Search PubMed⌕ Search

Biomedical subjects

J Casadesús

Publications and source records attributed to J Casadesús.

At least 19 recordsLinked to original sources
PubMed2001

Genetic mapping by duplication segregation in Salmonella enterica.

MudP and MudQ elements were used to induce duplications in Salmonella enterica by formation of a triple crossover between two transduced fragments and the host chromosome. The large size (36 kb) of MudP and MudQ is a favorable trait for duplication formation, probably because homology length is a limiting factor for the central crossover. Additional requirements are a multiplicity of infection of 2 or higher in the infecting phage suspensions (which reflects the need of two transduced fragments) and an exponentially growing recipient (which reflects the need of a chromosome replication fork). We describe a set of 11 strains of S. enterica, each carrying a chromosomal duplication with known endpoints. The collection covers all the Salmonella chromosome except the terminus. For mapping, a dominant marker (e.g., a transposon insertion in or near the locus to be mapped) is transduced into the 11-strain set. Several transductants from each cross are grown nonselectively, and haploid segregants are scored for the presence of the marker. If all the segregants contain the transduced marker, it maps outside the duplication interval. If the marker is found only in a fraction of the segregants, it maps within the duplicated region.

Alleles↗
PubMed2001

Daily time course of whole-shoot gas exchange rates in two drought-exposed Mediterranean shrubs.

Effects of drought on water relations, whole-shoot gas-exchange characteristics, and pigment and zeatin concentrations were investigated in the Mediterranean shrubs rosemary (Rosmarinus officinalis L.) and lavender (Lavandula stoechas L.). Two-year-old, greenhouse-grown plants were placed in a whole-shoot gas-exchange measurement system and subjected to 10 days of drought, resulting in severe water stress, and then re-watered for 5 days in order to study their recovery. Water stress resulted in a significant decline in maximum whole-shoot net CO2 assimilation rates (An) for both species that was associated with reductions in leaf area and stomatal conductance. Because shoot dark respiration rate (Rd) was less sensitive to water stress than An, shoot Rd/An ratio increased from about 15 to 95% during water stress. No major changes in chlorophyll and carotenoid concentrations of rosemary leaves were observed during the experiments, but chlorophyll and carotenoid concentrations fell significantly in water-stressed lavender leaves. Zeatin concentrations were higher in rosemary leaves than in lavender leaves during water stress. After re-watering, whole-shoot An and Rd rapidly recovered to their pre-drought rates.

Carbon Dioxide↗
PubMed2001

Salmonella enterica serovar Typhimurium response involved in attenuation of pathogen intracellular proliferation.

Salmonella enterica serovar Typhimurium proliferates within cultured epithelial and macrophage cells. Intracellular bacterial proliferation is, however, restricted within normal fibroblast cells. To characterize this phenomenon in detail, we investigated the possibility that the pathogen itself might contribute to attenuating the intracellular growth rate. S. enterica serovar Typhimurium mutants were selected in normal rat kidney fibroblasts displaying an increased intracellular proliferation rate. These mutants harbored loss-of-function mutations in the virulence-related regulatory genes phoQ, rpoS, slyA, and spvR. Lack of a functional PhoP-PhoQ system caused the most dramatic change in the intracellular growth rate. phoP- and phoQ-null mutants exhibited an intracellular growth rate 20- to 30-fold higher than that of the wild-type strain. This result showed that the PhoP-PhoQ system exerts a master regulatory function for preventing bacterial overgrowth within fibroblasts. In addition, an overgrowing clone was isolated harboring a mutation in a previously unknown serovar Typhimurium open reading frame, named igaA for intracellular growth attenuator. Mutations in other serovar Typhimurium virulence genes, such as ompR, dam, crp, cya, mviA, spiR (ssrA), spiA, and rpoE, did not result in pathogen intracellular overgrowth. Nonetheless, lack of either SpiA or the alternate sigma factor RpoE led to a substantial decrease in intracellular bacterial viability. These results prove for the first time that specific serovar Typhimurium virulence regulators are involved in a response designed to attenuate the intracellular growth rate within a nonphagocytic host cell. This growth-attenuating response is accompanied by functions that ensure the viability of intracellular bacteria.

Animals↗
PubMed2000

Regulation of septation: a novel role for SerC/PdxF in Salmonella?

The sfiW locus of Salmonella enterica, previously identified by mutations that suppress the cell division defect of His-constitutive (His(c)) strains, corresponds to serC, the bifunctional gene for phosphoserine-oxoglutarate aminotransferase (SerC) and 2-ketoerythroic acid 4-phosphate transaminase (PdxF). SerC- mutants form small, nearly spherical cells in a wild-type (His+) background, suggesting that the SerC/PdxF product acts as a septation antagonist. Suppression of His(c) filamentation by serC mutations may be explained by loss of the anti-septation activity of SerC/PdxF. The isolation of serC alleles that have lost their biosynthetic activities but are still able to inhibit septum formation suggests that the anti-septation activity of the SerC/PdxF product is unrelated to its known roles in serine and pyridoxine biosynthesis.

Aldehyde Oxidoreductases↗
PubMed1999

Lack of hotspot targets: a constraint for IS30 transposition in Salmonella.

IS30 is an insertion element common in E. coli strains but rare or absent in Salmonella. Transfer of the IS30-flanked transposon Tn2700 to Salmonella typhimurium was assayed using standard delivery procedures of bacterial genetics (conjugation and transduction). Tn2700 'hops' were rare and required transposase overproduction, suggesting the existence of host constraints for IS30 activity. Sequencing of three Tn2700 insertions in the genome of S. typhimurium revealed that the transposon had been inserted into sites with a low homology to the IS30 consensus target, suggesting that inefficient Tn2700 transposition to the Salmonella genome might be caused by a lack of hotspot targets. This view was confirmed by the introduction of an IS30 'hot target sequence', whose sole presence permitted Tn2700 transposition without transposase overproduction. Detection of IS30-induced DNA rearrangements in S. typhimurium provided further evidence that the element undergoes similar activities in E. coli and S. typhimurium. Thus, hotspot absence may be the main (if not the only) limitation for IS30 activity in the latter species. If these observations faithfully reproduce the scenario of natural populations, establishment of IS30 in the Salmonella genome may have been prevented by a lack of DNA sequences closely related to the unusually long (24 bp) IS30 consensus target.

Bacteriophage T4↗
PubMed1999

DNA adenine methylase mutants of Salmonella typhimurium show defects in protein secretion, cell invasion, and M cell cytotoxicity.

Mutants of Salmonella typhimurium lacking DNA adenine methylase are attenuated for virulence in BALB/c mice. LD(50) values of a DNA adenine methylation (Dam)(-) mutant are at least 10(3)- to 10(4)-fold higher than those of the parental strain when administrated by oral or intraperitoneal routes. Dam(-) mutants are unable to proliferate in target organs but persist in low numbers in these locations. Efficient protection to challenge with the virulent parental strain is observed in mice infected with a Dam(-) mutant. Use of the ileal loop assay shows that Dam(-) mutants are less cytotoxic to M cells and fail to invade enterocytes. In the tissue culture model, lack of DNA adenine methylation causes reduced ability to invade nonphagocytic cells. In contrast, no effect is observed either in intracellular proliferation within nonphagocytic cells or in survival within macrophages. The invasion defect of Dam(-) mutants is correlated with a distinct pattern of secreted proteins, which is observed in both PhoP(+) and PhoP(-) backgrounds. Altogether, our observations suggest a multifactorial role of Dam methylation in Salmonella virulence.

Animals↗
PubMed1999

Repression of IS200 transposase synthesis by RNA secondary structures.

The IS 200 transposase, a 16 kDa polypeptide encoded by the single open reading frame (ORF) of the insertion element, has been identified using an expression system based on T7 RNA polymerase. In wild-type IS 200, two sets of internal inverted repeats that generate RNA secondary structures provide two independent mechanisms for repression of transposase synthesis. The inverted repeat located near the left end of IS 200 is a transcriptional terminator that terminates read-through transcripts before they reach the IS 200 ORF. The terminator is functional in both directions and may terminate >80% of transcripts. Another control operates at the translational level: transposase synthesis is inhibited by occlusion of the ribosome-binding site (RBS) of the IS 200 ORF. The RBS (5'-AGGGG-3') is occluded by formation of a mRNA stem-loop structure whose 3' end is located only 3 nt upstream of the start codon. This mechanism reduces transposase synthesis approximately 10-fold. Primer extension experiments with AMV reverse transcriptase have provided evidence that this stem-loop RNA structure is actually formed. Tight repression of transposase synthesis, achieved through synergistic mechanisms of negative control, may explain the unusually low transposition frequency of IS 200.

Base Pairing↗
PubMed1999

Synthesis of FinP RNA by plasmids F and pSLT is regulated by DNA adenine methylation.

DNA adenine methylase mutants of Salmonella typhimurium contain reduced amounts of FinP, an antisense RNA encoded by the virulence plasmid pSLT. Lowered FinP levels are detected in both Dam- FinO+ and Dam- FinO- backgrounds, suggesting that Dam methylation regulates FinP production rather than FinP half-life. Reduced amounts of F-encoded FinP RNA are likewise found in Dam- mutants of Escherichia coli. A consequence of FinP RNA scarcity in the absence of DNA adenine methylation is that Dam- mutants of both S. typhimurium and E. coli show elevated levels of F plasmid transfer. Inhibition of F fertility by the S. typhimurium virulence plasmid is also impaired in a Dam- background.

Adenine↗
PubMed1999

The virulence plasmids of Salmonella.

Certain Salmonella serovars belonging to subspecies I carry a large, low-copy-number plasmid that contains virulence genes. Virulence plasmids are required to trigger systemic disease; their involvement in the enteric stage of the infection is unclear. Salmonella virulence plasmids are heterogeneous in size (50-90 kb), but all share a 7.8 kb region, spv, required for bacterial multiplication in the reticuloendothelial system. Other loci of the plasmid, such as the fimbrial operon pef, the conjugal transfer gene traT and the enigmatic rck and rsk loci, may play a role in other stages of the infection process. The virulence plasmid of Salmonella typhimurium LT2 is self-transmissible; virulence plasmids from other serovars, such as Salmonella enteritidis and Salmonella choleraesuis, carry incomplete tra operons. The presence of virulence plasmids in host-adapted serovars suggests that virulence plasmid acquisition may have expanded the host range of Salmonella.

Bacterial Adhesion↗
PubMed1998

Underground metabolism.

All enzymes are able to use alternative substrates. When these are naturally occurring metabolites, an 'underground reaction' takes place. Examples are presented in which underground metabolism of this sort produces an observable phenotype. Although biological processes can be remarkably accurate, evolution has selected error rates far from perfect. It is suggested here that a certain level of metabolic inaccuracy, in addition to saving energy, may also confer an evolutionary advantage, for example by providing metabolic plasticity. Since underground reactions are unpredictable from DNA sequence data, caution is in order when interpreting correlations between genetic disorders and pathological syndromes.

Alanine↗
PubMed1998

The P22 Erf protein and host RecA provide alternative functions for transductional segregation of plasmid-borne duplications.

A tandem DNA duplication carried on a ColE1-derived plasmid segregates at high frequency upon generalized transduction by phage P22 HT. Transductional segregation of the plasmid-borne duplication can be promoted either by RecA or by the Erf function of P22, indicating that transductional segregation is a consequence of the recombination events that re-circularize the plasmid in the recipient cell. RecA-mediated and Erf-mediated transduction give similar frequencies of duplication segregation and yield the same types of segregation products, indicating that two distinct recombination machineries (RecA + RecBCD and Erf + RecBCD) perform similar or identical recombination reactions on plasmid DNA substrates transduced by bacteriophage P22 HT.

Bacteriophage P22↗
PubMed1998

The sfiX, rfe and metN genes of Salmonella typhimurium and their involvement in the His(c) pleiotropic response.

Two loci involved in the pleiotropic response of His(c) strains of Salmonella typhimurium (sfiX and sfiY) have been characterized at the molecular level. The sfiX gene (CS 44) has been identified as a homolog of the E. coli gene sanA, located downstream of the cytidine deaminase gene (cdd). The cdd-sanA (or cdd-sfiX) operon shows a highly conserved structure in E. coli and Salmonella. Like its E. coli homolog, the sfiX gene of S. typhimurium is required for vancomycin resistance at high temperature. The dual effect of sfiX mutations (induction of vancomycin sensitivity and suppression of cell division inhibition) suggests a link between SfiX function and murein synthesis. The sfiY locus (CS 85), contains two genes arranged in a single transcriptional unit. The upstream gene is a homolog of the E. coli gene rfe; mutations in this gene suppress the cell division defect of His(c) strains. The suppressor effect of rfe mutations can be reproduced by tunicamycin, suggesting that suppression of filamentation results from an increase in the intracellular concentration of UDP-N-acetyl-D-glucosamine. The gene located downstream of rfe is also found in E. coli but its function is unknown. Insertions in rfe suppress the methionine requirement of His(c) strains of S. typhimurium by a polar effect on the downstream gene, tentatively designated metN. Complementation with a rfe+ clone indicates that the rfe gene is not involved in the methionine requirement of His(c) strains. Thus metN expression appears to cause methionine auxotrophy in a His(c) background.

Cell Division↗
PubMed1998

Cell division inhibition in Salmonella typhimurium histidine-constitutive strains: an ftsI-like defect in the presence of wild-type penicillin-binding protein 3 levels.

Histidine-constitutive (Hisc) strains of Salmonella typhimurium undergo cell division inhibition in the presence of high concentrations of a metabolizable carbon source. Filaments formed by Hisc strains show constrictions and contain evenly spaced nucleoids, suggesting a defect in septum formation. Inhibitors of penicillin-binding protein 3 (PBP3) induce a filamentation pattern identical to that of Hisc strains. However, the Hisc septation defect is caused neither by reduced PBP3 synthesis nor by reduced PBP3 activity. Gross modifications of peptidoglycan composition are also ruled out. D-Cycloserine, an inhibitor of the soluble pathway producing peptidoglycan precursors, causes phenotypic suppression of filamentation, suggesting that the septation defect of Hisc strains may be caused by scarcity of PBP3 substrate.

Aztreonam↗
PubMed1997

Conserved structure of IS200 elements in Salmonella.

Sequence analysis of three IS200 elements (two from Salmonella typhimurium, one from Salmonella abortusovis) reveals a highly conserved structure, with a length of 707-708 bp and absence of terminal repeats. IS200 contains an open-reading-frame (ORF) which potentially encodes a peptide of 151 amino acids, with a putative ribosome-binding-site properly placed upstream of the ORF. A potential RNA stem-loop structure that might occlude the ribosome-binding-site of the ORF is also found. Another conserved trait is a potential RNA hairpin which resembles a Rho-independent transcription terminator, located near one end of IS200. The junctions between IS200 and host DNA sequences are A+T-rich. Upon insertion, IS200 duplicates 1-2 bp of host DNA sequences. The observation that IS200 elements characterized as 'hops' are roughly identical to those residing in the Salmonella genome suggests that IS200 transposition is unlikely to generate inactive copies. If such is the case and many or all IS200 elements are active, the extremely low frequency of IS200 transposition may reflect the normal behavior of the element.

Amino Acid Sequence↗
PubMed1997

Cloning with Mud-P22 hybrid prophages: mapping of IS200 elements on the chromosome of Salmonella typhimurium LT2.

Specific DNA fragments from the chromosome of Salmonella typhimurium LT2 were packaged in P22 capsids by induction of "locked-in" Mud-P22 hybrid prophages. High yields of the packaged DNA were obtained upon capsid disruption. DNA hybridization using a fragment of insertion sequence IS200 as probe permitted physical mapping of IS200 elements on the chromosome of S. typhimurium LT2 within +/- 1 centisome (CS). IS200 copies were found at the following locations: CS 24 (copy VI), CS 53 (copy V), CS 63 (copy I), CS 80 (copy II) and CS 93 (copy III). Copy IV, previously mapped near fliA (CS 42), was not included in our study.

Bacteriophage P22↗
PubMed1997

Identification of Salmonella abortusovis by PCR amplification of a serovar-specific IS200 element.

Field and collection isolates of Salmonella abortusovis carry one IS200 element in a distinct chromosome location. IS200 is not found in the corresponding region of the chromosome of other Salmonella serovars. Sequencing of the boundaries of the S. abortusovis-specific IS200 insertion permitted the design of primers for the amplification of this IS200 element by PCR. Isolates of S. abortusovis are identified by the amplification of a DNA fragment of about 900 bp or larger. PCR amplification of DNA from salmonellae other than S. abortusovis yields either a fragment of about 200 bp or no product. The high specificity of the assay is confirmed by the absence of cross-reactivity with the following templates: (i) sheep DNA, (ii) DNAs from abortion-causing agents other than S. abortusovis, and (iii) DNAs from microorganisms that do not cause abortion but are common in flocks.

Animals↗
PubMed1996

recB recJ mutants of Salmonella typhimurium are deficient in transductional recombination, DNA repair and plasmid maintenance.

recB recJ mutants of Salmonella typhimurium are deficient in transduction of chromosomal markers and ColE1-derived plasmids, and also in the maintenance of ColE1 and F plasmids. Plasmid instability is less severe in recD recJ strains; ColE1 plasmid DNA preparations from these strains show an increased yield of high molecular weight (HMW) linear multimers and a concomitant reduction in plasmid monomers compared to the wild type. Plasmids remain unstable in recA recD recJ mutants; since these do not produce HMW linear concatemers, we propose that a decrease in monomer production leads to plasmid instability. recB recJ strains also display decreased viability, a component of which may be related to their deficiency in DNA repair. In contrast to their severe defects in recombination, DNA repair and plasmid maintenance, recB recJ mutants of S. typhimurium behave similarly to the wild type in the segregation of chromosome duplications. The latter observation suggests that neither RecBCD nor RecJ functions are required for chromosomal recombination events that do not involve the use of free ends as recombination substrates.

Bacterial Proteins↗