Search PubMed⌕ Search

Biomedical subjects

P Zuber

Publications and source records attributed to P Zuber.

At least 37 records · Page 2Linked to original sources
PubMed1998

Anaerobic growth of a "strict aerobe" (Bacillus subtilis).

There was a long-held belief that the gram-positive soil bacterium Bacillus subtilis is a strict aerobe. But recent studies have shown that B. subtilis will grow anaerobically, either by using nitrate or nitrite as a terminal electron acceptor, or by fermentation. How B. subtilis alters its metabolic activity according to the availability of oxygen and alternative electron acceptors is but one focus of study. A two-component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, occupies an early stage in the regulatory pathway governing anaerobic respiration. One of the essential roles of ResD and ResE in anaerobic gene regulation is induction of fnr transcription upon oxygen limitation. FNR is a transcriptional activator for anaerobically induced genes, including those for respiratory nitrate reductase, narGHJI.B. subtilis has two distinct nitrate reductases, one for the assimilation of nitrate nitrogen and the other for nitrate respiration. In contrast, one nitrite reductase functions both in nitrite nitrogen assimilation and nitrite respiration. Unlike many anaerobes, which use pyruvate formate lyase, B. subtilis can carry out fermentation in the absence of external electron acceptors wherein pyruvate dehydrogenase is utilized to metabolize pyruvate.

Anaerobiosis↗
PubMed1997

The modular organization of multifunctional peptide synthetases.

Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.

Amino Acid Isomerases↗
PubMed1997

Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth.

Bacillus subtilis can grow anaerobically by respiration with nitrate as a terminal electron acceptor. In the absence of external electron acceptors, it grows by fermentation. Identification of fermentation products by using in vivo nuclear magnetic resonance scans of whole cultures indicated that B. subtilis grows by mixed acid-butanediol fermentation but that no formate is produced. An ace mutant that lacks pyruvate dehydrogenase (PDH) activity was unable to grow anaerobically and produced hardly any fermentation product. These results suggest that PDH is involved in most or all acetyl coenzyme A production in B. subtilis under anaerobic conditions, unlike Escherichia coli, which uses pyruvate formate lyase. Nitrate respiration was previously shown to require the ResDE two-component signal transduction system and an anaerobic gene regulator, FNR. Also required are respiratory nitrate reductase, encoded by the narGHJI operon, and moaA, involved in biosynthesis of a molybdopterin cofactor of nitrate reductase. The resD and resDE mutations were shown to moderately affect fermentation, but nitrate reductase activity and fnr are dispensable for fermentative growth. A search for genes involved in fermentation indicated that ftsH is required, and is also needed to a lesser extent for nitrate respiration. These results show that nitrate respiration and fermentation of B. subtilis are governed by divergent regulatory pathways.

ATP-Dependent Proteases↗
PubMed1997

Regulation of Bacillus subtilis sigmaH (spo0H) and AbrB in response to changes in external pH.

The RNA polymerase sigma subunit, sigmaH, of Bacillus subtilis is required for the transcription of genes that are induced in late-growth cultures at high cell density, including genes that function in sporulation. The expression of sigmaH-controlled genes is repressed when nutrient broth sporulation medium (Difco sporulation medium [DSM]) is supplemented with high concentrations of glucose and glutamine (DSM-GG), preferred carbon and nitrogen sources of B. subtilis. Under these conditions, the pH of the DSM-GG medium decreases to approximately 5. Raising the pH by the addition of morpholinepropanesulfonic acid (MOPS) or Tris-HCl (pH 7.5) results in a dramatic increase in the expression of lacZ fusions to sigmaH-dependent promoters. Correspondingly, the level of sigmaH protein was higher in cells of late-growth DSM-GG cultures treated with a pH stabilizer. When sigmaH-dependent gene expression was examined in cells bearing a mutation in abrB, encoding the transition state regulator that negatively controls genes transcribed by the sigmaH form of RNA polymerase, derepression was observed as well as an increase in medium pH. Reducing the pH with acetic acid resulted in repression, suggesting that AbrB was not functioning directly in pH-dependent repression but was required to maintain the low medium pH in DSM-GG. AbrB protein levels were high in late-growth, DSM-GG cultures but significantly lower when the pH was raised by Tris-HCl addition. An active tricarboxylic acid (TCA) cycle was required to obtain maximum derepression of sigmaH-dependent transcription, and transcription of the TCA cycle enzyme gene citB was repressed in DSM-GG but derepressed when the pH was artificially raised. The negative effect of low pH on sigmaH-dependent lacZ expression was also observed in unbuffered minimal medium and appeared to be exerted posttranslationally with respect to spo0H expression. However, the addition of amino acids to the medium caused pH-independent repression of both sigmaH-dependent transcription and spo0H-lacZ expression. These results suggest that spo0H transcription or translation is repressed by a mechanism responding to the availability of amino acids whereas spo0H is posttranslationally regulated in response to external pH.

Aconitate Hydratase↗
PubMed1996

A new enzyme superfamily - the phosphopantetheinyl transferases.

BACKGROUND: All polyketide synthases, fatty acid synthases, and non-ribosomal peptide synthetases require posttranslational modification of their constituent acyl carrier protein domain(s) to become catalytically active. The inactive apoproteins are converted to their active holo-forms by posttranslational transfer of the 4'-phosphopantetheinyl (P-pant) moiety of coenzyme A to the sidechain hydroxyl of a conserved serine residue in each acyl carrier protein domain. The first P-pant transferase to be cloned and characterized was the recently reported Escherichia coli enzyme ACPS, responsible for apo to holo conversion of fatty acid synthase. Surprisingly, initial searches of sequence databases did not reveal any proteins with significant peptide sequence similarity with ACPS. RESULTS: Through refinement of sequence alignments that indicated low level similarity with the ACPS peptide sequence, we identified two consensus motifs shared among several potential ACPS homologs. This has led to the identification of a large family of proteins having 12-22 % similarity with ACPS, which are putative P-pant transferases. Three of these proteins, E. coli EntD and o195, and B. subtilis Sfp, have been overproduced, purified and found to have P-pant transferase activity, confirming that the observed low level of sequence homology correctly predicted catalytic function. Three P-pant transferases are now known to be present in E. coli (ACPS, EntD and o195); ACPS and EntD are specific for the activation of fatty acid synthase and enterobactin synthetase, respectively. The apo-protein substrate for o195 has not yet been identified. Sfp is responsible for the activation of the surfactin synthetase. CONCLUSIONS: The specificity of ACPS and EntD for distinct P-pant-requiring enzymes suggests that each P-pant-requiring synthase has its own partner enzyme responsible for apo to holo activation of its acyl carrier domains. This is the first direct evidence that in organisms containing multiple P-pant-requiring pathways, each pathway has its own posttranslational modifying activity.

Amino Acid Sequence↗
PubMed1996

Two-component regulatory proteins ResD-ResE are required for transcriptional activation of fnr upon oxygen limitation in Bacillus subtilis.

Bacillus subtilis can grow anaerobically in the presence of nitrate as a terminal electron acceptor. The two component regulatory proteins, ResD and ResE, and an anaerobic gene regulator, FNR, were previously shown to be indispensable for nitrate respiration in B. subtilis. Unlike Escherichia coli fnr, B. subtilis fnr transcription was shown to be highly induced by oxygen limitation. fnr is transcribed from its own promoter as well as from a promoter located upstream of narK, the first gene in the narK-fnr dicistronic operon. DNA fragments containing the narK promoter, the fnr promoter, and both of the promoters were used to construct three lacZ fusions to examine the transcriptional regulation of the narK-fnr operon. ResDE was found to be required for transcriptional activation of fnr from the fnr-specific promoter, and FNR was required for activation of narK-fnr transcription from the FNR-dependent narK operon promoter under anaerobiosis. In order to determine if the requirement for ResDE in nitrate respiration is solely to activate fnr transcription, fnr was placed under control of the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter, Pspac. The observed defect in anaerobic growth of a Pspac-fnr delta resDE mutant in the presence of IPTG indicated that resDE has an additional role in B. subtilis anaerobic gene regulation.

Anaerobiosis↗
PubMed1996

Oxygen-controlled regulation of the flavohemoglobin gene in Bacillus subtilis.

A gene, hmp, which encodes a ubiquitous protein homologous to hemoglobin was isolated among genes from Bacillus subtilis that are induced under anaerobic conditions. The hmp protein belongs to the family of two-domain flavohemoproteins, homologs of which have been isolated from various organisms such as Escherichia coli, Alcaligenes eutrophus, and Saccharomyces cerevisiae. These proteins consist of an amino-terminal hemoglobin domain and a carboxy-terminal redox active site domain with potential binding sites for NAD(P)H and flavin adenine dinucleotide. The expression of hmp is strongly induced upon oxygen limitation, and the induction is dependent on a two-component regulatory pair, ResD and ResE, an anaerobic regulator, FNR, and respiratory nitrate reductase, NarGHJI. The requirement of FNR and NarGHJI for hmp expression is completely bypassed by the addition of nitrite in the culture medium, indicating that fnr is required for transcriptional activation of narGHJI, which produces nitrite, leading to induction of hmp expression. In contrast, induction of hmp was still dependent on resDE in the presence of nitrite. A defect in hmp in B. subtilis has no significant effect on anaerobic growth.

Anaerobiosis↗
PubMed1996

Plasmid-amplified comS enhances genetic competence and suppresses sinR in Bacillus subtilis.

The establishment of genetic competence in Bacillus subtilis is controlled by a vast signal transduction network involving the products of genes that function in several postexponential-phase processes. Two of these proteins, SinR and DegU, serve as molecular switches that influence a cell's decision to undergo either sporulation or genetic competence development. In order to determine the roles of SinR and DegU in competence control, multicopy suppression experiments with plasmid-amplified comS, SinR, and degU genes were undertaken. Multicopy comS was found to elevate competence gene transcription and transformation efficiency in both wild-type and sinR mutant cells but not in degU mutant cells. Multicopy degU failed to suppress comS or sinR mutations. No suppression of comS or degU by multicopy sinR was observed. The expression of a comS'::'lacZ translational fusion and srf-lacZ operon fusion was examined in sinR cells and cells bearing plasmid-amplified sinR. The expression of comS'::'lacZ gene fusion was reduced by the sinR mutation, but both comS'::'lacZ and srf-lacZ were repressed by multicopy sinR. Cells bearing plasmid-amplified sinR were poorly competent. These results suggest that sinR is required for optimal comS expression but not transcription from the srf promoter and that SinR at high concentrations represses srf transcription initiation.

Bacillus subtilis↗
PubMed1995

Translation of the open reading frame encoded by comS, a gene of the srf operon, is necessary for the development of genetic competence, but not surfactin biosynthesis, in Bacillus subtilis.

A small open reading frame, comS of the srf operon, is the site of mutations that impair competence development in Bacillus subtilis. comS open reading frame translation was required for competence, as was confirmed by the suppression of a comS amber mutation [comS(Am)] by the nonsense suppressor sup-3. comS(Am), when introduced into the srf operon, eliminated late competence gene expression but had no significant effect on surfactin production.

Amino Acid Sequence↗
PubMed1995

Nitrogen regulation of nasA and the nasB operon, which encode genes required for nitrate assimilation in Bacillus subtilis.

The divergently transcribed nasA gene and nasB operon are required for nitrate and nitrite assimilation in Bacillus subtilis. The beta-galactosidase activity of transcriptional lacZ fusions from the nasA and nasB promoters was high when cells were grown in minimal glucose medium containing poor nitrogen sources such as nitrate, proline, or glutamate. The expression was very low when ammonium or glutamine was used as the sole nitrogen source. The repression of the genes during growth on good sources of nitrogen required wild-type glutamine synthetase (GlnA), but not GlnR, the repressor of the glnRA operon. Primer extension analysis showed that the -10 region of each promoter resembles those of sigma A-recognized promoters. Between the divergently oriented nasA and nasB promoters is a region of dyad symmetry. Mutational analysis led to the conclusion that this sequence is required in cis for the activation of both nasA and nasB. The derepression of these genes in a glnA mutant also required this sequence. These results suggest that an unidentified transcriptional activator and glutamine synthetase function in the regulation of nasA and the nasB operon.

Bacillus subtilis↗
PubMed1995

Identification and isolation of a gene required for nitrate assimilation and anaerobic growth of Bacillus subtilis.

The Bacillus subtilis narA locus was shown to include narQ and narA. The putative product of narQ is similar to FdhD, which is required for formate dehydrogenase activity in Escherichia coli. NarA showed homology to MoaA, a protein involved in biosynthesis of the molybdenum cofactor for nitrate reductase and formate dehydrogenase. Analysis of mutants showed that narA but not narQ is required for both nitrate assimilation and respiration.

Anaerobiosis↗
PubMed1995

[Epidemiology of toxoplasmosis: worldwide status].

A literature review of toxoplasmosis seroprevalence data available over the last 40 years has been conducted both among European women of reproductive age and general populations at the global level. Summary maps are presented. In Europe, the highest seroprevalences have been reported from France, both among women of reproductive age and the general population. There is a higher seroprevalence in Central Europe than in Scandinavia or the United Kingdom. At the world level, prevalences similar to those of Central Europe have been reported in sub-Saharan Africa and Latin America. Lower prevalences are found in North America, South-East Asia, and Oceania. The limitations of such comparisons are discussed.

Animals↗
PubMed1995

[The status of infection with Toxoplasma gondii in the Swiss population: contribution of a seroepidemiologic study from the Zurich canton].

To obtain information on the seroprevalence of specific antibodies to Toxoplasma gondii at the level of the general population, a study was performed using 4300 serum samples: 3500 samples collected at the blood donor center of Zürich (individuals between 18 and > 65 years old), 500 serum samples from children referred to the University Children's Hospital of Zürich (between 1 month and 17 years old) and 300 maternal sera, collected at delivery (from a mother-child sera collection of the University Children's Hospital of Zürich). Samples were systematically collected by age- (5 years) and sex-groups (sex ratio 1:1), until the desired number was obtained. The crude seroprevalence of specific IgG anti-T. gondii was 52.4% (confidence interval at 95% [p < 0.05]: 50.9-53.9). There were no significant differences between males and females. The seroprevalence of the group of women of childbearing age (between 20 and 40) was 40%. The comparison of this value with the results obtained from 300 maternal sera from the mother-child collection revealed no significant difference between the two groups. On the basis of these results and assuming that the information obtained at the level of the general population in the region of Zürich is representative of Switzerland, it was possible to calculate the required sample size to initiate a national study on seroprevalence of the infection with T. gondii among women at time of delivery.

Adolescent↗
PubMed1995

[Epidemiology of toxoplasmosis in Switzerland: national study of seroprevalence monitored in pregnant women 1990-1991].

A national study was performed to assess the seroprevalence of anti-Toxoplasma gondii IgG in pregnant women at the time of delivery. The study was organized between 1990 and 1991 in 23 out of 26 Swiss cantons. 9059 women, corresponding to 11.8% of the annual total of births in those cantons, were included. The global seroprevalence of specific IgG was 46.1% (95% confidence interval: 45.0-47.1%). There was no significant difference in seroprevalence between different cantons after adjustment according to age. At the national level, the seroprevalence was 46.0% for Swiss women and 45.8% for women of other nationality (information on nationality was available only for 8382 persons). The use of a model of linear regression according to age showed that the risk of seroconversion among seronegative women during their 9 months of pregnancy was 1.21%. In addition, a certain number of data were calculated from that value to "simulate" a theoretical situation with absence of specific serological screening during pregnancy at a national level and consequently, absence of appropriate treatment. It was estimated that 548 cases of seroconversion would occur annually during pregnancy. This would lead to 183 congenital transmissions of toxoplasmosis, among which, 75% would be asymptomatic at birth. The number of expected pathologies would be: 40 cases of chorioretinitis with impared vision, 18 cases of cerebral lesions, and 2.7 cases of perinatal death. We observed positive results for specific anti-T. gondii-IgM in 1.7% of persons tested. This result can be the source of medical interpretation difficulties if the serum sample is the first done during pregnancy.

Adolescent↗
PubMed1995

[Prevention of congenital toxoplasmosis in Europe].

To sound out prevalent opinions among health authorities in Europe concerning the control and prevention of congenital toxoplasmosis (CT), a questionnaire was sent to the 28 WHO member countries in Europe. The questionnaire was returned by 23 countries. Only 7 countries recommend systematic screening of pregnant women. The reasons given by the 14 countries which do not recommend systematic screening are diverse: recommendations are in preparation, unfavourable cost-benefit return, absence of satisfactory treatment, programme not possible, incidence level too low, etc. 11 countries have a surveillance system for CT, of which only 3 are among the countries which recommend systematic screening. However, the absence of a multidisciplinary approach does not permit proper surveillance of cases. It appears from this survey that control of CT is undertaken in a very heterogeneous manner in Europe and no country has a programme whose impact on CT can be measured. So far, the European experience does not permit conclusions either in favour of or against a programme for the systematic screening of CT. However, cost-benefit analysis plays a very important role in determining whether such a programme should be implemented. Parameters such as the security of diagnosis (standardization of methods, quality and experience of the laboratories) and the monitoring of cases (definition, multidisciplinary approach to the surveillance and long-term treatment of patients, national collection of case reports, evaluation of the programme) are indispensable for the implementation of an effective surveillance system.

Cost-Benefit Analysis↗
PubMed1994

Identification of comS, a gene of the srfA operon that regulates the establishment of genetic competence in Bacillus subtilis.

Genetic competence (the ability to internalize exogenous DNA) in Bacillus subtilis is dependent on a regulatory pathway that activates the expression of a battery of competence-specific genes. The srfA operon, encoding the subunits of surfactin synthetase, which catalyzes the nonribosomal synthesis of the peptide antibiotic surfactin, also functions in the competence regulatory pathway. The DNA encoding only one of the seven amino acid-activating domains of surfactin synthetase, the valine-activating domain (srfAB1), is necessary for competence. Deletion analysis revealed that a 569-bp fragment of srfAB1, fused to the srfA promoter, complements a srfA deletion mutation (delta srfA) with respect to competence. This fragment contains an open reading frame consisting of 46 amino acids (orf46), which is out of frame with srfAB1. A frameshift mutation in srfAB upstream of orf46 has no effect on competence but a frameshift and nonsense mutation in orf46 resulted in failure to complement the delt srfA mutation. These results indicate that orf46 encodes the srfA-associated competence regulatory factor. Computer-aided analysis of the putative orf46 product (ComS) shows similarity to the homeodomain of the POU domain class of eukaryotic transcriptional regulators.

Amino Acid Sequence↗