BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N-terminal protease domain and a C-terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone-like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the β-barrel assembly machinery (BAM) driving integration of β-barrel proteins into the outer membrane. However, the molecular mechanism of BepA function and its association with the BAM complex remains unclear. Here, we determined the crystal structure of the BepA TPR domain, which revealed the presence of two subdomains formed by four TPR motifs. Systematic site-directed in vivo photo-cross-linking was used to map the protein-protein interactions mediated by the BepA TPR domain, showing that this domain interacts both with a substrate and with the BAM complex. Mutational analysis indicated that these interactions are important for the BepA functions. These results suggest that the TPR domain plays critical roles in BepA functions through interactions both with substrates and with the BAM complex. Our findings provide insights into the mechanism of biogenesis and quality control of the outer membrane.

No abstract

E , an alternative factor that governs a major signaling pathway in envelope stress responses in Gram-negative bacteria, is essential for growth of Escherichia coli not only under stressful conditions, such as elevated temperature, but also under normal laboratory conditions. A mutational inactivation of the hicB gene has been reported to suppress the lethality caused by the loss of E . hicB encodes the antitoxin of the HicA-HicB toxin-antitoxin (TA) system; overexpression of the HicA toxin, which exhibits mRNA interferase activity, causes cleavage of mRNAs and an arrest of cell growth, while simultaneous expression of HicB neutralizes the toxic effects of overproduced HicA. To date, however, how the loss of HicB rescues the cell lethality in the absence of E and, more specifically, whether HicA is involved in this process remain unknown. Here we showed that simultaneous disruption of hicA abolished suppression of the E essentiality in the absence of hicB, while ectopic expression of wild-type HicA, but not that of its mutant forms without mRNA interferase activity, restored the suppression. Furthermore, HicA and two other mRNA interferase toxins, HigB and YafQ, suppressed the E essentiality even in the presence of chromosomally encoded cognate antitoxins when these toxins were overexpressed individually. Interestingly, when the growth media were supplemented with low levels of antibiotics that are known to activate toxins, E. coli cells with no suppressor mutations grew independently of E . Taken together, our results indicate that the activation of TA system toxins can suppress the E essentiality and affect the extracytoplasmic stress responses. IMPORTANCEE is an alternative factor involved in extracytoplasmic stress responses. Unlike other alternative factors, E is indispensable for the survival of E. coli even under unstressed conditions, although the exact reason for its essentiality remains unknown. Toxin-antitoxin (TA) systems are widely distributed in prokaryotes and are composed of two adjacent genes, encoding a toxin that exerts harmful effects on the toxin-producing bacterium itself and an antitoxin that neutralizes the cognate toxin. Curiously, it is known that inactivation of an antitoxin rescues the E essentiality, suggesting a connection between TA systems and E function. We demonstrate here that toxin activation is necessary for this rescue and suggest the possible involvement of TA systems in extracytoplasmic stress responses.

Mycoplasma species cannot be identified by routine bacteriological culture methods and are resistant to common antimicrobial agents. Mycoplasma hominis usually colonizes the lower urogenital tract and causes pyelonephritis, pelvic inflammatory disease, chorioamnionitis, rupture of fetal membranes, preterm labor, postpartum fever, postabortal fever, and neonatal infection. This organism is highly prevalent in cervicovaginal cultures of sexually active women. M. hominis, M. genitalis, Ureaplasma urealyticum, and U. parvum may invade and infect placental and fetal tissues, leading to adverse pregnancy outcomes. M. hominis occasionally causes nongenitourinary infection of the blood, wounds, central nervous system, joints, or respiratory tract. We present a case of a 27-year-old woman who developed abdominal wound hematoma and abscess after cesarean section. The wound was drained, but her high fever persisted, in spite of antibiotic treatment using flomoxef sodium and imipenem·cilastatin sodium. Because the exudate exhibited M. hominis growth in an anaerobic environment, we administered the quinolone ciprofloxacin. This therapy resolved her fever, and her white blood cell count and C-reactive protein level diminished to the normal ranges. To our knowledge, there are four published articles regarding the isolation of M. hominis from postcesarean incisions. Based on the current study and the literature, infection by this pathogen may cause hematoma formation with or without abscess after cesarean section or in immunosuppressed postoperative patients. In such cases, physicians may need to suspect Mycoplasma infection and initiate appropriate antibacterial treatment as soon as possible in order to avoid persistent fever.

Escherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246–mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.