Sublethal effects of ultraviolet A radiation on Enterobacter cloacae.

We report the sublethal effects of ultraviolet A (UVA) on Enterobacter cloacae in comparison with those produced in Escherichia coli. UVA-induced sublethal effects were investigated in either bacterial membrane and at tRNA level. Limited dependence on oxygen concentration for photoinduced inhibition of biochemical membrane functions and low levels of oxidative damage during the irradiation period were found in En. cloacae. On the other hand, ultraviolet spectroscopy and reversed-phase HPLC analysis of hydrolysed tRNA showed that radio induced damage to tRNA is similar in En. cloacae and E. coli. Nevertheless, growth delay induced by UVA in En. cloacae was shorter than that found in E. coli submitted to the same experimental conditions. A limited post-irradiation ppGpp accumulation and the absence of any influence of the membrane damage on the growth delay extent seem to be responsible for the shortness of this effect in En. cloacae. Most of the differences between En. cloacae and E. coli could be attributed to an increased ability of En. cloacae to overcome oxidative stress during UVA exposure.

In vivo effects of anti-inducers of the cysteine regulon in Salmonella typhimurium.

Growth on readily utilizable sulfur sources reduces expression of the cysteine regulon in Salmonella typhimurium. Inhibition of serine transacetylase by cysteine and direct actions of the anti-inducers sulfide and thiosulfate are responsible for reduction of expression. In order to evaluate individual contributions of each mechanism, the inhibitory effects of Na2S and Na2S2O3 were studied in strains with or without the capacity to synthesize cysteine from these compounds, using a transcriptional fusion to the cysDNC operon. In a cysK cysM strain, although cysteine synthesis from sulfide and thiosulfate was blocked, Na2S and Na2S2O3 efficiently reduced expression of the cysDNC operon. The inhibitory effect observed in this mutant was equivalent to 70-100% of that found in a strain carrying the fusion in a wild-type context grown in the same conditions. The actions of sulfide and thiosulfate as anti-inducers seem therefore to be responsible for most of the reduction of expression caused by these agents in vivo.

Involvement of cysB and cysE genes in the sensitivity of Salmonella typhimurium to mecillinam.

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.

Increased susceptibility to beta-lactam antibiotics and decreased porin content caused by envB mutations of Salmonella typhimurium.

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.

Contact area of bovine somatotropin dimer: involvement of tyrosine 142.

The presence of tyrosine residues in the contact area between protomers of bovine somatotropin dimers (Fernandez & Delfino, Biochem. J. 209, 107-115, 1983) was investigated taking advantage of the impaired self-associating ability of molecules iodinated at such residues. Reaction of bovine somatotropin dissolved in 8 M urea with the NaI-Chloramine T couple (2.1 x 10(-4) M) rendered a preparation with 3.1 iodine atoms per molecule which, by stepwise elimination of the denaturant and gel filtration through Sephadex G-100, originated two distinguishable populations: one able (iododerivatives I), the other unable (iododerivatives II) to self-associate. After frontal analysis, iododerivatives II were found to be unable to interact even with native molecules. Identification of the reacting tyrosine residues indicated that iodination of tyrosine 142 was responsible for the loss of the ability to form dimers in iododerivatives II. Iodohormones retained the ability to bind to somatogenic mouse hepatocyte receptors--the relative potency for iododerivatives I and II being 0.60 (0.34-1.03) and 0.71 (0.41-1.22) respectively.

Hydrophobic residues involved in the interaction between protomers of the bovine growth hormone dimer. Methionine and tyrosine residues.

The bovine growth hormone dimeric form covalently stabilized by cross-linking with dimethyl suberimidate (DMS) and the hormone modified by DMS without forming covalent links with other hormone molecules (DMS-bGH) were oxidized with chloramine-T at molar-ratios of 2 and 50 with respect to methionine content. The extent of oxidation undergone by each methionine residue, estimated on the purified tryptic peptides, closely resembled that obtained for the native hormone, thus suggesting that methionine residues are not involved in the protomers interaction area. Evaluation of the reactivity of tyrosine residues toward tetranitromethane indicated that, in both the covalent dimer and DMS-bGH, tyrosine residues 35, 174 and 142 are the more susceptible to undergo reaction. Net charges can be induced in the iodotyrosine residues in the iodinated hormone, by setting the pH at 10.5. At this pH, dissociation of a fraction of uniformly iodinated hormone was observed in the derivatives containing 2 or more iodine atoms, indicating that tyrosine residues might integrate the contact area between protomers.

Hydrophobic residues involved in the interaction between protomers of the bovine growth hormone dimer. Methionine and tyrosine residues.

The bovine growth hormone dimeric form covalently stabilized by cross-linking with dimethyl suberimidate (DMS) and the hormone modified by DMS without forming covalent links with other hormone molecules (DMS-bGH) were oxidized with chloramine-T at molar-ratios of 2 and 50 with respect to methionine content. The extent of oxidation undergone by each methionine residue, estimated on the purified tryptic peptides, closely resembled that obtained for the native hormone, thus suggesting that methionine residues are not involved in the protomers interaction area. Evaluation of the reactivity of tyrosine residues toward tetranitromethane indicated that, in both the covalent dimer and DMS-bGH, tyrosine residues 35, 174 and 142 are the more susceptible to undergo reaction. Net charges can be induced in the iodotyrosine residues in the iodinated hormone, by setting the pH at 10.5. At this pH, dissociation of a fraction of uniformly iodinated hormone was observed in the derivatives containing 2 or more iodine atoms, indicating that tyrosine residues might integrate the contact area between protomers.