RESUMO
A transcriptional fusion (opgG1::MudJ) to the opgGH operon of Salmonella enterica serovar Typhimurium (S. Typhimurium) LT2, isolated by resistance to mecillinam, was used to study the influence of global regulators RpoS, ppGpp, and cAMP/cAMP-receptor protein (CRP) on expression of the opgGH operon and osmoregulated periplasmic glucan (OPG) content. Neither high growth medium osmolarity nor absence of ppGpp or CRP had important effects on opgG1::MudJ expression in exponential cultures. However, under the same conditions, OPG content was strongly decreased by high osmolarity or cAMP/CRP defectiveness, and reduced to a half by lack of ppGpp. In stationary cultures, high osmolarity as well as CRP loss caused significant descents in opgG1::MudJ expression that were compensated by inactivation of RpoS sigma factor. No effect of RpoS inactivation on OPG content was observed. It is concluded that opgGH expression in S. Typhimurium is only slightly affected by high osmolarity, but is inversely modulated by RpoS level. On the other hand, osmolarity and the cAMP/CRP global regulatory system appear to control OPG content, either directly or indirectly, mainly at the post-transcriptional level.
Assuntos
Proteínas de Bactérias/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Glucanos/metabolismo , Polissacarídeos Bacterianos/metabolismo , Salmonella typhimurium/metabolismo , Proteínas de Bactérias/genética , Glucanos/análise , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Óperon , Periplasma/química , Periplasma/metabolismo , Polissacarídeos Bacterianos/análise , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Fator sigma/metabolismo , Equilíbrio HidroeletrolíticoRESUMO
By screening for high-level mecillinam resistant derivatives of a low-level resistant strain (cysB403 galE1922 relA21::Tn10) of Salmonella enterica serovar Typhimurium, a MudJ insertion in the gene for soluble lytic transglycosylase (slt) was isolated. This insertion (slt-1::MudJ) increased the resistance to mecillinam of cysB and cysE strains (MIC: about 20-40 microg mL(-1)) to a strikingly high level (MIC: 160 microg mL(-1)). As in Escherichia coli K-12, the slt mutation slightly increased the sensitivity of the wild type and of several strains that carried mutations that did not increase mecillinam resistance. All the strains acquired a spherical cell shape when treated with mecillinam. The effect of slt-1::MudJ was limited to mecillinam, the response to several other antibiotics remaining unaltered by the insertion. The results presented in this paper demonstrate that soluble lytic transglycosylase performs an important role in the response to mecillinam, which only becomes evident when failure of CysB/CysE function causes medium-level resistance. The results also suggest that soluble lytic transglycosylase interacts with, and is partially inhibited by normal lipopolysaccharide.
Assuntos
Andinocilina/farmacologia , Glicosiltransferases/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/enzimologia , Acetiltransferases/genética , Andinocilina/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Proteínas de Escherichia coli , Glicosiltransferases/genética , Testes de Sensibilidade Microbiana , Mutagênese Insercional , Mutação , Serina O-AcetiltransferaseRESUMO
Insertion of factor MudJ in the intergenic region between divergent genes yrfF and yrfE, at centisome 76 in the genome of Salmonella enterica serovar Typhimurium LT2, confers the characteristics recently described for mucM mutants, i.e. mucoidy and resistance to mecillinam. Cloning of the intergenic region plus either the yrfF or the yrfE gene in a multicopy plasmid showed that only the plasmid carrying the yrfF gene complemented mucM mutants, thus suggesting that mucM mutations are in fact yrfF mutations. A null yrfF mutation obtained by insertion of a kanamycin cassette into the yrfF open reading frame (yrfF28::Kan) produced abortive colonies when transduced to a wild-type strain but was normally accepted by rcsB, rcsC or yojN strains. Neither mutations preventing synthesis of the capsular exopolysaccharide colanic acid (cps, galE) nor rcsA mutations, which reduce expression of cps genes, conferred tolerance to the lethal yrfF28::Kan mutation. Spontaneous suppressor mutations arose very frequently in abortive yrfF28::Kan colonies, and all of them affected either rcsC, yojN, or rcsB genes. Thus, the lethal effect caused by inactivation of gene yrfF appears to be mediated by a function that is dependent on the rcsC-yojN-rcsB phosphorelay system but does not involve synthesis of colanic acid.
