A dominant sulfhydryl-containing protein in the outer membrane of Neisseria gonorrhoeae

Abstract

By using a method that labels sulfhydryl-containing proteins in situ, we (6,14,15). It reacts selectively with the thiol anion of sulfhydryls, crosses biological membranes, and does not significantly alter the molecular weight of proteins (6,14,15).We have used mB to label the sulfhydryl-containing proteins of the outer membrane of Neissena gonorrhoeae. By labeling during growth, we have eliminated the effect of oxidative changes that occur during harvesting and preparation of fractions, thereby detecting the sulfhydryl status of proteins in situ.A nonpiliated, opaque colonial variant of N. gonorrhoeae 1384 (22, 23) was used for all studies. Growth conditions were as previously described (23) (Pharmacia, Piscataway, N.J.) with 0.9 mM dithiothreitol for 30 min and 5.4 mM mB for 20 min. The treated standards were diluted into sample buffer with mercaptoethanol (ME).Electrophoretic analysis of outer membranes from labeled samples revealed multiple fluorescent bands, with the two major fluorescent bands having apparent molecular masses of 41 and 31.5 kDa (Fig. 1A and B). On the basis of its intense fluorescence, the protein at 41 kDa is the major sulfhydryl-containing protein in the outer membrane. Gels stained for protein also showed the 41-kDa protein to be a major protein band in labeled fractions (Fig. 1D, lanes 3 and 5), with an intensity equivalent to that of PIII as seen in outer membrane fractions prepared by conventional methods. However, unlabeled fractions that were not reduced by ME showed no evidence of this protein (Fig. 1D, lanes 1 and 4). When unlabeled samples were reduced, only a lightly staining protein band at 41 kDa was seen (Fig. 1C, lanes 1 and 4). Samples obtained from different experiments (Fig. 1, lanes 1 to 3 versus lanes 4 and 5) confirmed the differences in the 41-kDa protein between labeled and unlabeled samples. Although there was some variation in the intensity of the 41-kDa protein between the two unlabeled, reduced samples, in both cases there was significantly less protein than in labeled samples.There are several possible reasons for the difference between the labeled and unlabeled samples. A likely explanation, given the nature of sulfhydryl compounds, the nature of the reaction with mB, and the effect of ME on the 41-kDa protein in the unlabeled samples, is that in the unlabeled samples artifacts of distribution occur during preparation of the fractions and result in decreases of the 41-kDa protein in the outer membrane; i.e., the protein undergoes autoxidation, with a change in conformation and loss from the membrane, whereas in labeled samples the 41-kDa protein is stable because of its reaction with mB. Another possibility is

Controls on Bacterial Cell Envelope Sulfhydryl Site Concentrations: The Effect of Glucose Concentration During Growth

“…32−34 In the present study, we found that increasing the concentration of glucose, a common carbon source that can be utilized by a wide range of bacteria, in the M9 minimal medium significantly increases the concentration of sulfhydryl sites within the cell envelopes of the three bacterial species studied (Figure 2b), suggesting that carbon utilization is required for the synthesis of bacterial sulfhydryl sites. Although sulfhydryl sites themselves do not contain carbon, some previous studies suggest that sulfhydryl sites are predominantly present attached to proteins within cell envelopes, 14,35,36 and carbon is an essential element in the framework of proteins. It is likely that the cells require a certain concentration of carbon source in order to have enough bioavailable carbon to construct sulfhydryl-containing proteins.…”

Role of bacterial cell surface sulfhydryl sites in cadmium detoxification by Pseudomonas putida

“…Although it is not clear why the P. putida cells contain 466 such high concentrations of cell surface sulfhydryl sites when they are cultured in the LB10 467 medium, Choi et al (2014) found that P. putida cells cultured in the LB10 medium have more than a three-fold higher protein content within their EPS matrix compared to P. putida cells grown in 469 two other minimal media. Proteins are likely the primary hosts of sulfhydryl sites both within cell envelopes and on EPS material (Norrod et al, 1993;Fein et al, 2019). Therefore, our findings suggest that the energy source and perhaps other environmental factors during cell growth play important roles in the production of surface proteins and hence cell surface sulfhydryl binding sites by P. putida (Choi et al, 2014).…”