No abstract

The efficient export of a subset ofEscherichia coli envelope proteins is dependent upon the product of the secB gene. Previous studies indicated that SecB promotes the export of the periplasmic maltose-binding protein (MBP) by preventing premature folding of the precursor MBP in the cytoplasm into an export-incompetent form. In this study, SecB has been purified to homogeneity and shown to be a soluble, cytoplasmic, multimeric protein composed of identical 17-kDa subunits. SecB was required for efficient in vitro translocation of MBP into inverted membrane vesicles. The addition of purified SecB to an in vitro system prepared from SecB-cells significantly enhanced MBP translocation. The purified protein also quantitatively retarded folding of precursor MBP into a stable, protease-resistant conformation in the absence of membranes. Finally, the inclusion of excess purified SecB in a SecB+ in vitro system significantly prolonged the time in which precursor MBP remained competent for posttranslational import into membrane vesicles.A number of components of the protein export machinery of Escherichia coli have been identified by genetic approaches [reviewed by Oliver (1)] and by biochemical approaches using in vitro protein translocating systems (2-4). The export of a subset of envelope proteins, including the periplasmic maltose-binding protein (MBP), is adversely affected by mutations in secB, a nonessential gene that maps near min 81 on the E. coli chromosome (5, 6). We have presented (7) evidence that the SecB protein functions as an antifolding factor that specifically interacts with the mature region of the precursor MBP (pre-MBP) to prevent its premature folding in the cytoplasm into a translocation-incompetent form. Kumamoto and Gannon (8) reached a similar conclusion by using a different experimental approach. Thus a specific biochemical function was assigned to the product of an E. coli sec gene, which complemented earlier work by Randall and Hardy (9) correlating the folding of pre-MBP into its stable tertiary structure in the cytoplasm with the loss ofexport competence. The essential role of protein conformation during membrane translocation has been recognized in eukaryotic cells as well (10). For example, unfolding factors that facilitate translocation of proteins into the endoplasmic reticulum and mitochondria have been described (11,12).An understanding ofprotein export in E. coli will be greatly aided by the purification and characterization of the individual components that mediate this process and eventual reconstitution of a complete protein translocating system in vitro. This study was undertaken to demonstrate the SecB dependence of MBP translocation in vitro and to purify biologically active SecB capable of interacting with newly synthesized pre-MBP to retard its folding and promote its import into membrane vesicles. MATERIALS AND METHODSIn Vitro MBP Synthesis and Translocation. MBP was synthesized in vitro by using a coupled E. coli transcriptiontranslation system, in the presence o...

Chlamydia trachomatis and Neisseria gonorrhoeae are universally acknowledged as urethral pathogens, yet the etiology in the majority of cases of urethritis is unclear. Our case-control study assessed the association of Mycoplasma genitalium, Ureaplasma urealyticum, and other potential pathogens with acute nongonococcal urethritis (NGU) in heterosexual men presenting to an urban sexually transmitted diseases clinic. M. genitalium was detected in 27 (22%) of 121 NGU case patients and in 5 (4%) of 117 control subjects (P<.01). Although C. trachomatis was detected in 36 (30%) of 121 NGU case patients and in 4 (3%) of 117 control subjects (P<.01), only 3 men with NGU were infected with both C. trachomatis and M. genitalium. U. urealyticum was not associated with NGU. By multivariate analyses, controlling for age, race, history of prior urethritis, and chlamydial infection, M. genitalium was associated with a 6.5-fold increased risk of urethritis (95% confidence interval, 2.1-19.5), which supports a role of this organism in the etiology of NGU.

The authors found that HSV-2 is a more common etiology of GUD than has been suggested by previous studies conducted in South Africa; serologic evidence of HSV-2 infection and current cases of genital herpes are strongly associated with HIV infection among men who present to STD clinics with GUD or urethritis.

A multiplex polymerase chain reaction (M-PCR) assay for Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV) was compared with clinical and standard laboratory methods for the diagnosis of genital ulcer disease (GUD) in 105 patients; 36% were human immunodeficiency virus (HIV)-seropositive. Chancroid (80%), syphilis (8%), and genital herpes (8%) were the most frequent diagnoses. H. ducreyi and HSV were isolated from ulcers of 43% and 18% of patients, respectively; in 35%, all cultures were negative and the laboratory diagnosis indeterminate. M-PCR detected H. ducreyi, T. pallidum, and HSV in 56%, 23%, and 26% of patients, respectively; (no definitive diagnosis, 6%). The proportion of patients with more than one agent was 4% by culture and 17% by M-PCR (P = .002). Resolved sensitivities of M-PCR for H. ducreyi and HSV cultures were 95% and 93%, respectively. The sensitivities of H. ducreyi and HSV cultures were 75% and 60%, respectively. HSV, detected in 47% of specimens from HIV-infected versus 16% from HIV-uninfected patients (P < .001), may be emerging as a more frequent cause of GUD.