elastomers, and isoprenoid medicines (17, 39). Isoprene has several advantages over ethanol. First, it is much easier to separate from the fermentation broth, since it is present in the upper gas phase of a fermentor due to its low boiling point (34°C) and low solubility in water. Second, bacteria are more tolerant to isoprene than to ethanol (the MIC of isoprene for Bacillus subtilis strain DSM 10 is more than 10%, which is higher than the MIC of ethanol according to our unpublished data). Third, isoprene is a highly versatile molecule that can be biochemically and thermochemically re-formed into more complicated products.Isoprene is produced both by eukaryotes and by prokaryotes, including humans, plants, yeasts, and bacteria (18). Two pathways in isoprene biosynthesis have been discovered: the mevalonate (MVA) pathway and the nonmevalonate route, or the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway ( Fig. 1) (13, 39). The MVA pathway is present in eukaryotes, archaea, and the cytosol of higher plants; the DXP pathway is present in most bacteria, green algae, and the chloroplasts of higher plants (18). B. subtilis, the host in the present study, uses the DXP pathway to produce isoprene ( Fig. 1) (37). The DXP pathway initiates with the glycolytic intermediates pyruvate and glyceraldehyde-3-phosphate (G3P). Pyruvate and G3P are condensed to form DXP by 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), and subsequently, 6 enzymes, encoded by the ispC (dxr), ispD, ispE, ispF, ispG, and ispH genes, catalyze sequential reactions converting DXP to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (39). IPP and its isomer DMAPP can be interconverted by isopentenyl pyrophosphat...

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The phosphotransferase system regulation domain (PRD)-containing activator, ManR, is required for glucose-controlled transcription of the mannose permease two (mpt) operon in Listeria innocua. His-871 in ManR PRD-II is needed for mpt repression in glucose-free media. His-506 in PRD-I is needed for mpt induction by glucose.

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A focal defect of the mastoid bone shell overlying the transverse-sigmoid sinus, with no abnormality of the sinus itself, may be a new cause of pulsatile tinnitus. Surgical reconstruction of the mastoid bone shell overlying the transverse-sigmoid sinus can provide lasting symptom relief for patients with pulsatile tinnitus and computed tomographic evidence of the defect.

To identify genes that are important for class IIa bacteriocin interaction and resistance in Listeria species, transposon Tn917 knockout libraries were constructed for Listeria innocua strain Lin11 and screened for mutants that are resistant to pediocin AcH. A highly resistant mutant (G7) (MIC > 20 g/ml; 1,000-fold less susceptible than the wild type), in which the transposon integrated into the putative promoter of the lin0142 gene, was isolated. lin0142 is located immediately upstream of the mpt operon (mptA/mptC/mptD) that encodes the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system permease EII t Man , which serves as a docking protein for class IIa bacteriocins. The transcription of the mpt operon is known to be positively controlled by 54 factor and ManR (a 54 -associated activator). Transcripts for lin0142 and mpt were undetectable in the G7 mutant, based on quantitative real-time reverse transcriptase PCR analysis. When the wild-type lin0142 gene was expressed at a 7.9-fold-elevated level in the mutant via a multicopy-number plasmid, the level of mpt mRNA became 70% higher than that in the wild-type strain. In addition, the complementation strain reverted back to the pediocin AcH-susceptible phenotype. The levels of manR and rpoN ( 54 ) mRNAs were not directly influenced by the level of lin0142 transcription. lin0142 is the only one of the three mpt regulatory genes whose transcription is induced, albeit slightly (1.2-fold), by glucose. The combined results show that the lin0142 gene encodes a novel activator of the mpt operon. The Lin0142 protein contains a winged-helix DNA-binding motif and is distantly related to the Crp-Fnr family of transcription regulators.

The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes, as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding system level regulation and control of the pathway. To address these limitations, we examined Bacillus subtilis grown under multiple conditions and determined the relationship between altered isoprene production and gene expression patterns. We found that with respect to the amount of isoprene produced, terpenoid genes fall into two distinct subsets with opposing correlations. The group whose expression levels positively correlated with isoprene production included dxs, which is responsible for the commitment step in the pathway, ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome-wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. These analyses showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model that accurately predicts production of this secondary metabolite across many simulated environmental conditions.

Chemical compositions of soil samples are multivariate in nature and provide datasets suitable for the application of multivariate factor analytical techniques. One of the analytical techniques, the positive matrix factorization (PMF), uses a weighted least square by fitting the data matrix to determine the weights of the sources based on the error estimates of each data point. In this research, PMF was employed to apportion the sources of heavy metals in 104 soil samples taken within a 1-km radius of a lead battery plant contaminated site in Changxing County, Zhejiang Province, China. The site is heavily contaminated with high concentrations of lead (Pb) and cadmium (Cd). PMF successfully partitioned the variances into sources related to soil background, agronomic practices, and the lead battery plants combined with a geostatistical approach. It was estimated that the lead battery plants and the agronomic practices contributed 55.37 and 29.28%, respectively, for soil Pb of the total source. Soil Cd mainly came from the lead battery plants (65.92%), followed by the agronomic practices (21.65%), and soil parent materials (12.43%). This research indicates that PMF combined with geostatistics is a useful tool for source identification and apportionment.