SSP2, a sporulation-specific gene necessary for outer spore wall assembly in the yeast Saccharomyces cerevisiae.

Sporulation in yeast consists of two highly coordinated processes. First, a diploid cell that is heterozygous at the mating-type locus undergoes meiosis, in which one round of DNA replication is followed by two rounds of nuclear division. Second, the meiotic products are packaged into spore cells that remain within the mother cell. A large number of genes are induced specifically during sporulation, and their products carry out different sporulation-specific events. Expression of these sporulation-specific genes is controlled by several regulators which function at different stages of the sporulation program, resulting in a cascade of gene expression following induction of meiosis. Here we describe one sporulation-specific gene, SSP2, which is induced midway through meiosis. Ssp2 shows significant homology to the predicted product of a hypothetical ORF in Candida albicans. Homozygous mutant ssp2 diploid cells fail to sporulate. In the mutant background, meiotic recombination and nuclear divisions remain normal; however, viability declines rapidly. Following meiosis, ssp2 cells form the prospore membrane, but fail to form the outer layer of the spore wall. The Ssp2 protein localizes to the spore wall after meiosis II. In addition, the ssp2 defect is also associated with delayed and reduced expression of late sporulation-specific genes. Our results suggest that SSP2 function is required after meiosis II and during spore wall formation.

Identification and characterization of mycobacterial proteins differentially expressed under standing and shaking culture conditions, including Rv2623 from a novel class of putative ATP-binding proteins.

The environmental signals that affect gene regulation in Mycobacterium tuberculosis remain largely unknown despite their importance to tuberculosis pathogenesis. Other work has shown that several promoters, including acr (also known as hspX) (alpha-crystallin homolog), are upregulated in shallow standing cultures compared with constantly shaking cultures. Each of these promoters is also induced to a similar extent within macrophages. The present study used two-dimensional gel electrophoresis and mass spectrometry to further characterize differences in mycobacterial protein expression during growth under standing and shaking culture conditions. Metabolic labeling of M. bovis BCG showed that at least 45 proteins were differentially expressed under standing and shaking culture conditions. Rv2623, CysA2-CysA3, Gap, and Acr were identified from each of four spots or gel bands that were specifically increased in bacteria from standing cultures. An additional standing-induced spot contained two comigrating proteins, GlcB and KatG. The greatest induction was observed with Rv2623, a 32-kDa protein of unknown function that was strongly expressed under standing conditions and absent in shaking cultures. Analysis using PROBE, a multiple sequence alignment and database mining tool, classified M. tuberculosis Rv2623 as a member of a novel class of ATP-binding proteins that may be involved in M. tuberculosis's response to environmental signals. These studies demonstrate the power of combined proteomic and computational approaches and demonstrate that subtle differences in bacterial culture conditions may have important implications for the study of gene expression in mycobacteria.

Functional classification of cNMP-binding proteins and nucleotide cyclases with implications for novel regulatory pathways in Mycobacterium tuberculosis.

We have analyzed the cyclic nucleotide (cNMP)-binding protein and nucleotide cyclase superfamilies using Bayesian computational methods of protein family identification and classification. In addition to the known cNMP-binding proteins (cNMP-dependent kinases, cNMP-gated channels, cAMP-guanine nucleotide exchange factors, and bacterial cAMP-dependent transcription factors), new functional groups of cNMP-binding proteins were identified, including putative ABC-transporter subunits, translocases, and esterases. Classification of the nucleotide cyclases revealed subtle differences in sequence conservation of the active site that distinguish the five classes of cyclases: the multicellular eukaryotic adenylyl cyclases, the eukaryotic receptor-type guanylyl cyclases, the eukaryotic soluble guanylyl cyclases, the unicellular eukaryotic and prokaryotic adenylyl cyclases, and the putative prokaryotic guanylyl cyclases. Phylogenetic distribution of the cNMP-binding proteins and cyclases was analyzed, with particular attention to the 22 complete archaeal and eubacterial genome sequences. Mycobacterium tuberculosis H37Rv and Synechocystis PCC6803 were each found to encode several more putative cNMP-binding proteins than other prokaryotes; many of these proteins are of unknown function. M. tuberculosis also encodes several more putative nucleotide cyclases than other prokaryotic species.

Intracellular passage within macrophages affects the trafficking of virulent tubercle bacilli upon reinfection of other macrophages in a serum-dependent manner.

SETTING: The interaction of tubercle bacilli with macrophages is central to understanding of tuberculosis disease. OBJECTIVE: The objective was to determine whether prior passage within macrophages affects the behavior of Mycobacterium tuberculosis (Mtb) upon re-entry into other macrophages. DESIGN: Transmission electron microscopy was used to monitor fusion of bacterial phagosomes with late endosomal/lysosomal compartments using thoria as a fluid phase marker. Two-dimensional polyacrylamide gel electrophoresis was used to study bacterial protein expression within macrophages. RESULTS: H37Rv and BCG expressed novel proteins within macrophages. H37Rv also underwent less fusion after intracellular (IC) (24.2+/-7.7%) than extracellular (XC) (67.4+/-5.5%) passage when the bacteria entered new macrophages in small clusters. These effects were inhibited by serum, and were not observed with H37Ra or BCG bacteria (78.9+/-1.6% fused for all conditions). In addition, vacuoles which contained single bacilli were less likely to acquire markers (26.9+/-2.6%) than those that contained multiple bacilli (77.3+/-2.8%). CONCLUSION: These results indicate that phagolysosomal fusion patterns can be modulated by a variety of factors and that virulent Mtb bacteria may express proteins within macrophages that alter their interaction with these host cells.

Fluorescence-based detection of lacZ reporter gene expression in intact and viable bacteria including Mycobacterium species.

A variety of fluorescein di-beta-D-galactopyranoside (FDG)-based substrates were evaluated for measuring beta-galactosidase expression in bacteria. One substrate, 5-acetylamino-FDG (C2FDG), performed well in all bacteria tested, including the slow growing mycobacterium, Mycobacterium bovis BCG. The sensitivity of C2FDG in intact, viable BCG was similar to that of o-nitrophenyl-beta-D-galactopyranoside in cell lysates when used to measure lacZ reporter gene activity. C2FDG was approximately 70-fold more sensitive than green fluorescent protein (GFP) in BCG when assayed in a fluorescence plate reader, and comparable to GFP when measured by flow cytometry. These assays provide an important new alternative for the rapid measurement of reporter gene expression in viable bacteria.

High-frequency RecA-dependent and -independent mechanisms of Congo red binding mutations in Yersinia pestis.

Yersinia pestis, which causes bubonic and pneumonic plague, forms pigmented red colonies on Congo red (CR) dye agar. The hmsHFRS genes required for CR binding (Crb(+)) are genetically linked to virulence-associated genes encoding a siderophore uptake system. These genes are contained in a 102-kb chromosomal pgm locus that is lost in a high-frequency deletion event, resulting in loss of the Crb(+) phenotype. We constructed a recA mutant strain of Y. pestis KIM10+ (YPRA) to test whether the high frequency Crb mutants result from a RecA-mediated deletion of the IS100-flanked pgm locus. Two Pgm-associated phenotypes (Crb(+) and pesticin sensitivity [Pst(s)]) were used as markers for the presence of the pgm locus in the RecA(+) KIM10+ and RecA(-) YPRA strains. In KIM10+, both phenotypes were lost at a very high (2 x 10(-3)) frequency, due to the deletion of the entire pgm locus. In YPRA, the Crb(+) phenotype was still lost at a high frequency (4.5 x 10(-5)), although the loss of the Pst(s) phenotype occurred at spontaneous antibiotic resistance mutation frequencies (2 x 10(-7)). These RecA-independent Crb(-) mutants were caused by mutations in both the hmsHFRS locus and in a newly identified gene, hmsT. Nonpigmented Yersinia pseudotuberculosis and Escherichia coli strains transformed with both hmsT and hmsHFRS became Crb(+). This study demonstrates that in a laboratory culture, the Crb(+) phenotype is unstable, independent of the pgm locus deletion. We propose that a lack of selection for the CR-binding ability of Y. pestis in vitro may contribute to the mutation frequencies observed at the hmsHFRS and hmsT loci.

Independent acquisition and insertion into different chromosomal locations of the same pathogenicity island in Yersinia pestis and Yersinia pseudotuberculosis.

We show that Yersinia pestis and pesticin-sensitive isolates of Y. pseudotuberculosis possess a common 34 kbp DNA region that has all the hallmarks of a pathogenicity island and is inserted into different asparaginyl tRNA genes at different chromosomal locations in each species. This pathogenicity island (YP-HPI) is marked by IS100, has a G + C content different from its host, is flanked by 24 bp direct repeats, encodes a putative, P4-like integrase and contains the iron uptake virulence genes from the pgm locus of Y. pestis. These findings indicate independent horizontal acquisition of this island by Y. pestis and Y. pseudotuberculosis. The two YP-HPI locations and their possession of an integrase gene support a model of site-specific integration of the YP-HPI into these bacteria.

Homology with a repeated Yersinia pestis DNA sequence IS100 correlates with pesticin sensitivity in Yersinia pseudotuberculosis.

We have identified IS100 sequences in a specific subset of Yersinia pseudotuberculosis isolates that were also sensitive to the Y. pestis-produced bacteriocin, pesticin. In contrast, Y. pseudotuberculosis strains which did not contain IS100 sequences were not sensitive to pesticin. We propose that IS100 serves as a molecular marker that identifies a subset of Y. pseudotuberculosis isolates that have a particularly close evolutionary and/or ecological relationship with Y. pestis.

Cytotoxicity for lung epithelial cells is a virulence-associated phenotype of Mycobacterium tuberculosis.

Dissemination of viable tubercle bacilli from the lung is a critical event in the establishment of Mycobacterium tuberculosis infection. We examined the possibility that M. tuberculosis bacteria could infect and damage lung epithelial cells to determine whether direct penetration of the alveolar epithelium is a plausible route of M. tuberculosis infection. While both virulent H37Rv tubercle bacilli and the attenuated Mycobacterium bovis BCG vaccine strain were able to enter A549 human lung epithelial cells in culture, only the virulent tubercle bacilli were cytotoxic for both polarized and nonpolarized epithelial monolayers and macrophages. In addition, bacterial entry into epithelial cells, but not macrophages, was increased by intracellular passage through macrophages, suggesting enhancement of a bacterially mediated cell entry mechanism in bacteria grown within macrophages. These findings suggest that M. tuberculosis bacteria might have the ability to gain access to the host lymphatics and circulatory system by directly penetrating the alveolar epithelial lining of an infected lung.

Mutation in the pla gene of Yersinia pestis alters the course of the plague bacillus-flea (Siphonaptera: Ceratophyllidae) interaction.

Yersinia pestis possesses a unique gene (pla) encoding coagulase and fibrinolysin which is implicated in the transmission of plague by fleas. This gene is encoded on the highly conserved but poorly characterized 'pesticin' plasmid pKYP1. The role of the pKYP1-encoded gene, pla, in plague transmission was addressed by feeding fleas on blood containing avirulent Y. pestis strain EV76-6 and three derivatives of this strain (K10-2, K10-3, and K10-5) carrying Tn801 insertions in pKYP1. One of these mutant strains, K10-5, contains an insertion within the pla gene that eliminates both coagulase and fibrinolysin activities, whereas strains K10-3 and K10-2 retain both pla-associated phenotypes. After feeding, it was found that flea mortality at 4 d after infection associated with strain K10-5 (26%) was significantly lower than the mortality observed with other strains (53-64%). These results suggest that expression of the pla gene product may contribute to the deleterious effects of plague bacilli on fleas that have been associated with flea blockage and plague transmission. This increased mortality is not caused simply by an increased bacterial load in fleas containing pla+ bacteria because fleas ingesting pla+ strains contained no more bacteria by flea blot hybridization analysis than did those that ingested the pla- strain K10-5. It is anticipated that further work in this area will clarify the mechanism by which pla acts and will reveal additional genetic loci in the plague bacillus which are required for transmission by fleas.

Pathogenesis of tuberculosis: interaction of Mycobacterium tuberculosis with macrophages.

Central to understanding the pathogenesis of tuberculosis is the interaction between the pathogen and mononuclear phagocytes. A key question about that interaction is whether Mycobacterium tuberculosis exerts an effect on phagolysosome fusion. We have reexamined the dynamics of phagolysosome fusion and its effect on intracellular bacterial replication in M. tuberculosis-infected macrophages by performing an extensive study at the electron microscopic level. Thoria-labelled murine and human macrophages were infected with a virulent (H37Rv) or avirulent (H37Ra) strain of M. tuberculosis or with Mycobacterium bovis BCG vaccine for times ranging from 2 h to 7 days. In all cases, by 2 h postinfection, approximately 85% of the bacteria clearly resided in fused vacuoles. However, at 4 days postinfection, fusion levels for viable H37Rv and H37Ra were reduced by half, whereas the fusion profiles of BCG and of heat-killed H37Rv and H37Ra were unchanged. A comparison of the numbers of bacteria per fused and nonfused vacuoles suggests both a net transfer of bacteria out of fused vacuoles and preferential bacterial multiplication in nonfused vacuoles. H37Rv and H37Ra appeared to bud from the phagolysosomes into tightly apposed membrane vesicles that did not fuse with secondary lysosomes. In some cases, no such membrane was seen and the bacteria appeared to be free in the cytoplasm. Only viable H37Rv showed a significant increase in bacterial counts during the course of infection. Thus, both of the attenuated strains we examined differed from the virulent strain H37Rv in their abilities to replicate successfully within macrophages, but each diverged from H37Rv at a different point in the process. Viable tubercle bacilli H37Rv and H37Ra had the capacity to escape from fused vesicles as the infection progressed; BCG did not. After extrusion from the phagolysosome, H37Rv, but not H37Ra, was able to multiply. These results suggest a novel mechanism by which virulent M. tuberculosis eludes the microbicidal mechanisms of macrophages by escaping from fused phagolysosomes into nonfused vesicles or the cytoplasm.

Use of DNA hybridizations probes for detection of the plague bacillus (Yersinia pestis) in fleas (Siphonaptera: Pulicidae and Ceratophyllidae).

The detection of active plaque in nature relies primarily on demonstration of the etiologic agent of the disease. Yersinia pestis, in the flea vectors and susceptible mammalian hosts. A live animal assay is currently used for identification of a Y. pestis virulence antigen that is not expressed in the flea. We have found that DNA hybridization probes specific for Y. pestis, used in very simple sample preparation schemes, allow detection of Y. pestis in three species of fleas as well as tissues of experimentally infected mice at minimum concentrations of 1 x 10(6) bacilli/ml. We detected Y. pestis in 22 of 90 (24%) experimentally infected Xenopsylla cheopis (Rothschild), 13 of 25 (52%) Thrassis bacchi (Rothschild), and 9 of 25 (36%) Diamanus montanus (Baker), but no hybridization signals were observed from fleas that had fed on normal mice. The probe technique indicated infection in 9 of 10 potentially infected liver and spleen samples and none of the 5 control samples. Our techniques permit definitive diagnosis in 48 h.

A Yersinia pestis-specific DNA fragment encodes temperature-dependent coagulase and fibrinolysin-associated phenotypes.

The effect of temperature on coagulase and fibrinolysin expression (Pla) by Yersinia pestis has been implicated in the transmission of plague by fleas. In an attempt to improve our understanding of this process, we have cloned, sequenced and characterized the gene encoding the Pla phenotypes in Y. pestis, and examined its temperature-dependent regulation. The coding region for this gene overlaps a 900bp Y. pestis-specific DNA fragment that we have previously shown to be capable of detecting plague bacilli in fleas. The pla gene contains a single open reading frame encoding 312 amino acids with a predicted molecular weight of 34.7 kD and a putative signal sequence of 20 amino acids. This coding region appears to be sufficient for both coagulase and fibrinolytic activities. In Y. pestis, modulation between coagulase and fibrinolytic activities is temperature-dependent: coagulase activity is most evident at temperatures below 30 degrees C but fibrinolytic activity increases with higher temperatures (greater than 30 degrees C), regardless of the temperature at which the bacteria are grown. Our results lead us to believe that this regulation occurs post-translationally. It is possible that the alternative forms of the Pla protein are essential to 'flea blockage' and subsequent transmission of the plague bacillus to animals.

Identification of a Yersinia pestis-specific DNA probe with potential for use in plague surveillance.

A 900-base-pair DNA fragment derived from a 9.5-kilobase plasmid in Yersinia pestis hybridized specifically with Y. pestis DNA. We demonstrated the feasibility of using this DNA fragment to detect plague bacilli directly in fleas, suggesting that this Y. pestis-specific DNA probe may be used for plague surveillance in the field. Additional applications for this DNA probe may include plague diagnosis and pathogenesis research.