(Kcccived October 25, 1991) -EJB 91 1438In addition to the 50-kDa (a) and 40-kDa (0) subunits, a n 11-kDa polypeptide has been discovered in highly purified Desulfovihrio vulguris (Hildenborough) dissimilatory sulfite reductase. This is in contrast with the hitherto generally accepted u2p2 tetrameric subunit composition. Purification, high-ionic-strength gel-filtration, native electrophoresis and isoelectric focussing d o not result in dissociation of the 11-kDa polypeptide from the complex. Densitometric scanning of SDS gels and denaturing gel-filtration indicate a stoichiometric occurrence. A similar 1 I-kDa polypeptide is present in the desulfoviridin of D. vulgaris oxarnicus (Monticello), D . gigas and D. dcsulfuricuns ATCC 27774. We attribute an cc,D,y, subunit structure to desulfoviridin-type sulfite reductases. N-terminal sequences of the a, / 3 and y subunits are reported.A key enzyme in dissimilatory sulfate reduction is sulfite reductase, a complex redox enzyme containing both Fe/S and siroheme prosthetic groups. Sulfite reductases that are supposed to have a dissimilatory function have been observed in, and isolated from, over 20 species of microorganisms [ l -41. Kinetic parameters and subunit structure of dissimilatory sulfite reductases are markedly different from assimilatory sulfite reductases.Dissimilatory enzymes are x 2 P 2 proteins (1 50-230 kDa) that have a millimolar K,,, for sulfite, a slightly acidic pH optimum and reduce their substrate to SJOzSp and S20:-(1, 4-81, The physiological relevance of the in vitro observed product composition is a matter of controversy. A number of explanations has been put forward: loss of interaction with the cytoplasmic membrane [9], in vitro assay conditions [lo], and the possibility that the observed products are in vivo intermediates [I 11. Partial demctallation of the siroheme is observed in desulfoviridins [2,12] and is presumably an intrinsic feature of some dissimilatory enzymes. It probably has no bearing on the formation of S 3 0 i -and SzO:-since desulfoviridin, desulfofuscidin, P582 and desulforubidin-type dissimilatory enzymes have a comparable product composition and specific activity [2-7, 131.Contrarily, assimilatory sulfite reductases cleanly perform the full six-electron reduction to sulfide. They have a slightly basic pH optimum with a submillimolar K,, for sulfite. The subunit composition, however, is non-uniform: ugp4 [14], a4-6 1351, or cc2 [16]. This probably reflects differences in the source of reducing equivalents.A distinct, third, group of sulfitc reductases comprises the so-called low-molecular-mass sulfite reductases. These enzymes arc presumably assimilatory. They differ from the other two groups in two aspects: they are monomeric (20 -30 kDa), We report here that D . vulgaris (Hildenborough) desulfoviridin contains a small, hitherto unnoted, y-subunit in addition to the usual a2P2 motive of dissimilatory enzymes. Nterminal sequences, size and stoichiometry of the subunits have been determined. An immunological comparison of d...

A Cornamonas acidooovans strain, designated NBA-10, was isolated on 4-nitrobenzoate as sole carbon and energy source. When grown on 4-nitrobenzoate, it was simultaneously adapted to 4-nitrosobenzoate and 4-hydroxylaminobenzoate but not to 4-hydroxybenzoate or 4-aminobenzoate. In cell extracts with NADPH present, 4-nitrobenzoate was degraded to 4-hydroxylaminobenzoate and 3,4-dihydroxybenzoate. Partial purification of the 4-nitrobenzoate reductase revealed that 4-nitrobenzoate is degraded via 4-nitrosobenzoate to 4-hydroxylaminobenzoate. The substrate specificity of the enzyme was narrow and NADPH was 15 times more effective as a cofactor than NADH. The results provide evidence for a novel pathway for aerobic degradation of 4-nitrobenzoate, since neither 4-hydroxybenzoate nor 4-aminobenzoate were involved in the degradative pathway.

Mucosal tolerance prevents the body from eliciting productive immune responses against harmless Ags that enter the body via the mucosae, and is mediated by the induction of regulatory T cells that differentiate in the mucosa-draining lymph nodes (LN) under defined conditions of Ag presentation. In this study, we show that mice deficient in FcγRIIB failed to develop mucosal tolerance to OVA, and demonstrate in vitro and in vivo a critical role for this receptor in modulating the Ag-presenting capacity of dendritic cells (DC). In vitro it was shown that absence of FcγRIIB under tolerogenic conditions led to increased IgG-induced release of inflammatory cytokines such as MCP-1, TNF-α, and IL-6 by bone marrow-derived DC, and increased their expression of costimulatory molecules, resulting in an altered immunogenic T cell response associated with increased IL-2 and IFN-γ secretion. In vivo we could show enhanced LN-DC activation and increased numbers of Ag-specific IFN-γ-producing T cells when FcγRIIB−/− mice were treated with OVA via the nasal mucosa, inferring that DC modulation by FcγRIIB directed the phenotype of the T cell response. Adoptive transfer of CD4+ T cells from the spleen of FcγRIIB−/− mice to naive acceptor mice demonstrated that OVA-responding T cells failed to differentiate into regulatory T cells, explaining the lack of tolerance in these mice. Our findings demonstrate that signaling via FcγRIIB on DC, initiated by local IgG in the mucosa-draining LN, down-regulates DC activation induced by nasally applied Ag, resulting in those defined conditions of Ag presentation that lead to Tr induction and tolerance.

Chloramphenicol is frequently used for better visualization of the Escherichia coli nucleoid. Here, we show that chloramphenicol causes not only rounding off of the nucleoid but also fusion of as many as four separated nucleoids. Nucleoid fusion occurred in fast-growing cells and in filaments obtained by dicF antisense RNA induction or in ftsZ84(Ts) and pbpB(Ts) mutants. Thus, treatment with chloramphenicol erroneously suggests that DNA segregation is inhibited.

The notion that the mucosal immune system maintains a tolerogenic response to harmless Ags while continually being challenged with microbial products seems an enigma. The aim of this study was to unravel mechanisms that are involved in regulating the development of tolerance under constant microbial pressure. The tolerogenic response to Ags administered via the nasal mucosa is dependent on the organized lymphoid tissue of the cervical lymph nodes (LN). We show that cervical LN differentially express secretory leukoprotease inhibitor (SLPI) compared with peripheral LN. SLPI was expressed by dendritic cells (DCs) and because SLPI is known to suppress LPS responsiveness, it was hypothesized that its expression in mucosal DCs may be required to regulate cellular activation to microbial products. Indeed, compared with wild-type controls, bone marrow-derived DCs from SLPI−/− mice released more inflammatory cytokines and enhanced T cell proliferation after stimulation with low dose LPS. This increased sensitivity to LPS was accompanied by increased NF-κB p65 activation in SLPI−/− DCs. In vivo, nasal application of OVA with LPS to SLPI−/− mice resulted in enhanced DC activation in the cervical LN reflected by increased costimulatory molecule expression and release of inflammatory cytokines. This led to failure to maintain tolerance to nasal OVA application in the presence of low doses of LPS. We propose that expression of SLPI functions as a rheostat by controlling the level of bacterial stimuli that induce mucosal DC activation. As such, it regulates the quality of the ensuing Ag-specific immune response in the mucosa draining LN.