RESUMO
Mucoid mutants of Salmonella enterica serovar Typhimurium isolated by resistance to mecillinam include lon (27%) and rcsC (8%) mutants but the most frequent class (65%) is affected in a new gene (mucM) located at centisome 76. mucM cells are shorter than mucM+ cells and rcsB mutations normalize size and response to mecillinam. Expression of ftsA1p, the ftsA-ftsZ promoter submitted to RcsB stimulation, is greatly increased in mucM mutants, and this expression is dependent on RcsB and ftsA1p. It is proposed that the mucM product interferes with RcsB activation. Mucoidy results from the activation of cps genes and mecillinam resistance from ftsA-ftsZ overexpression, both traits caused by the increased activity of the RcsB effector. The same mechanism seems to be responsible for the resistance of mucoid rcsC mutants to mecillinam but the resistance of lon mutants is not dependent on RcsB and so responds to a different cause.
Assuntos
Andinocilina/farmacologia , Proteínas de Bactérias/genética , Proteínas do Citoesqueleto , Penicilinas/farmacologia , Salmonella enterica/genética , Proteínas de Bactérias/fisiologia , Resistência Microbiana a Medicamentos/genética , Expressão Gênica , Testes de Sensibilidade Microbiana , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/fisiologiaRESUMO
Rapid growth of Salmonella typhimurium LT2 in rich LB-broth caused the induction of the cysteine regulon when the culture reached an optical density at 650 nm of 1.5. Addition of 0.05 mM L-cystine to LB-broth abolished induction, while 0.025 mM L-cystine delayed it for a doubling time (30 min). Induction did not occur when lack of oxygen or a temperature shift to 19 degrees C slowed down growth in LB-broth. Uninduced cultures reached growth yield similar to that of induced cultures after overnight incubation. Growth on solid LB-medium also brought about induction, but to a lower level than in liquid medium. Leaky cysB mutants, which are sensitive to the beta-lactam antibiotic mecillinam, displayed partial induction, whereas mecillinam-resistant cysB and cysE mutants showed no induction. It is suggested that induction develops in these mutants when constitutive transport systems fail to satisfy the high uptake of cysteine demanded by fast-growing cultures. The behavior of cysB mutants agrees with the hypothesis that, under some conditions, mecillinam action would be dependent on expression of the cysteine regulon.
Assuntos
Andinocilina/farmacologia , Proteínas de Bactérias/metabolismo , Cisteína/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulon/genética , Salmonella typhimurium/genética , Proteínas de Bactérias/genética , Meios de Cultura , Testes de Sensibilidade Microbiana , Mutação , Penicilinas/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/crescimento & desenvolvimentoRESUMO
Round-cell (rodA, mre, divD) derivatives of a conditional alaS mutant of Salmonella typhimurium were studied under conditions allowing expression of tolerance to lethal cell shape mutations (41 degrees C), and under nontolerant conditions (30 degrees C). The rodA22::Tn10d(Kan) derivative grew normally (OD650 nm) in LB-broth at 30 degrees C; however, doubling of total cell count took much longer (130 min) than at 41 degrees C (57 min). Although the cells were able to divide in LB-broth at 30 degrees C, viable count on LB-agar at 30 degrees C was 10(3)-fold lower than on LB-agar at 41 degrees C. Phase-contrast microscopy of rodA cells incubated under different conditions showed that their size increased on LB-soft agar at 30 degrees C, but they failed to divide and finally lysed. In contrast, division occurred in LB-broth at 30 degrees C and also in LB-broth and LB-soft agar at 41 degrees C. The mre-17::Tn10d(Kan) derivative acted like the rodA strain whereas the divD135::Tn10d(Kan) mutant behaved normally both at 30 degrees C and 41 degrees C. It is concluded that rodA and mre mutations delay cell division, but are lethal only on solid medium. Mutations conferring tolerance to "lethal" rodA and mre mutations improve division performance both in liquid and solid media.
Assuntos
Meios de Cultura , Proteínas de Escherichia coli , Proteínas de Membrana , Mutação/fisiologia , Salmonella typhimurium/genética , Andinocilina/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Divisão Celular , Resistência Microbiana a Medicamentos , Temperatura Alta , Microscopia de Contraste de Fase , Salmonella typhimurium/citologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/crescimento & desenvolvimento , Fatores de TempoRESUMO
cysB and cysE strains were obtained as spontaneous mecillinam-resistant mutants of Salmonella typhimurium. The resistance to mecillinam was caused by the cys mutations which also conferred tolerance to lethal cell shape mutations. Most, but not all, cysB and cysE mutations from other origins displayed the same behavior. Resistance was abolished by O- and N-acetylserine in cysE mutants; by thiosulfate, sulfite, and sulfide in cysB mutants; and by cysteine in both types of mutants. It is concluded that an event involved in mecillinam action requires the inducer and the activator protein of the cysteine regulon.
Assuntos
Acetiltransferases , Andinocilina/farmacologia , Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Salmonella typhimurium/genética , Cisteína/genética , Cisteína/metabolismo , Proteínas de Escherichia coli , Mutação , Regulon , Salmonella typhimurium/efeitos dos fármacos , Serina/análogos & derivados , Serina/metabolismo , Serina O-Acetiltransferase , Sulfetos/metabolismo , Sulfitos/metabolismo , Tiossulfatos/metabolismoRESUMO
Lipopolysaccharide (LPS), spoT, and cya or crp mutations individually do not affect the minimum inhibitory concentration of mecillinam on Salmonella typhimurium. However, when mutations of two of these types were combined in the same strain, high-level resistance appeared, and increased even further when all three types of mutations were present. Most mutations affecting LPS (rfa, rfb, rfc) showed this behaviour, although to different degrees. The highest resistance to mecillinam was caused by galE and rfc mutations whereas almost no effect was noticed with rfaB or rfaK mutations. This phenomenon appears to be specific for mecillinam since none of several other antibiotics elicited it. Reduction of guanosine tetraphosphate (ppGpp) levels by introduction of a relA mutation did not significantly affect the MIC of mecillinam on strains carrying different combinations of spoT, galE, and cya or crp mutations. All the strains produced spherical cells in medium with a low concentration (0.05 microgram ml-1) of the antibiotic. These results suggest that the antibacterial action of mecillinam on S. typhimurium is somehow dependent on the interaction of LPS, cyclic AMP/cyclic AMP receptor protein (cAMP/CRP), and SpoT. The reported resistance to mecillinam of cya and crp mutants of Escherichia coli K-12 is probably due to the natural LPS defectiveness of this strain.
Assuntos
Adenilil Ciclases/genética , Andinocilina/farmacologia , Proteína Receptora de AMP Cíclico/genética , Lipopolissacarídeos/biossíntese , Pirofosfatases/genética , Salmonella typhimurium/efeitos dos fármacos , UDPglucose 4-Epimerase , Adenilil Ciclases/fisiologia , Antibacterianos/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Sequência de Carboidratos , Proteínas de Transporte , Conjugação Genética , Proteína Receptora de AMP Cíclico/fisiologia , Guanosina Tetrafosfato/metabolismo , Hexosiltransferases/genética , Lipopolissacarídeos/química , Dados de Sequência Molecular , Mutagênese , Pirofosfatases/fisiologia , Salmonella typhimurium/genética , Salmonella typhimurium/ultraestrutura , Resistência beta-Lactâmica/genéticaRESUMO
Thirty-three insertions of transposon Tn10 delta 16 delta 17 into genes involved in the control of rod cell shape were isolated in Salmonella typhimurium by the characteristic glossy appearance of colonies composed of spherical cells. Genetic tests demonstrated that 25 (76%) were insertions in the rodA gene, 7 (21%) were mre mutants, and 1 (3%) was a divD mutant. No insertion in the pbpA gene were found. Insertions in cell shape genes only appeared when strains displaying resistance to mecillinam (not caused by beta-lactamase production) were employed. Neither rodA nor mre insertions could be transduced to wild-type strains but they were normally accepted by mecillinam-resistant derivatives and by cya and crp mutants, which, unlike the corresponding Escherichia coli strains, did not display resistance to mecillinam. On the other hand, the divD insertion could be efficiently transduced to any strain. It is concluded that the rodA, mre, and divD genes are involved in the control of rod cell shape but, in addition, the RodA and Mre products perform some function(s) that is essential for wild-type cells but dispensable for some mecillinam-resistant strains, and for cya and crp mutants.
Assuntos
Proteínas de Escherichia coli , Genes Letais/genética , Proteínas de Membrana , Morfogênese/genética , Mutagênese Insercional , Salmonella typhimurium/genética , Andinocilina , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Resistência Microbiana a Medicamentos , Salmonella typhimurium/citologia , Transdução GenéticaRESUMO
Isogenic derivatives carrying envB6, envB9, or envB+ alleles were obtained from a strain of Salmonella typhimurium that was partially resistant to mecillinam, a beta-lactam antibiotic specific for penicillin-binding protein 2 (PBP 2). Testing of the isogenic strains with several antibacterial agents demonstrated that envB mutations either increased resistance (mecillinam) or did not affect the response (imipemen) to beta-lactams that act primarily on PBP 2, while susceptibilities to beta-lactams that act on PBP 1B, PBP 3, or both were increased. Furthermore, the susceptibilities of envB strains to hydrophobic compounds such as rifampin, novobiocin, or chloramphenicol were not modified, even though their susceptibilities to deoxycholate and crystal violet were enhanced. Outer cell membranes of envB mutants presented a 50% reduction in protein content compared with that of the isogenic envB+ strains, and OmpF and OmpD porins were particularly affected by the reduction. No alteration in the amount or pattern of periplasmic proteins was noticed, and lipopolysaccharides from envB mutants appeared to be normal by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. By using derivatives that produced a plasmid-encoded beta-lactamase, it was demonstrated that envB cells are slightly less permeable to cephalothin than envB+ bacteria are. It is concluded that the high susceptibility of envB mutants to beta-lactams is due to the increased effectiveness of the antibiotics on PBP 1B, PBP 3, or both.
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Salmonella/genética , Proteínas da Membrana Bacteriana Externa/genética , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Testes de Sensibilidade Microbiana , Permeabilidade , Porinas , Salmonella/efeitos dos fármacos , Salmonella/enzimologia , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-LactamasRESUMO
Incubation of streptomycin-resistant (rpsL) mutants of Salmonella typhimurium in alkaline nutrient medium containing streptomycin brought about an inhibition of cell growth that was readily reversed by removing the antibiotic or neutralizing the medium. Growth inhibition was maximal at pH 8.2 and a streptomycin concentration of 800 micrograms/ml. A similar amount of dihydrostreptomycin had a negligible effect, and 10-times-higher concentrations of this antibiotic were required to reproduce the streptomycin action. Addition of streptomycin (400 micrograms/ml) to rpsL cells in alkaline (pH 8.2) nutrient medium caused inhibition of protein and DNA synthesis and also, but to a lower degree, of RNA synthesis. This effect on macromolecular synthesis was not due to ATP deprivation, since ATP content rose after addition of the antibiotic. At pH 8.2, the rate of entrance of streptomycin increased fourfold with respect to the rate at pH 7.0, leading to a large accumulation of streptomycin into rpsL cells. Uptake of the antibiotic was halted by addition of KCN or chloramphenicol. Equal uptake was obtained with 800 micrograms of dihydrostreptomycin or 400 micrograms of streptomycin per ml, yet the former did not affect cell growth at that concentration. It is concluded that high pH stimulates streptomycin and dihydrostreptomycin uptake by rpsL strains but only streptomycin accumulation causes growth inhibition in cells lacking the high-affinity ribosomal site.