Biomedical subjects
Publications and source records attributed to C Bonamy.
We report the fourth case of combined D-and L-2-hydroxyglutaric aciduria presenting with neonatal encephalopathy and subependymal cysts.
Integration of plasmid pCGL320 into a Corynebacterium glutamicum ATCC21086 derivative led to tandem amplification of the inserted plasmid (Labarre et al., 1993). One amplification event was associated with integration of an insertion sequence that we have named IS1206. Hybridizing sequences were only found in C. glutamicum strains and at various copy numbers. IS1206 is 1290 bp long, carries 32 bp imperfect inverted repeats and generates a 3 bp duplication of the target DNA upon insertion. IS1206 presents the features characteristic of the IS3 family and part of the DNA sequence centering on the putative transposase region (orfB) is similar to those of IS3 and some other related elements. Phylogenetic analysis of orfB deduced protein sequences from IS1206 and IS3-related elements contradicts the phylogeny of the species, suggesting that evolution of these elements might be complex. Horizontal transfer could be invoked but other alternatives like ancestral polymorphism or/and different rates of evolution could also be involved.
A pBR322-derived plasmid (pCGL107) that carries the Corynebacterium melassecola ATCC17965 analogue of Escherichia coli gdhA gene (encoding glutamate dehydrogenase), was introduced into the related strain, Brevibacterium lactofermentum CGL2002, by electroporation and integrated into its chromosome by homologous recombination. However, pCGL107 cannot integrate into C. melassecola, since the host restriction prevents successful electroporation by E. coli-modified DNA. Nevertheless, B. lactofermentum-modified replicative plasmid DNA can be transformed by electroporation into C. melassecola; thus pCGL519-2, a shuttle plasmid that carries the C. melassecola analogue of E. coli gltA (encoding citrate synthase), was extracted from the former host and electroporated into the latter. Rare restriction sites conveniently placed in pCGL519-2 were used to recover a replicon-less cartridge called 'integron', that contains a selectable marker and gtlA within a single fragment. Integron prepared from pCGL519-2 DNA which had been extracted from C. melassecola, and thus, was capable of eluding the C. melassecola restriction barrier(s), was successfully electroporated into this host. The molecular analysis of the resulting transformants suggests that they result from the integration of a single circular integron molecule by homologous recombination between the gltA regions of the host genome and the integron. These transformants were stable for 30 generations in the absence of selection.
Plasmid DNA was efficiently electro-transformed into intact cells of nine Corynebacteria strains belonging to Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium glutamicum and Corynebacterium melassecola. Relationships were explored between transformation efficiency and parameters such as electric field strength and pulse length, DNA concentration, physiological state and concentration of the cells. In optimal conditions, more than 10(7) transformants per microgram of DNA could be obtained. Electro-transformation with plasmid DNA isolated from different sources indicates that DNA modification may play a role in transformation efficiency.
During sporulation, Bacillus subtilis undergoes successive morphological changes that can be arrested at various stages by mutations in many genes. One of these, spoIIGB, encodes a transcriptional factor, sigma E, which is necessary to proceed beyond stage II and to differentiate the cell in two compartments, the forespore and the mother cell. Mutations were introduced in an open reading frame located immediately downstream of spoIIGB. They block sporulation at stage III and define a new gene, spoIIIG, encoding a 260-amino-acid polypeptide highly similar to bacterial sigma-factors. A promoter was identified in the spoIIGB-spoIIIG interval by transcriptional fusion to lacZ. It is turned on 1 hr after the start of sigma E synthesis and is specifically activated in the forespore. The tandemly arranged spoIIGB and spoIIIG genes appear to encode homologous proteins that modulate transcription in a sequential fashion during sporulation.
Sporulation of Bacillus subtilis is a primitive example of coupling between morphological changes and timing of gene expression during development. A major early control of transcriptional activity is dependent on a new sigma factor, sigma E, which is encoded by the sigE gene and synthesized as an inactive precursor, pro-sigma E. We show that mutations in the spoIIGA gene block the processing of pro-sigma E. Moreover, synthesis of both spoIIGA and sigE products in vegetative cells leads to expression of a sigma E-controlled promoter during growth, suggesting that SpoIIGA has pro-sigma E processing activity. The SpoIIGA polypeptide, which contains five potential transmembrane domains, is synthesized during sporulation 1 hr before processing activity can be detected. We propose that SpoIIGA processing activity is triggered by the presence of the sporulation septum, which is itself dependent on the spoIIAA and spoIIE products. These proteins are normally needed for pro-sigma E processing during sporulation but can be bypassed in vegetative cells. According to this model, a morphological structure would directly control the synthesis of a developmental sigma factor and would modify gene expression.
Evidence is presented that the sporulation-essential locus spoIIG codes for both sigma 29 and a structurally related protein, P31. This demonstrates that at least one specific Bacillus subtilis RNA polymerase binding protein provides a critical function in endospore formation. spoIIG-specific RNA is present in B. subtilis cultures that are synthesizing P31 and sigma 29 and is absent in those that are not. A monoclonal antibody specific for an antigenic determinant on P31/sigma 29 detected crossreacting proteins (P25/P21) but not P31 or sigma 29 in a Spo- B. subtilis strain with a mutation at the spoIIG locus (spoIIG41). The appearance of P25 and P21 occurs in this mutant at a time when P31 and sigma 29 would normally appear and suggests that they are homologous proteins. Transformation of the spoIIG41 strain with plasmid DNA carrying the structural gene for spoIIG complements the Spo- phenotype and results in the synthesis of P31, sigma 29, P25, and P21 at the appropriate times during sporulation. In Escherichia coli, the cloned spoIIG sequence encoded a protein that reacted with the anti-P31/sigma 29 monoclonal antibody and had the electrophoretic mobility of authentic P31.
The spo0B gene is one of the genes involved in initiation of sporulation of Bacillus subtilis. This gene, previously cloned into the pHV33 shuttle vector, is expressed in Escherichia coli and B. subtilis. We have determined the sequence of 1118 base pairs (bp) of the DNA insert carrying the spo0B gene. The promoter sequence of this gene shows the canonical T-A-T-A-A-T region at 10 bp from the transcriptional start (-10 region) but an unusual sequence, T-T-T-T-C-T-, in the -35 region. The nucleotide sequence shows an open reading frame encoding a 192-amino-acid polypeptide of Mr 22,542, which is close to the molecular weight of the spo0B product synthesized in E. coli minicells. To investigate the regulation of the spo0B gene under a variety of physiological conditions, we constructed an in-frame fusion between the spo0B promoter proximal region and the lacZ gene of E. coli. This hybrid gene was subsequently integrated into the B. subtilis chromosome, and the beta-galactosidase activity was measured. It was found that the spo0B gene is preferentially expressed during exponential growth; it is not induced by exhaustion of the growth medium nor repressed by glucose.
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The present paper describes an investigation, at the transcription level, in a thermosensitive sporulation mutant of Bacillus subtilis (ts-4) grown at the permissive (30 degrees C) or restrictive (42 degrees C) temperature where sporulation capacity is respectively expressed or arrested. These studies were carried out by analysing the ribonucleic acid from vegetative and stationary phase cells (t3 cells) grown under both conditions, by hybridization-competition experiments.
The incorporation of radioactive uracil into 50s and 30s ribosomal subunits and ribosomal ribonucleic acid (rRNA) was studied during the growth cycle of different sporogenic and asporogenic strains of Bacillus subtilis. It was found that partially synchronized cultures of the strains examined incorporated labeled uracil into the two ribosomal subunit species and rRNA during sporulation and during the stationary phase of the asporogenic strains. Kinetic studies have shown that, compared to vegetative cells, the percentage of uracil incorporated into the ribosomal subunits of cells taken 30 min after the end of exponential growth was decreased by about 25 to 35%. This decrease, however, appeared to be a general characteristic of stationary-phase cells and seems to depend on the nature of the sporulation medium and to some extent on the nature of the strain but not on the sp(+) or sp(-) phenotype of the strain. Moreover, by use of actinomycin D it was shown that the labeled uracil incorporated, in the presence of the drug, during the sporulation period was located in the ribosomal subunits (stable RNA). Based on these results, we concluded that during sporulation ribosomal genes are transcribed and consequently rRNA continues to be synthesized, although to a lesser extent than during vegetative growth. These results are discussed in the light of those obtained by Hussey et al.
A thermosensitive sporulation mutant (t(s)-4) of Bacillus subtilis was isolated, and its morphological, physiological, and enzymatic properties were investigated. This mutant is able to grow equally well at 30 and 42 C, but is unable to sporulate at the higher temperature. Electron microscope studies have shown that the t(s)-4 mutant is blocked at stage zero of spore development. This was further confirmed by its inability to produce antibiotic when grown at the restrictive temperature and by the relatively low ribonucleic acid (RNA) and protein turnover during the stationary growth phase, characteristic for stage zero asporogenic mutants. At the permissive temperature, however, antibiotic production and RNA and protein turnover took place at the rate normally found in sporogenic strains of B. subtilis. The above properties were not altered in the parent strain when grown at either 30 or 42 C. By shifting cultures of the t(s)-4 mutant from 30 to 42 C and from 42 to 30 C at different stages of growth, we have been further able to show that the event affected at the high temperature takes place at a very early stage of spore development. As a consequence of this early block in the sporulation process, the t(s)-4 mutant grown at 42 C became defective in the late spore-specific enzymes involved in the biosynthesis of dipicolinic acid. This study suggests that the sporulation process is mediated by a regulatory protein which is altered in the thermosensitive mutant when grown at the restrictive temperature. As a result of this alteration, a pleiotropic phenotype is produced which has lost the ability to catalyze the late biochemical reactions required for spore formation.
Sporulation of Bacillus subtilis involves sequential morphological and biochemical changes and is regulated by specific genes (spo genes) estimated to occupy more than 30 loci. A mutation in any one of these genes blocks the sporulation process at the corresponding developmental stage. Despite intensive genetic studies, the nature and function of the spo gene products remain unknown. Vegetative B. subtilis RNA polymerase core enzyme may interact with several sigma factors and discriminate among different classes of promoters. During sporulation, new polypeptides are associated with the core enzyme which may have a central role in modifying its promoter recognition specificity. As a first step to understanding their function in the switch from vegetative to sporulation mode, several early sporulation genes have been cloned and analysed. Here we report the cloning and nucleotide sequence of the spoIIG gene of B. subtilis. This gene encodes a polypeptide with a predicted relative molecular mass of 27,652 which contains a 65-amino acid region highly homologous to an internal part of the Escherichia coli sigma factor.
In this study, the authors surveyed the chief nurses of 152 Veterans' Health Administration Medical Centers to determine which medical centers based their nursing practice on one or more nursing theories or models. Of the 76 medical centers responding, 24 (35%) stated theory-based practice was in use in their institutions. The greatest number (16 of the 24) reported use of Orem's Self-Care Deficit theory or a combination of Orem with other theories. Most of the 24 chief nurses agreed that theory-based practice: 1) improves patient outcomes; 2) maximizes patient health; and 3) provides a consistent approach to care. However, they were less convinced that theory-based practice reduces nursing staff turnover or improves job satisfaction. They also stated that theory-based practice is more important to nursing administrators than to staff nurses. A similar survey of staff nurses is recommended.