Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labelled DNA probes using a hybridisation protection assay.

DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5' end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×10(2)1×10(3) CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories.

The herpes simplex virus type 1 ICP0 promoter is activated by viral reactivation stimuli in trigeminal ganglia neurons of transgenic mice.

Herpes simplex virus type 1 (HSV-1) causes a latent infection in sensory ganglia neurons in humans and in the mouse model. The ability of the virus to latently infect neurons and reactivate is central to the ability of HSV-1 to remain in the human population and spread to new hosts. It is possible that neuronal transcriptional proteins control latency and reactivation by modulating activation of the HSV-1 immediate-early (IE) gene ICP0. We have previously shown that factors in trigeminal ganglia neurons can differentially activate the IE ICP0 promoter and the IE ICP4 promoter in developing trigeminal ganglia neurons of transgenic mice. Ultraviolet (UV) irradiation and hyperthermic stress have been shown to result in HSV-1 reactivation from sensory neurons in the mouse model. Reporter transgenic mice were exposed to UV irradiation or hyperthermia to test whether stimuli that are known to reactivate HSV-1 could activate viral IE promoters in the absence of viral proteins. Measurement of beta-galactosidase activity in trigeminal ganglia from these transgenic mice indicated that the ICP0 promoter activity was significantly increased by both UV irradiation and hyperthermia. The IE genes ICP4 and ICP27 and the late gene gC reporter transgenes failed to be activated in parallel experiments. These results suggest that the ICP0 promoter is a target for activation by host transcription factors in sensory neurons that have undergone damage. It further suggests the possibility that activation of ICP0 gene expression by neuronal transcription factors may be important in reactivation of HSV-1 in neurons.

The transgenic ICP4 promoter is activated in Schwann cells in trigeminal ganglia of mice latently infected with herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) establishes a latent infection in neurons of sensory ganglia, including those of the trigeminal ganglia. Latent viral infection has been hypothesized to be regulated by restriction of viral immediate-early gene expression in neurons. Numerous in situ hybridization studies in mice and in humans have shown that transcription from the HSV-1 genome in latently infected neurons is limited to the latency-associated transcripts. In other studies, immediate-early gene (ICP4) transcripts have been detected by reverse transcription-PCR (RT-PCR) in homogenates of latently infected trigeminal ganglia of mice. We used reporter transgenic mice containing the HSV-1(F) ICP4 promoter fused to the coding sequence of the beta-galactosidase gene to determine whether neurons in latently infected trigeminal ganglia activated the ICP4 promoter. Mice were inoculated via the corneal route with HSV-1(F). At 5, 11, 23, and 37 days postinfection (dpi), trigeminal ganglia were examined for beta-galactosidase-positive cells. The numbers of beta-galactosidase-positive neurons and nonneuronal cells were similar at 5 dpi. The number of positive neurons decreased at 11 dpi and returned to the level of mock-inoculated transgenic controls at 23 and 37 dpi. The number of positive nonneuronal cells increased at 11 and 23 dpi and remained elevated at 37 dpi. Viral proteins were detected in neurons and nonneuronal cells in acutely infected ganglia, but were not detected in latently infected ganglia. Colabeling experiments confirmed that the transgenic ICP4 promoter was activated in Schwann cells during latent infection. These findings suggest that the cells that express the HSV-1 ICP4 gene in latently infected ganglia are not neurons.

Increased severity of herpes simplex virus type 1-induced keratitis in Hox A5 transgenic mice.

PURPOSE: Herpes simplex virus type 1 is a major cause of stromal keratitis and blindness in humans. Understanding of the role of host genes in the pathogenesis of herpes stromal keratitis is limited. We used a transgenic mouse model to examine the effect of a host gene, Hox A5 (which binds to the TAATGARAT sequence in the promoter regions of HSV-1 immediate early genes and increases HSV-1 replication), on the pathogenesis of HSV-1 induced stromal keratitis. METHODS: Corneas of wildtype and Hox A5 transgenic mice were infected with HSV-1 strain F following corneal scarification. Clinical severity of keratitis was evaluated using slit-lamp biomicroscopy. Histologic severity of keratitis was determined by light microscopic evaluation and by computerized morphometry. Ocular viral replication was measured via plaque assay. RESULTS: Clinical lesions of stromal keratitis were more severe at 17 and 23 days post infection in Hox A5 transgenic mice than in wildtype mice. Histological evaluation and morphometric analysis confirmed that keratitis lesions were more severe in the transgenic mice. HSV-1 replication was approximately100-fold greater in the corneas of transgenic mice than in wildtype mice. CONCLUSIONS: Our results demonstrate that a host gene (Hox A5) can increase ocular replication of HSV-1 and alter the pathogenesis of herpetic stromal keratitis.

Characterization of a maltose transport system in Clostridium acetobutylicum ATCC 824.

The utilization of maltose by Clostridium acetobutylicum ATCC 824 was investigated. Glucose was used preferentially to maltose, when both substrates were present in the medium. Maltose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity was detected in extracts prepared from cultures grown on maltose, but not glucose or sucrose, as the sole carbon source. Extract fractionation and PTS reconstitution experiments revealed that the specificity for maltose is contained entirely within the membrane in this organism. A putative gene system for the maltose PTS was identified (from the C. acetobutylicum ATCC 824 genome sequence), encoding an enzyme II(Mal) and a maltose 6-phosphate hydrolase.

Pathogenesis of herpes simplex virus type 1-induced corneal inflammation in perforin-deficient mice.

Herpetic stromal keratitis (HSK) is an inflammatory disease of the cornea that often results in blindness. It is mediated by a host immune response which is triggered by herpes simplex virus (HSV) infection. Immune effector mechanisms are hypothesized to be important in disease development. We investigated, in a mouse model, whether perforin-dependent cytotoxicity is an important effector mechanism in the production of HSK. Wild-type (C57BL/6) and perforin-deficient (PKO) mice were infected intracorneally with HSV-1 strain F. Clinical disease and histologic lesions of the cornea at 23 days postinfection (p.i.) were significantly less severe in HSV-1-infected PKO mice than in infected wild-type mice. mRNA for the chemokine macrophage inflammatory protein 1alpha (MIP-1alpha) was detected by reverse transcription-PCR in the corneas of infected wild-type mice but not in the corneas of infected PKO mice at 23 days p.i. Adoptive transfer of wild-type HSV-1 immune T-cell-enriched splenocytes into HSV-1-infected PKO mice restored the disease phenotype which was seen in infected wild-type mice. In contrast, mice carrying a null-function mutation in the Fas ligand, which is involved in an alternative cytotoxic mechanism, developed clinical disease and histologic lesions which were comparable to those in wild-type mice. Viral clearance from the eyes of PKO mice was not impaired. There was no significant difference between the infectious viral titers isolated from the eyes of PKO and wild-type mice. Our findings show that perforin is important in the pathogenesis of HSK.

Reliability and validity of a DSM-IV based ADHD screener.

The Diagnostic Rating Scale (DRS) was completed by the parents and teachers of 82 children referred for clinical evaluations, 73 referred children seen twice, and 218 non-referred children from the community. The DRS, which uses a categorical rather than a dimensional rating approach, was 70% to 90% sensitive to diagnoses of Attention Deficit/Hyperactivity Disorder (ADHD) made by blind clinical teams. In research and clinical applications, the DRS could improve screening efficiency, especially in situations where it would be desirable to exclude all children who might have ADHD or identify all children with Hyperactive-Impulsive symptoms. Because of its objectivity and consistency with the Diagnostic and Statistical Manual (DSM)-IV criteria, the DRS could facilitate comparison of participant samples across studies.

Analysis of a catabolic operon for sucrose transport and metabolism in Clostridium acetobutylicum ATCC 824.

The utilization of sucrose by Clostridium acetobutylicum ATCC 824 was investigated. Sucrose was found to be transported via a phosphoenol-pyruvate (PEP)-dependent phosphotransferase system (PTS) and a metabolic pathway identical to that previously identified in C. beijerinckii, was established. The genes encoding the proteins of this pathway were identified from the C. acetobutylicum genome sequence, in the order scrAKB encoding Enzyme II of the sucrose PTS, fructokinase and sucrose 6-phosphate hydrolase respectively. While the pathway for sucrose metabolism is conserved between C. acetobutylicum and C. beijerinckii, the operons show considerable differences in organization and regulatory elements. The C. acetobutylicum scr operon contains the elements of an antiterminator-mediated regulation mechanism, typical of the BgIG family of regulators. The scrT gene, located upstream of scrA encodes an antiterminator that is preceded by a transcription terminator, which is overlapped by a classical ribonucleic antiterminator (RAT) sequence. We also propose the existence of a new variant RAT-like sequence which overlaps a terminator between scrT and the downstream structural genes.

Persistence of herpes simplex virus type 1 DNA in chronic conjunctival and eyelid lesions of mice.

Herpes simplex virus type 1 (HSV-1) causes chronic blepharitis and conjunctivitis as well as keratitis in humans. The pathogenesis of these inflammatory ocular and dermal lesions is not well understood. We have examined the persistence of HSV-1 DNA and its relationship to inflammatory lesions in the conjunctiva and eyelid skin of mice which were inoculated with HSV-1 by the corneal route. Viral DNA was detected by in situ PCR in the conjunctiva and eyelid tissue of infected mice at 5, 11, 23, and 37 days postinfection (p.i.). This DNA was localized in the epithelial cells of the conjunctiva and hair follicles and in the epidermal cells of the eyelid skin. Viral proteins were not detected in the conjunctiva or the eyelid skin after 5 days p.i., even though histopathological lesions were found at 23 and 37 days p.i. in both tissues. The DNA-containing cells were adjacent to sites of inflammation in the chronic lesions in both the conjunctiva and the eyelid skin. A similar temporal and spatial relationship between HSV-1 DNA and inflammatory lesions has been previously reported for the cornea. Our data suggest that the lesions in the cornea, conjunctiva, and eyelid skin progress similarly. Further studies are required to determine whether the long-term presence of HSV-1 is involved in the mechanism by which these chronic inflammatory lesions develop. The presence of HSV-1 DNA in these extraocular tissues for extended periods may constitute persistent viral infection of nonneuronal cells.

Note: sucrose transport and metabolism in Clostridium beijerinckii NCIMB 8052.

Sucrose is the major carbon source in molasses, the traditional substrate employed in the industrial acetonebutanol-ethanol (ABE) fermentation by solventogenic clostridia. The utilization of sucrose by Clostridium beijerinckii NCIMB 8052 was investigated. Extracts prepared from cultures grown on sucrose (but not xylose or fructose) as the sole carbon source possessed sucrose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity. Extract fractionation and reconstitution experiments revealed that the entire sucrose Enzyme II complex resides within the membrane in this organism. Sucrose-6-phosphate hydrolase and fructokinase activities were also detected in sucrose grown cultures. The fructokinase activity, which is required specifically during growth on sucrose, was shown to be inducible under these conditions. A pathway for sucrose metabolism in this organism is proposed.

A gene system for glucitol transport and metabolism in Clostridium beijerinckii NCIMB 8052.

The gutD gene of Clostridium beijerinckii NCIMB 8052 encoding glucitol 6-phosphate dehydrogenase was cloned on a 5.7-kbp chromosomal DNA fragment by complementing an Escherichia coli gutD mutant strain and selecting for growth on glucitol. Five open reading frames (ORFs) in the order gutA1 gutA2 orfX gutB gutD were identified in a 4.0-kbp region of the cloned DNA. The deduced products of four of these ORFs were homologous to components of the glucitol phosphotransferase system (PTS) and glucitol 6-phosphate dehydrogenase from E. coli, while the remaining ORF (orfX) encoded an enzyme which had similarities to members of a family of transaldolases. A strain in which gutD was inactivated by targeted integration lacked glucitol 6-phosphate dehydrogenase activity. The gutA1 and gutA2 genes encoded two polypeptides forming enzyme IIBC of the glucitol PTS comprising three domains in the order CBC. Domain IIA of the glucitol PTS was encoded by gutB. Glucitol phosphorylation assays in which soluble and membrane fractions of cells grown on glucose (which did not synthesize the glucitol PTS) or cells grown on glucitol were used confirmed that there is a separate, soluble, glucitol-specific PTS component, which is the product of the gutB gene. The gut genes were regulated at the level of transcription and were induced in the presence of glucitol. Cells grown in the presence of glucose and glucitol utilized glucose preferentially. Following depletion of glucose, the glucitol PTS and glucitol 6-phosphate dehydrogenase were synthesized, and glucitol was removed from the culture medium. RNA analysis showed that the gut genes were not expressed until glucose was depleted.

Physiology of carbohydrate to solvent conversion by clostridia.

The solvent-forming clostridia have attracted interest because of their ability to convert a range of carbohydrates to end-products such as acetone, butanol and ethanol. Polymeric substrates such as cellulose, hemicellulose and starch are degraded by extracellular enzymes. The majority of cellulolytic clostridia, typified by Clostridium thermocellum, produce a multi-enzyme cellulase complex in which the organization of components is critical for activity against the crystalline substrate. A variety of enzymes involved in degradation of hemicellulose and starch have been identified in different strains. The products of degradation, and other soluble substrates, are accumulated via membrane-bound transport systems which are generally poorly characterized. It is clear, however, that the phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in solute uptake in several species. Accumulated substrates are converted by intracellular enzymes to end-products characteristic of the organism, with production of ATP to support growth. The metabolic pathways have been described, but understanding of mechanisms of regulation of metabolism is incomplete. Synthesis of extracellular enzymes and membrane-bound transport systems is commonly subject to catabolite repression in the presence of a readily metabolized source of carbon and energy. While many genes encoding cellulases, xylanases and amylases have been cloned and sequenced, little is known of control of their expression. Although the mechanism of catabolite repression in clostridia is not understood, some recent findings implicate a role for the PTS as in other low G-C Gram-positive bacteria. Emphasis has been placed on describing the mechanisms underlying the switch of C. acetobutylicum fermentations from acidogenic to solventogenic metabolism at the end of the growth phase. Factors involved include a lowered pH and accumulation of undissociated butyric acid, intracellular concentration of ATP and reduced pyridine nucleotides, nutrient limitation, and the interplay between pathways of carbon and electron flow. Genes encoding enzymes of solvent pathways have been cloned and sequenced, and their expression correlated with the pattern of end-product formation in fermentations. There is evidence that the initiation of solvent formation may be subject to control mechanisms similar to other stationary-phase phenomena, including sporulation. The application of recently developed techniques for genetic manipulation of the bacterium is improving understanding of the regulatory circuits, but a complete molecular description of the control of solvent formation remains elusive. Experimental manipulation of the pathways of electron flow in other species has been shown to influence the range and yield of fermentation end-products. Acid-forming clostridia can, under appropriate conditions, be induced to form atypical solvents as products. While the mechanisms of regulation of gene expression are not at all understood, the capacity to adapt in this way further illustrates the metabolic flexibility of clostridial strains.

Molecular analysis of the amy gene locus of Thermoanaerobacterium thermosulfurigenes EM1 encoding starch-degrading enzymes and a binding protein-dependent maltose transport system.

A gene of Thermoanaerobacterium thermosulfurigenes EM1 encoding a protein with similarity to the maltose-binding protein of Escherichia coli was cloned and sequenced. It was located in the amy gene region of the chromosome downstream of the pullulanase-encoding amyB gene and upstream of amyDC, encoding membrane components of an ABC transport system, and the alpha-amylase gene amyA. The gene was designated amyE. Analysis of mRNA by Northern (RNA) blotting revealed that expression of the amy gene region is repressed during growth on glucose. Maximum levels of mRNA were detected with maltose as a substrate. An operon which was transcribed in the order amyBEDC was identified. However, an additional transcription start point was found in front of amyE. The amyA gene represented a monocistronic operon. Putative -35 and -10 promoter sites were deduced from the three transcription start sites of the amy gene region, and possible regulatory regions mediating induction by maltose and catabolite repression by glucose were identified by sequence analysis and comparison. The biochemical characterization of maltose uptake in T. thermosulfurigenes EM1 revealed two transport systems with Km values of 7 microM (high affinity) and 400 microM (low affinity). We conclude that the high-affinity system, which is specific for maltose and maltotriose, is a binding-protein-dependent transporter encoded by amyEDC. The gene for the putative ATP-binding protein has not yet been identified, and in contrast to similar systems in other bacteria, it is not located in the immediate vicinity of the chromosome.

Glucose Transport in Stationary-Phase Cultures of an Asporogenous Strain of Bacillus licheniformis.

The sporulation-deficient industrial organism Bacillus licheniformis HWL10 possesses two distinct glucose transport systems in log-phase cells, a glucose phosphotransferase system (PTS) and a non-PTS mechanism. The strain continues to take up glucose at a significant though reduced rate during prolonged stationary-phase incubation, but only the PTS is active.

Genetic transfer of lactate-utilizing ability in the rumen bacterium Selenomonas ruminantium.

Matings between the lactate-utilizing, tetracycline-sensitive Selenomonas ruminantium strains 5521C1 and 5934e and the lactate-non-utilizing, tetracycline-resistant strain FB322 resulted in putative recombinant strains capable of growth on lactate. Analysis of total protein by SDS-PAGE and chromosomal DNA by hybridization, indicated that the recombinants were derived from strain FB322. DNA hybridization produced no evidence that plasmid transfer occurred, leaving chromosomal DNA transfer as the most likely mechanism for the altered phenotype. Analysis of strains 5934e, FB322 and the resulting recombinant TC3 indicated that all three strains contained D-nLDH and L-nLDH activities. In addition strains 5934e and TC3 possessed D-iLDH activity when grown on DL-lactate. The ability of strain FB322 to grow on pyruvate but not lactate suggested that the lactate-utilizing recombinant had acquired the ability to synthesize D-iLDH.

Optimization of mosquitocidal toxin synthesis from Bacillus sphaericus using gene fusions.

ß-Galactosidase gene fusions have been used to monitor the progress of mosquito-larvicidal-toxin gene expression in Bacillus sphaericus strain 2362. ß-Galactosidase estimation in cells from late-growth-phase batch cultures was compared with larvicidal toxicity after incubation for 48 h. Conditions which promoted efficient sporulation, such as plentiful trace elements and relatively crude protein sources (soybean or cottonseed flours), enhanced reporter gene expression and provided high toxicity. However, acetate, which repressed sporulation, similarly repressed binary toxin yield. Gene fusions to the binary and 100-kDa toxin genes of B. sphaericus could be useful for the rapid screening of fermentation conditions for the local production of this larvicidal bacterium but, in view of the poor correlation with toxicity at high toxicity levels, such experiments should be confirmed with bioassays.

Neurons differentially control expression of a herpes simplex virus type 1 immediate-early promoter in transgenic mice.

The immediate-early proteins of herpes simplex virus control the cascade of viral gene expression during lytic infection. It is not known which viral or host proteins control the reactivation of the viral genome in latently infected neurons. To determine whether neuronal proteins can regulate a herpes simplex virus immediate-early promoter in vivo, transgenic mice containing the promoter regulatory region of the herpes simplex virus type 1 immediate-early gene (ICP4) fused to the bacterial beta-galactosidase gene were generated. Two lines of mice, in the absence of viral proteins, displayed ICP4 promoter activity in neurons in specific locations in the central nervous system. The anatomic locations of these neurons were the hippocampus, cerebellar cortex, superior colliculus, indusium griseum, mammillary nucleus, cerebral cortex, and the dorsal laminae of the dorsal horns of the spinal cord. Additional subsets of neurons expressed the ICP4 promoter at lower levels; these included trigeminal ganglia and retinas. In a third line of mice, lower levels of expression were present in many of the above-described neurons. Many types of neurons, nearly all nonneuronal cells in the central nervous system, and some non-nervous system tissues were negative. Viral proteins including VP16 are not necessary to induce transcription from the ICP4 promoter in many neurons and some other cell types but may be required in most cells in vivo. An approximately 100-fold-greater number of neurons in the trigeminal ganglia expressed ICP4 promoter activity in newborn mice compared with adults. These data provide direct evidence that host proteins are sufficient to activate a herpes simplex virus immediate-early promoter in neurons in vivo and that a differential expression pattern for this promoter exists within different neuronal phenotypes and between the same neurons in different ages of mice.

Targeting and gene expression in spinal cord motor neurons following intramuscular inoculation of an HSV-1 vector.

One problem in devising strategies of gene transfer to the nervous system is targeting specific neuronal populations. To evaluate the potential for using herpes simplex virus (HSV) as a vector for gene transfer to spinal cord motor neurons, the HSV-1 mutant LAT-LTR in which the E. coli beta-galactosidase gene is expressed under control of the HSV LAT core promoter (LAT) and the Moloney murine leukemia virus long terminal repeat (LTR) was inoculated unilaterally into the gastrocnemius muscle. Infectious virus was isolated from the spinal cord on days 3-7 post inoculation (PI). Immunocytochemical labeling of HSV antigen was detected in ipsilateral ventral horn neurons in the spinal cord at day 3 PI and had spread to contiguous spinal cord regions by day 6 PI. No viral antigen was detected at 14 or 28 DPI. beta-galactosidase expression (driven by the LAT-LTR promoter) was detected in neurons of the ventral horn on days 3, 6, 14, and 28 PI. Histological analysis showed mild lesions in the ventral horn on day 3 PI which progressed through days 6, 14 and 28 PI. This study demonstrates the feasibility of gene delivery into spinal motor neurons after injection of an HSV vector at a peripheral muscular site. This approach should prove useful in neurobiological investigations and it suggests a possible application to development of gene therapy for heritable diseases affecting motor neurons.