crB ((r37) is a secondary species of RNA polymerase sigma factor found in the gram-positive bacterium Bacillus subtilis. To study the function of (rB genetically, we sought mutations that block the expression of a gene (ctc) known to be transcribed by B-containing RNA polymerase in vitro. One such mutation, called crl, was found to map in or near the structural gene (sigB) for (yB. To determine directly whether mutations in sigB would prevent transcription of ctc, we replaced sigB in the B. subtilis chromosome with insertion and deletion mutations that disrupted the cJB coding sequence. Like crl, these in vitro-constructed mutations blocked expression of ctc, but had little or no effect on viability, sporulation, expression of the sporulation gene spoVG, or production of sporulation-associated alkaline protease. Using fusions of ctc to the reporter genes xylE and lacZ, we also identified mutations that enhanced ctc expression. One such mutation, called socB, was found to be located in an open reading frame immediately downstream of sigB.The DNA-dependent RNA polymerase of the grampositive bacterium Bacillus subtilis is a highly heterogenous enzyme that exists in several different holoenzyme forms (4, 13). Each form of holoenzyme consists of core RNA polymerase and one of several different species of sigma factor that confers on the core enzyme the capacity to recognize and initiate transcription from a particular class of promoters. We have been interested in a secondary species of sigma factor called aB (formerly J7; 9, 10). This 29.9-kilodalton polypeptide (1, 7), which is found in vegetative and earlystationary-phase cells, directs the transcription in vitro of certain genes that are activated at the end of the exponential phase of growth (9-12). However, little is known about the contribution of arB to the expression of these genes in vivo or its role in B. subtilis gene expression. The recent identification of the structural gene for crJB (sigB; 1, 7) and the availability of plasmid and bacteriophage vectors bearing fusions of genes transcribed in vitro by cB_containing RNA polymerase to the reporter genes cat, xylE, and lacZ (11,17,21) have made it possible to study the function of this minor sigma factor genetically. In this paper we describe the identification and characterization of mutations in and near sigB that enhance or inhibit the expression of a B. subtilis gene, ctc, which is known to be actively transcribed by aB-containing RNA polymerase in vitro. One such mutation that significantly stimulates ctc transcription was found to be located in an open reading frame immediately adjacent to and downstream from sigB. We also describe the construction of insertion and deletion mutations in sigB and report, in confirmation and extension of previous work (1, 7), that they have little or no effect on cell viability, sporulation, or sporulation-associated gene expression.

During infection with Chlamydia trachomatis, CD8 ؉ T cells are primed, even though the bacteria remain confined to a host cell vacuole throughout their developmental cycle. Because CD8 ؉ T cells recognize antigens processed from cytosolic proteins, the Chlamydia antigens recognized by these CD8 ؉ T cells very likely have access to the host cell cytoplasm during infection. The identity of these C. trachomatis proteins has remained elusive, even though their localization suggests they may play important roles in the biology of the organism. Here we use a retroviral expression system to identify Cap1, a 31-kDa protein from C. trachomatis recognized by protective CD8 ؉ T cells. Cap1 contains no strong homology to any known protein. Immunofluorescence microscopy by using Cap1-specific antibody demonstrates that this protein is localized to the vacuolar membrane. Cap1 is virtually identical among the human C. trachomatis serovars, suggesting that a vaccine incorporating Cap1 might enable the vaccine to protect against all C. trachomatis serovars. The identification of proteins such as Cap1 that associate with the inclusion membrane will be required to fully understand the interaction of C. trachomatis with its host cell.

ABSTRACTaJ37 is a minor species of RNA polymerase cr factor found in the Gram-positive bacterium Bacillus subtilis. 37 governs the transcription in vitro of genes that are turned on at an early stage in spore formation, as well as other genes that are switched on at the end of the exponential phase of growth but that are not under sporulation control. To study the role of O37 in B. subtilis gene expression, we have cloned the gene for this minor species of (F factor in Escherichia coli by using as a hybridization probe a synthetic oligonucleotide that was designed on the basis of the NH2-terminal amino acid sequence of o'37 protein. We determined the nucleotide sequence of the entire o.37 gene, which was found to encode a 262-amino acid residue polypeptide of 29.9 kDa. The predicted amino acid sequence of a37 showed significant homology to that of other (r proteins in a region that has been proposed to be the site ofbinding of these factors to core RNA polymerase. holoenzyme forms in vitro, is induced at the end of the exponential phase of growth but under nutritional conditions that do not support spore formation (38). Unlike spoVG transcription, ctc RNA synthesis is not dependent on the products ofspoO genes but is instead stimulated by mutations in these regulatory genes. Studies on the interaction of E&37 with the ctc and spoVG promoters indicate that promoter recognition by this minor holoenzyme form is determined by contacts with conserved bases in the "-35" and "-10" regions of its cognate promoters (refs. 19 and 21-23; P. Zuber, J. Healy, and R.L., unpublished results).Although much has been learned about the interaction of Eoa37 with spoVG and ctc from in vitro studies, it has not yet been possible to assess the contribution of this minor holoenzyme form to the expression and differential regulation of these genes in vivo or to investigate the general role of Eo-37 in the overall pattern of B. subtilis gene expression.As a way of approaching this problem, we have cloned in Escherichia coli the structural gene for a37 and we have determined the location of its coding sequence on the B. subtilis chromosome. We show that the predicted product of the 37 gene, which we designate sigB, contains a region of significant homology to other B. subtilis and E. coli a factors and is evidently not essential for growth or sporulation, as judged by the construction of a sigB insertion mutation. MATERIALS AND METHODSBacterial Strains and Plasmids. Cloning of DNA from B. subtilis strain 168 (trpC2) was carried out in E. coli strains MM294 and HB101 using the plasmid vectors pBR322 and pJH101 (24). Mapping experiments were carried out in B. subtilis strain QB928 (aroI, purB33, dal, and trpC2; see ref. 25).Abbreviations: kb, kilobase(s); bp, base pair(s). 5943The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

BackgroundWhile families of polymorphic membrane protein (pmp) genes have been identified in several Chlamydia species, their function remains mostly unknown. These proteins are of great interest, however, because of their location in the outer membrane and possible role in chlamydial virulence.Methodology/Principal FindingWe analyzed the relative transcription of the pmpD gene, a member of the pmp gene family in C. trachomatis serovar L2, and its protein product translation and processing during the chlamydial developmental cycle. By real-time reverse transcription polymerase chain reaction, the pmpD gene was found to be upregulated at 16 to 24 four hours after infection. Using polyclonal antibodies generated against the predicted passenger domain of PmpD, we demonstrated that it is initially localized on the surface of reticulate bodies, followed by its secretion outside Chlamydia starting at 24 hours after infection. In elementary bodies, we found a ≈157 kDa PmpD only inside the cell. Both events, the upregulation of pmpD gene transcription and PmpD protein processing and secretion, are coincidental with the period of replication and differentiation of RBs into EBs. We also demonstrated that, in the presence of penicillin, the cleavage and secretion of the putative passenger domain was suppressed.Conclusion/SignificanceOur results are in agreement with the general concept that PmpD is an autotransporter protein which is post-translationally processed and secreted in the form of the putative passenger domain outside Chlamydia at mid- to- late point after infection, coinciding with the development of RBs into EBs.

To study the responses of the host cell to chlamydial infection, differentially transcribed genes of the host cells were examined. Complementary DNA (cDNA) probes were made from messenger RNAs of HeLa cells infected with Chlamydia trachomatis and were hybridized to a high-density human DNA microarray of 15,000 genes and expressed sequence tags. C. trachomatis alters host cell transcription at both the early and middle phases of its developmental cycle. At 2 h after infection, 13 host genes showed mean expression ratios >/=2-fold. At 16 h after infection, 130 genes were differentially transcribed. These genes encoded factors inhibiting apoptosis and factors regulating cell differentiation, components of the cytoskeleton, transcription factors, and proinflammatory cytokines. This indicates that chlamydial infection, despite its intravacuolar location, alters the transcription of a broad range of host genes in diverse cellular pathways and provides a framework for future studies.