The Dienes phenomenon: competition and territoriality in Swarming Proteus mirabilis.

When two different strains of swarming Proteus mirabilis encounter one another on an agar plate, swarming ceases and a visible line of demarcation forms. This boundary region is known as the Dienes line and is associated with the formation of rounded cells. While the Dienes line appears to be the product of distinction between self and nonself, many aspects of its formation and function are unclear. In this work, we studied Dienes line formation using clinical isolates labeled with fluorescent proteins. We show that round cells in the Dienes line originate exclusively from one of the swarms involved and that these round cells have decreased viability. In this sense one of the swarms involved is dominant over the other. Close cell proximity is required for Dienes line formation, and when strains initiate swarming in close proximity, the dominant Dienes type has a significant competitive advantage. When one strain is killed by UV irradiation, a Dienes line does not form. Killing of the dominant strain limits the induction of round cells. We suggest that both strains are actively involved in boundary formation and that round cell formation is the result of a short-range killing mechanism that mediates a competitive advantage, an advantage highly specific to the swarming state. Dienes line formation has implications for the physiology of swarming and social recognition in bacteria.

Rapid drug susceptibility testing of mycobacteria by culture on a highly porous ceramic support.

BACKGROUND: Phenotypic, culture-based methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis are relatively simple and may be particularly appropriate for resource-limited settings where tuberculosis (TB) is most prevalent. However, these methods can be slow and generate significant amounts of infectious waste. Low-cost digital imaging and a unique porous ceramic support for cell culture (Anopore) may offer opportunities to improve this situation. OBJECTIVE: To test a rapid DST method based on fluorescence microscopy of mycobacteria grown for a few generations on Anopore. DESIGN: Mycobacteria were cultured with and without drugs, and the resulting microcolonies were heat-killed and stained with the fluorogenic dye Syto16. Microscopy, image-capture with a charge-coupled device camera and digital processing were used to quantify the inhibition of growth by drugs. Rapid DST for rifampicin and isoniazid was performed for clinical isolates. RESULTS: Mycobacteria could be cultured, killed, stained and imaged on Anopore. For DST, the Anopore method gave an accurate result in 3 days. CONCLUSION: This is an unprecedented speed for culture-based DST for this group of organisms and results in minimal infectious waste (<20,000 colony forming units). Analysis of mycobacteria by fluorescence and electron microscopy on Anopore also opens up research possibilities.

Evidence of a breakdown of corticostriatal connections in Parkinson's disease.

Dendritic spines are important structures which receive synaptic inputs in many regions of the CNS. The goal of this study was to test the hypothesis that numbers of dendritic spines are significantly reduced on spiny neurones in basal ganglia regions in Parkinson's disease as we had shown them to be in a rat model of the disease [Exp Brain Res 93 (1993) 17]. Postmortem tissue from the caudate and putamen of patients suffering from Parkinson's disease was compared with that from people of a similar age who had no neurological damage. The morphology of Golgi-impregnated projection neurones (medium-sized spiny neurones) was examined quantitatively. The numerical density of dendritic spines on dendrites was reduced by about 27% in both nuclei. The size of the dendritic trees of these neurones was also significantly reduced in the caudate nucleus from the brains of PD cases and their complexity was changed in both the caudate nucleus and the putamen. Dendritic spines receive crucial excitatory input from the cerebral cortex. Reduction in both the density of spines and the total length of the remaining dendrites is likely to have a grave impact on the ability of these neurones to function normally and may partly explain the symptoms of the disorder.

Newborn screening compared to clinical identification of biochemical genetic disorders.

A group of 28 patients with inherited metabolic disease (homocystinuria galactosaemia, maple syrup urine disease and biotinidase deficiency) diagnosed by screening were compared with a group of 17 similar patients identified clinically. The rate of hospitalization was similar for the two groups. The patients diagnosed clinically showed a higher incidence of mental retardation and their parents experienced greater stress and found greater difficulty in meeting their child's needs.

Mutations in the ss subunit of the Bacillus subtilis RNA polymerase that confer both rifampicin resistance and hypersensitivity to NusG.

Mutations conferring resistance to the antibiotic rifampicin (Rif(r)) occur at specific sites within the ss subunit of the prokaryotic RNA polymerase. Rif(r) mutants of Escherichia coli are frequently altered in the elongation and termination of transcription. Rif(r) rpoB mutations were isolated in Bacillus subtilis and their effects on transcription elongation factor NusG and Rho-dependent termination were investigated. RNase protection assay, Northern analysis and the expression of nusG-lacZ fusions in cells with an inducible NusG suggested the B. subtilis nusG gene was autoregulated at the level of transcription. Rif(r) mutations that changed residue Q469 to a basic residue (Q469K and Q469R) enhanced autoregulation of nusG. A mutant expressing a truncated form of NusG, due to a nonsense mutation within the nusG gene, was isolated on the basis of the loss of autoregulation. The mechanism of autoregulation was found to be independent both of transcription termination factor Rho and of the promoter transcribing nusG. Autoregulation required sequences within the 5' coding sequence of the nusG gene or immediately upstream. This is the first evidence from any bacterium that Rif(r) RNA polymerases can display altered transcription regulation by NusG.

Dopamine and synaptic plasticity in the neostriatum.

After the unilateral destruction of the dopamine input to the neostriatum there are enduring changes in rat behaviour. These have been ascribed to the loss of dopamine and the animals are often referred to as 'hemiparkinsonian'. In the denervated neostriatum, we have shown that not only are the tyrosine hydroxylase positive boutons missing, but also the medium sized densely spiny output cells have fewer spines. Spines usually have asymmetric synapses on their heads. In a recent stereological study we were able to show that there is a loss of approximately 20% of asymmetric synapses in the lesioned neostriatum by 1 mo after the lesion. Current experiments are trying to establish the specificity of this loss. So far we have evidence suggesting that there is no obvious preferential loss of synapses from either D1 or D2 receptor immunostained dendrites in the neostriatum with damaged dopamine innervation. These experiments suggest that dopamine is somehow necessary for the maintenance of corticostriatal synapses in the neostriatum. In a different series of experiments slices of cortex and neostriatum were maintained in vitro in such a way as to preserve at least some of the corticostriatal connections. In this preparation we have been able to show that cortical stimulation results in robust excitatory postsynaptic potentials (EPSPs) recorded from inside striatal neurons. Using stimulation protocols derived from the experiments on hippocampal synaptic plasticity we have shown that the usual consequence of trains of high frequency stimulation of the cortex is the depression of the size of EPSPs in the striatal cell. In agreement with similar experiments by others, the effect seems to be influenced by NMDA receptors since the unblocking of these receptors with low Mg++ concentrations in the perfusate uncovers a potentiation of the EPSPs after trains of stimulation. Dopamine applied in the perfusion fluid round the slices has no effect but pulsatile application of dopamine, close to the striatal cell being recorded from, and in temporal association with the cortical trains, leads to a similar LTP like effect. The reduction of K+ channel conductance in the bath with TEA also has the effect of making cortical trains induce potentiation of corticostriatal transmission. TEA applied only to the cell being recorded from has no similar effect; the cortical stimulation again depresses the EPSP amplitude, so the site of action of TEA may well be presynaptic to the striatal cell. The morphological and physiological experiments may not necessarily be related but it is tempting to suggest that dopamine protects some corticostriatal synapses by potentiating them but that in the absence of dopamine others simply disconnect and are no longer detectable on electron microscopy.

Synapse loss associated with abnormal PrP precedes neuronal degeneration in the scrapie-infected murine hippocampus.

Numbers of neurones, synapses and axon terminals were quantified in a murine scrapie model with severe hippocampal pyramidal cell loss, in which definite clinical scrapie is evident from 226 days post-infection (dpi) and death occurs around 250 dpi. Disease-specific PrP accumulations were first seen at 70 dpi (28% of the incubation period (IP)) in thalamus and as sparse foci within the stratum pyramidale of CA1. By 98 dpi (39% IP), PrP was seen in the stratum radiatum and was found at later stages throughout all levels of the hippocampus. At the ultrastructural level in the stratum radiatum of CA1, a decrease in the numbers of simple synapses from 84 dpi (34% IP) and in perforated synapses from 98 dpi (42% IP) was found using an unbiased stereological method, the disector analysis. Degeneration of axon terminals was found from 98 dpi (39% IP) onwards. Neuronal loss was detected in CA1 from 180 dpi (72% IP). The results suggest that the fundamental lesion in the hippocampus of ME7-infected mice is associated with PrP release from CA1 pyramidal neurones, which perturbs synaptic function and leads to degeneration of preterminal axons, and that subsequent pathological changes including neurone loss are sequelae to this initial insult.

Characterisation of the enzymatic and RNA-binding properties of the Rhodobacter sphaeroides 2.4.1. Rho homologue.

The Escherichia coli Rho is a transcription termination factor with complex enzymatic properties. Rho is a near-universal prokaryotic transcription factor, but very few non-enteric Rho factors have been studied. The expression and enzymatic activity of Rho from the GC-rich, Gram-negative bacterium Rhodobacter sphaeroides was characterised. Poly(C)-activated ATP hydrolysis, multimerisation and the abundance of the R. sphaeroides Rho were similar to the E. coli Rho. The R. sphaeroides Rho was a DNA:RNA helicase. The R. sphaeroides Rho was unique in Rho factors characterised to date in that it did not interact with the lambdatR1 terminator transcript and ATP hydrolysis was unusually weakly activated by poly(U) RNA. A chimeric Rho (RhoER), with the RNA-binding domain from the E. coli Rho and the ATPase domain of the R. sphaeroides Rho, was activated by RNA co-factors in a similar fashion to the E. coli Rho. The activity of RhoER suggests functional interactions between the N- and C-terminal domains of Rho monomers are highly conserved between Rho factors. The main differences between Rho factors from different bacteria is in the specificity of RNA binding although this does not appear to be necessarily dependent on the GC bias of target RNA as has been previously suggested.

Double anterograde tracing of outputs from adjacent "barrel columns" of rat somatosensory cortex. Neostriatal projection patterns and terminal ultrastructure.

The sensory input to the neostriatum from groups of cortical cells related to individual facial vibrissae has been investigated at both light- and electron-microscopic resolution. The purpose of the study was to establish the extent to which corticostriatal input maintains the anatomical coding of spatial information that is present in cortex. A double anterograde tracing method was used to identify the output projections from groups of adjacent neurons in different barrel columns, so that the anatomical relationships between two groups could be studied throughout their length. Adjacent whiskers are represented in adjoining cortical barrels and an examination of corticostriatal projections from these reveals two patterns of projection. In one, the anatomical topography is partially preserved; the barrels are represented in adjoining, discrete, areas of the somatosensory neostriatum. In the second projection pattern, the neostriatal innervation is diffuse and adjacent barrels are represented in overlapping regions of the neostriatum. Moreover, the fibres are thinner, have smaller boutons, and are present in both the ipsilateral and contralateral neostriatum. The two systems also enter the neostriatal neuropile separately. The discrete topographic system enters the adjacent neostriatum as collaterals which leave the descending corticofugal fibres at right angles, while the diffuse system enters directly from the corpus callosum at an acute angle. Examination of the neostriatal terminal fields by correlated light and electron microscopy, shows that characteristic axospinous terminals on spiny neurons are made by both groups of cortical fibres, although they differ in their size and morphology. It is concluded that at least two corticostriatal pathways arise from the barrel cortex. One connection maintains some of the anatomical code implicit in the barrel pattern of primary somatosensory cortex, but another, more diffuse, system is overlaid upon it which may carry different information from this complex area of cortex.

Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis.

The expression and activity of transcription termination factor Rho and the requirement for transcription elongation factors NusA and NusG was investigated in Bacillus subtilis. Rho was present at < 5% of the level found in Escherichia coli, but Rho factors from these two bacteria had similar properties as RNA-activated ATPases and in vitro termination of transcription on the lambda tR1 terminator. The B. subtilis rho gene was autoregulated at the level of transcription; autoregulation required sequences within the rho mRNA leader region and gene. To date, the B. subtilis rho is the only gene from a Gram-positive bacterium found to be regulated by Rho. Rho was not involved in bulk mRNA decay in B. subtilis. The E. coli elongation factors NusA and NusG target Rho, and the importance of these proteins in B. subtilis was examined by gene disruption. The B. subtilis NusG was inessential for both the viability and the autoregulation of Rho, whereas NusA was essential, and the requirement for NusA was independent of Rho. This contrasts with E. coli in which NusG is essential but NusA becomes dispensable if Rho terminates transcription less efficiently.

Plasticity of synapses in the rat neostriatum after unilateral lesion of the nigrostriatal dopaminergic pathway.

In the 6-hydroxydopamine model of Parkinson's disease in the rat, there is a significant reduction in the number of dendritic spines on the principal projection neurons in the neostriatum, presumably attributable to loss of the nigrostriatal dopamine input. These spines invariably receive input from terminals forming asymmetric synapses that originate mainly from the cortex. The object of the present study was to determine the fate of those terminals after the loss of dendritic spines. Unbiased estimates of synaptic density and absolute numbers of synapses in a defined volume of the neostriatum were made using the "disector" and Cavalieri techniques. Numerical synaptic density of asymmetric synaptic contacts was 17% lower in the neostriatum deprived of dopamine innervation and, in absolute terms, there were 3 billion (19%) fewer contacts. The numerical density of a subpopulation of asymmetric contacts on dendritic spines that have complex or perforated synaptic specializations and normally make up 9% of the asymmetric population was 44% higher on the experimental side. Asymmetric synapses were found to be enriched in glutamate using postembedding immunogold labeling. The present observations demonstrate that the loss of spines previously reported after 6-hydroxydopamine lesions is accompanied by a loss of asymmetric synapses rather than by the movement of synapses from spines to other postsynaptic targets. The study also demonstrates that there is an increase in complex synaptic interactions that have been implicated in synaptic plasticity in other regions of the CNS after experimental manipulations.

Plasticity of striatopallidal terminals following unilateral lesion of the dopaminergic nigrostriatal pathway: a morphological study.

In Parkinson's disease the dopaminergic nigrostriatal pathway degenerates, resulting in an imbalance in activity of two pathways of information flow through the basal ganglia. In animal models of the disease, the striatonigral pathway becomes underactive and the striatopallidal pathway becomes overactive. In the present study immunocytochemistry for enkephalin and GABA and anterograde labelling were used to investigate whether morphological plasticity occurs in striatopallidal terminals following unilateral removal of the nigrostriatal dopaminergic pathway. Pallidal terminals were immunostained to reveal enkephalin and examined in the electron microscope (n=399). Immunoreactive synaptic bouton profiles were on average 64% larger on the experimental side 26 days after the lesion. Analysis of their shape revealed that those on the dopamine-depleted side of the brain were more irregular in profile and that their synaptic specialisations were more complex in shape but not significantly different in length. Striatopallidal terminals were also identified by GABA immunocytochemistry combined with anterograde labelling (n=20). Double-labelled boutons were significantly larger in cross-sectional area on the experimental side (57%). Analysis of terminals that were simply labelled by the immunogold method to reveal GABA (n=278) showed no significant differences in size between terminals from the dopamine-depleted and control side. This suggests that a substantial number of GABAergic terminals in the globus pallidus do not belong to the striatopallidal population of terminals. These morphological changes correlate with previous studies suggesting striatopallidal boutons are more active after destruction of dopaminergic input to the neostriatum.

Structural plasticity of optic synapses in the rat suprachiasmatic nucleus: adaptation to long-term influence of light and darkness.

Synapses of optic afferents (optic synapses) in the suprachiasmatic nucleus of hooded rats were morphometrically evaluated after exposing the animals to 12 h, 14 days, 2 months, and 8 months of constant light (light rats) and darkness (dark rats). Compared with dark rats, optic synapses from light rats have larger boutons with larger mitochondria, more clear vesicles, fewer dense-core vesicles and front-line vesicles, smaller presynaptic dense projections, a smaller amount of postsynaptic density material, a smaller relative number of Gray-type I (asymmetric) junctions, a greater relative number of Gray-type II (symmetric) junctions, as well as more and larger mitochondria in the postsynaptic dendrites. Junctions of optic synapses are mostly straight, but the small number of positively curved contacts are more flattened in light rats than in dark rats. An age-related increase in the size of presynaptic dense projections was also observed. There are no changes in the sizes of clear and dense-core vesicles, in the size of synaptic junctions and their numerical density in area, and in the unspecific contact area between pre- and postsynaptic elements. The changes in optic boutons are characteristic for activated and relatively disused synapses with a slow, tonic firing rate. It appears that (1) the amount of postsynaptic density material is proportional to the strength of Gray-type I synapses, and that (2) some excitatory optic synapses become inhibitory after long-term activity, whereas some inhibitory synapses turn into excitatory contacts after long-term disuse.

Isolation and sequencing of the rho gene from Streptomyces lividans ZX7 and characterization of the RNA-dependent NTPase activity of the overexpressed protein.

The gene for transcription termination factor Rho was isolated from Streptomyces lividans ZX7. It encoded a 77-kDa polypeptide (Rho 77) with considerable homology to known Rho factors. An atypical hydrophilic region of 228 residues was found within the N-terminal RNA-binding domain. Only Rho from Micrococcus luteus and Mycobacterium leprae (closely related GC-rich Gram-positive bacteria) had an analogous sequence. Rho 77 was overexpressed in Escherichia coli and purified using an N-terminal hexahistidine-tag. Rho 77 displayed a broad RNA-dependent ATPase activity, with poly(C) RNA being no more than 4-fold more effective than poly(A). This contrasts with the ATPase activity of Rho from E. coli which is stimulated primarily by poly(C) RNA. Rho 77 was a general RNA-dependent NTPase, apparent Km values for NTPs were: GTP 0.13 mM, ATP 0.17 mM, UTP 1.1 mM, and CTP >2 mM. Rho 77 poly(C)-dependent ATPase activity was inhibited by heparin, unlike the E. coli Rho. The antibiotic bicyclomycin inhibited the in vitro RNA-dependent ATPase activity of Rho 77, did not inhibit growth of streptomycetes but delayed the development of aerial mycelia. N-terminal deletion analysis to express a truncated form of Rho (Rho 72, 72 kDa) indicated that the first 42 residues of Rho 77 were not essential for RNA-dependent NTPase activity and were not the targets of inhibition by heparin or bicyclomycin.

Morphological changes in met(5)-enkephalin-immunoreactive synaptic boutons in the rat neostriatum after haloperidol decanoate treatment.

The morphological plasticity of an identified population of synaptic boutons in the rat neostriatum was investigated 24 h (short-term treatment) or 14 days (long-term treatment) after administration of the depot neuroleptic, haloperidol decanoate. Specific methionine(5)-enkephalin antiserum was used to label bouton profiles in the dorsal neostriatum. The size and shape of these boutons was subsequently analysed with quantitative methods at the ultrastructural level. Immunoreactive synaptic bouton profiles were found to have a larger cross-sectional area, to be less circular in shape and to have a longer maximum diameter after long-term neuroleptic treatment. These parameters were not significantly affected by short-term neuroleptic treatment. The morphological parameters indicate that methionine(5)-enkephalin-immunoreactive boutons become enlarged, probably by elongating. This suggests that boutons containing methionine(5)-enkephalin increase their potential synaptic efficacy in the long term after neuroleptic treatment.

Control of lytic development in the Streptomyces temperate phage phi C31.

The repressor gene, c, is required for maintenance of lysogeny in the Streptomyces phage phi C31. The c gene expresses three in-frame N-terminally different protein isoforms at least one of which is thought to bind to a 17bp highly conserved inverted repeat (CIR) sequence found at 18 (or more) loci throughout the phi C31 genome. Here we present evidence that one of these loci, CIR6, and its interaction with the products of the repressor gene are critical in the control of the lytic pathway in phi C31. To the right of CIR6, according to the standard map of phi C31, an 'immediate-early' promoter, ap1, was discovered after insertion of a fragment containing CIR6 upstream of a promoterless kanamycin-resistance gene, aphII, to form pCIA2. pCIA2 conferred kanamycin resistance upon Streptomyces coelicolor A3(2) but not upon a phi C31 lysogen of S. coelicolor. Operator-constitutive (Oc) mutants of pCIA2 were isolated and the mutations lay in CIR6, i.e. CIR6:G14T and CIR6:C2A. Primer extension analysis of RNA prepared from an induced, temperature-sensitive lysogen of S. coelicolor localized a mRNA 5' endpoint 21 bp to the right of CIR6. The importance of the ap1/CIR6 region in the regulation of lytic growth was demonstrated by the analysis of a virulent mutant, phi C31 vir1, capable of forming plaques on an S. coelicolor phi C31 lysogen. phi C31vir1 contained a DNA inversion with the breakpoints lying within the integrase gene (which lies approximately 7 kbp to the right of CIR6) and in the essential early region between CIR6 and the -10 sequence for ap1. The separation of ap1 from its operator was thought to be the basis for the virulent phenotype in phi C31 vir1. Band-shift assays and DNase I footprinting experiments using purified 42 kDa repressor isoform confirmed that CIRs 5 and 6 were indeed the targets for binding of this protein. The 42 kDa repressor bound to CIR6 with higher affinity than to CIR5 in spite of their identical core sequences. Repressor bound at CIR6 facilitated binding at CIR5. The high-affinity binding to CIR6 was abolished with the Oc mutant, CIR6:G14T. Hydroxyl radical footprinting and dimethyl sulphate methylation protection of the 42 kDa repressor-CIR6 interaction suggested that the protein bound in the major groove and to one face of the DNA.

Rho-independent terminators without 3' poly-U tails from the early region of actinophage øC31.

Previous work has identified three intergenic regions from the early region of actinophage øC31 where transcription was either terminated or the mRNA was processed. Here we show using in vivo and in vitro approaches that these regions contain rho-independent terminators designated eta, etb and etc. Transcripts through eta-c would be expected to form stable RNA stem-loops but would lack poly-U tails. Eta-c contained part or all of the conserved sequences 5' AGCCCC and 5' GGGGCTT. A Streptomyces 'terminator probe' vector, pUGT1, was constructed and used to assay the efficiency of termination of transcription by eta-c from the thiostrepton-inducible tipA promoter by measuring the expression of a downstream reporter gene (aphII). In pUGT1 etb was at best a minor terminator in vivo whilst eta and etc exhibited strong termination activity. In vitro termination was assayed using templates containing a synthetic promoter recognised by E.coli RNA polymerase and fragments containing eta-c inserted downstream. All three terminators stimulated the formation of 3' ends in the promoter-distal arm of the inverted repeats with efficiencies eta > etc > etb. As all three terminators either overlap with or lie close to sequences which interact with phage repressor proteins (conserved inverted repeats, CIRs) and these can potentially form stem-loop structures in RNA, the effect of CIRs on termination was also investigated. Termination at etb was unaffected by the presence or absence on the transcription template of CIR3. CIR4 forms the central 17 bp of etc and a 37 nt deletion which eliminated this stem-loop abolished termination in vivo and in vitro. Eta was investigated using an antisense oligonucleotide interference assay; an oligo designed to bind the 5' arm of eta inhibited termination whilst an oligo antisense to CIR5 was ineffective and an oligo targeted further upstream enhanced termination. Taken together these data show that eta-c are intrinsic, rho-independent terminators of varying efficiencies despite the absence of a poly-U tail.

An operator associated with autoregulation of the repressor gene in actinophage phiC31 is found in highly conserved copies in intergenic regions in the phage genome.

Previous reports have suggested that the repressor gene, c, of phiC31 is autoregulated and that likely operators are conserved inverted repeat sequences (CIRs1&2) located just upstream of the promoters, cp1 and cp2. Evidence is now presented that the CIRs 1&2 are indeed binding sites for one of the three inframe, N-terminally different protein isoforms of 42, 54 and 74 kDa produced by the c gene. A cp1-aphII fusion was repressed in a Streptomyces coelicolor A3(2) phiC31 lysogen and characterisation of an operator-constitutive (Oc) mutant showed a single mutation in CIR-1. CIR-1 containing fragments were retarded in electrophoresis gels by the 42 kDa repressor protein isoform and this retardation was inhibited by the addition of competing DNA fragments containing either CIR-1 or CIR-2. Using a combination of Southern blotting and analysis of available DNA sequence we also show that at least 18 copies of the CIRs are present throughout the phiC31 genome. Alignment of 9 CIR sequences showed that 8 contained a perfectly conserved 17 bp core whilst the exception had a single mismatch. The core includes a 16 bp inverted repeat (IR), and is usually part of a more extensive and less highly conserved palindrome. When superimposed on a previously derived transcription map of the early region, the CIRs lie in intergenic regions associated with transcription initiation and/or termination.